WO1996001323A1 - Alpha amylase from thermomyces lanuginosus - Google Patents
Alpha amylase from thermomyces lanuginosus Download PDFInfo
- Publication number
- WO1996001323A1 WO1996001323A1 PCT/EP1995/002607 EP9502607W WO9601323A1 WO 1996001323 A1 WO1996001323 A1 WO 1996001323A1 EP 9502607 W EP9502607 W EP 9502607W WO 9601323 A1 WO9601323 A1 WO 9601323A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- recombinant
- amylase
- enzyme
- nucleotide sequence
- seq
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
- C12N9/242—Fungal source
-
- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
- A21D8/00—Methods for preparing or baking dough
- A21D8/02—Methods for preparing dough; Treating dough prior to baking
- A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
- A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
Definitions
- the present invention relates to an ⁇ -amylase enzyme.
- the present invention also relates to the use of the ⁇ -amylase enzyme in the manufacture of foodstuffs, in particular foodstuff based on starch, and especially a bakery product, such as bread and similar baked products based on wheat flour.
- ⁇ -Amylases are also traditionally added to the wheat flour used in bread making to improve the baking quality of the flour.
- the fungal ⁇ -amylases used for this purpose is the fungal ⁇ -amylase from Aspergillus oryzae, Drapon, R. and Godon, B. in Enzymes And Their Role In Cereal Technology, (1987), Edited by Kinger, J.E. et al, 281-234, and known as the TAKA-amylase.
- the TAKA-amylase is far too thermolabile.
- the gelatinisation of starch i.e. the melting of starch crystals
- Bacterial ⁇ -amylases are too thermally stable so that they are not properly inactivated by the baking process and may lead to a gummy product - see Leloup et al [(1993) Bioconversion of Cereal Products, pp 79-127, B.Godon Ed.].
- ⁇ -amylase has been isolated from Thermomyces lanuginosus (T. lanuginosus) and was found to have a half-life of 10 minutes at 70°C (Enzyme Microbiol. Technol. (1992), 14, 112-116). This ⁇ -amylase was not suggested for use in baking.
- EP-A-0579360 and WO 90/11352 disclose ⁇ -amylases obtained from Pyrococcus and from the bacterium P. fiiriosus, respectively. These ⁇ -amylases were not suggested for use in baking.
- an ⁇ -amylase should have at least two main criteria. First, the enzyme should be active during the starch gelatinisation stage, which occurs from about 60°C to about 70°C. Second, the enzyme should be inactivated during the baking stage, which occurs from about 80°C to about 100°C. Thus, an ⁇ -amylase with intermediate heat stability will be suitable for baking purposes.
- the present invention seeks to provide an ⁇ -amylase that can be beneficial for baking and which can be prepared easily and in large amounts.
- a recombinant enzyme having the following characteristics: i. ⁇ -amylase activity over the range from about 60°C to about 80°C; ii. a molecular weight of about 54 - 60 kDa when measured by SDS- PAGE, or of about 55265 Da +/- 100 Da when measured by mass spectrometry;
- a recombinant nucleotide sequence comprising the coding nucleotide sequence shown in SEQ. I.D. No. 1. or a variant, homologue or fragment thereof.
- a recombinant nucleotide sequence comprising the nucleotide sequence shown as SEQ. I.D. No. 1. or a variant, homologue or fragment thereof.
- a construct comprising the recombinant nucleotide sequence according to the present invention.
- a vector comprising the recombinant nucleotide sequence according to the present invention or the construct according to the present invention.
- a cell, tissue or organism comprising the recombinant nucleotide sequence according to the present invention or the construct according to the present invention or the vector according to the present invention.
- a transgenic organism comprising the recombinant nucleotide sequence according to the present invention or the construct according to the present invention or the vector according to the present invention or the cell, tissue or organism according to the present invention.
- NCIMB deposit No. 40655 NCIMB deposit No. 40656, NCIMB deposit No. 40657 or NCIMB deposit No. 40658.
- a method of preparing a bakery product comprising forming a mixture by contacting a wheat-flour dough with an ⁇ -amylase and baking the mixture, wherein the ⁇ -amylase is a recombinant ⁇ -amylase according to the present invention, or is a recombinant ⁇ - amylase expressed by the nucleotide sequence of the present invention, which may be present in any of the afore-mentioned nucleotide aspects of the present invention.
- a bakery product obtained by use of the recombinant ⁇ -amylase according to the present invention, or a recombinant ⁇ -amylase expressed by the nucleotide aspect of the present invention.
- a recombinant ⁇ -amylase having ⁇ -amylase activity over the range from about 60°C to about 80°C in baking.
- a method of preparing a foodstuff comprising forming a mixture by contacting starch or a starch based ingredient with an ⁇ -amylase and optionally processing the mixture, wherein the ⁇ -amylase is a recombinant ⁇ -amylase as defmed above or is a recombinant ⁇ - amylase expressed by the nucleotide aspect of the present invention.
- a foodstuff obtained by use of an ⁇ -amylase enzyme wherein the enzyme is a recombinant ⁇ - amylase as defined above or is a recombinant ⁇ -amylase expressed by the nucleotide aspect of the present invention.
- a fourteenth aspect of the present invention there is provided the use of a recombinant ⁇ -amylase having ⁇ -amylase activity over the range from about 60°C to about 80°C in preparing a foodstuff.
- Other aspects of the present invention include inserting the recombinant ⁇ -amylase nucleotide sequence of the present invention into a suitable vector and/or transforming a suitable host organism with the recombinant ⁇ -amylase nucleotide sequence of the present invention and/or expressing that recombinant ⁇ -amylase nucleotide sequence gene within the host and/or collecting the expressed recombinant ⁇ -amylase and/or isolating the recombinant ⁇ -amylase.
- a recombinant nucleotide sequence which may be either an RNA or DNA sequence, that encodes for a recombinant protein according to the first aspect of the present invention, and in particular the DNA sequence shown in SEQ I.D. No. 1; ii) a nucleotide sequence that is functionally equivalent to the DNA sequence shown in SEQ I.D. No. 1, that is to say a DNA sequence showing substantial (at least 90%) homology (similarity) therewith and coding for a recombinant ⁇ -amylase having the same properties of the recombinant ⁇ -amylase having the amino acid sequence as set out in SEQ I.D.
- the enzyme comprises an N-terminal sequence shown as SEQ. I.D. No. 11.
- the enzyme comprises the sequence shown as SEQ. I.D. No. 1 or SEQ. I.D. No. 2, or a variant, homologue or fragment thereof.
- the enzyme is expressed by a nucleotide sequence comprising the coding sequence shown in SEQ. I.D. No. 1, or a variant, homologue or fragment thereof.
- the enzyme is expressed by a nucleotide sequence comprising the nucleotide sequence shown as SEQ. I.D. No. l, or a variant, homologue or fragment thereof.
- the enzyme is obtained or obtainable from T. lanuginosus.
- the enzyme is expressed by a member of the Aspergillus family.
- the enzyme is expressed by Aspergillus niger.
- the enzyme is obtainable from any one of NCIMB deposit No. 40655, NCIMB deposit No. 40656, NCIMB deposit No. 40657 and NCIMB deposit No. 40658.
- the transgenic organism is a filamentous fungi, preferably an Aspergillus fungus, more preferably Aspergillus niger.
- the recombinant ⁇ -amylase is incorporated into the dough in an amount sufficient to provide from 50 to 400 units of ⁇ -amylase activity per kg of wheat flour used to form the dough.
- the recombinant ⁇ -amylase is incorporated into the mixture in an amount sufficient to provide from 50 to 400 units of ⁇ -amylase activity per kg of starch or starch based ingredient to form the mixture.
- the foodstuff or bakery product is bread.
- the recombinant ⁇ -amylase is a recombinant ⁇ -amylase according to the first aspect of the present invention or is a recombinant ⁇ -amylase expressed by the nucleotide aspects of the present invention.
- the recombinant ⁇ -amylase enzyme of the present invention has a protein sequence as set out in SEQ.I.D. No. 1 and SEQ. I.D.
- the recombinant ⁇ -amylase enzyme of the present invention is coded by the nucleotide sequence as set out in SEQ.I.D. No. 1.
- the recombinant nucleotide sequence is the nucleotide sequence as set out in SEQ.I.D. No. 1.
- One of the advantages of the recombinant enzyme of the present invention is that when it is used in baking it improves the shelf-life of bread and other products made from a wheat-flour dough. In this regard, it improves the resistance of bread, and other products made from a wheat-flour dough, to staling.
- the recombinant ⁇ -amylase has a beneficial thermal activity profile for use in baking.
- the recombinant enzyme of the present invention can be beneficially used in combination with other enzymes useful in the preparation of bakery products.
- Other advantages are that the recombinant enzyme can be prepared easily, reliably and cheaply and in large amounts.
- expressing the gene in Aspergillus niger has the advantage that purification and recovery procedures of the recombinant ⁇ -amylase are simpler.
- expression in Aspergillus niger would obviate the need to remove the intron sequences prior to transformation of that host organism with the recombinant nucleotide sequence of the present invention.
- recombinant is used in its normal sense.
- the term “recombinant enzyme” does not include an enzyme when prepared by expression of the genomic nucleotide sequence coding for the enzyme when that nucleotide sequence is in its natural environment.
- the term covers the enzyme when prepared by expression of a recombinant nucleotide sequence coding for the enzyme when that nucleotide sequence is not in the natural environment for the genomic nucleotide coding sequence.
- recombinant nucleotide includes recombinant DNA and recombinant RNA.
- it means recombinant DNA, and in some instances it preferably means cDNA.
- the recombinant enzyme can be used on its own or in combination with one or more other enzymes.
- variant or variant or fragment include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid or amino acid from or to a respective sequence providing the resultant sequence has the respective ability to code for or act as an enzyme according to the present invention, preferably having at least the same activity of the enzyme comprising the sequence shown as the sequence listing SEQ. I.D. No. 1.
- homologue covers homology with respect to similarity of structure and/or similarity of function providing the resultant nucleotide sequence has the ability to code for an enzyme according to the present invention.
- sequence homology preferably there is more than 80% homology, more preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95% homology, more preferably at least 98% homology.
- the expressions also cover any codon replacement or substitution with another codon coding for the same amino acid or any addition or removal thereof providing the resultant enzyme has ⁇ -amylase activity.
- the present invention also covers a modified nucleotide sequence in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted so as to encode an enzyme having the activity of an ⁇ -amylase, preferably having an increased ⁇ -amylase activity.
- the above terms are also synonymous with allelic variations of the sequences.
- complementary means that the present invention also covers recombinant nucleotide sequences that can hybridise to the recombinant nucleotide sequences.
- construct which is synonymous with terms such as “conjugate”, “cassette” and “hybrid” - includes the nucleotide sequence according to the present invention directly or indirectly attached to the promoter.
- An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention.
- fused in relation to the present invention which includes direct or indirect attachment.
- the terms do not cover the natural combination of the wild type ⁇ -amylase gene ordinarily associated with the wild type gene promoter and the wild type promoter and when they are both in their natural environment.
- the construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a fungus or a plant cell into which it has been transferred.
- a marker which allows for the selection of the genetic construct in, for example, a fungus or a plant cell into which it has been transferred.
- Various markers exist which may be used in, for example, plants - such as mannose.
- Other examples of markers include those that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
- the construct of the present invention preferably comprises a promoter.
- promoter is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression.
- suitable promoters are those that can direct efficient expression of the nucleotide sequence of the present invention and/or in a specific type of cell.
- the promoter could additionally include conserved regions such as a Pribnow Box or a TATA box.
- the promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention.
- suitable examples of such sequences include the Shl-intron or an ADH intron.
- T. lanuginosus include products obtained directly from cultures of transformed T. lanuginosus and products obtained by expression of a recombinant ⁇ -amylase gene of the present invention obtainable from T. lanuginosus when the gene is in another cellular organism.
- the terms cover expression of the recombinant ⁇ -amylase gene of the present invention in another cellular organism, preferably Aspergillus niger.
- vector includes an expression vector and a transformation vector.
- expression vector means a construct capable of in vivo or in vitro expression.
- transformation vector means a construct capable of being transferred from one species to another - such as from an E.Coli plasmid to a fungus or a plant cell, or from an Agrobacterium to a plant cell.
- cell include cell, tissue and organ per se and when within an organism.
- organ in relation to the present invention includes any organism that could comprise the recombinant nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein the recombinant nucleotide sequence according to the present invention can be expressed when present in the organism.
- the organism is an ⁇ -amylase producing organism such as any one of a plant, algae, fungi, yeast and bacteria, as well as cell lines thereof.
- the organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.
- transgenic orgamsm in relation to the present invention includes any organism that comprises the recombinant nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein the recombinant nucleotide sequence according to the present invention can be expressed within the organism.
- the recombinant nucleotide sequence is incorporated in the genome of the organism.
- the transgenic organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.
- the host organism can be a prokaryotic or a eukaryotic organism.
- suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press). If a prokaryotic host is used then the recombinant nucleotide sequence of the present invention may need to be suitably modified before transformation - such as by removal of introns.
- a preferred host organism is of the genus Aspergillus, such as Aspergillus niger.
- the transgenic Aspergillus according to the present invention can be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-393), Davis R.W. 1994 (Heterologous gene expression and protein secretion in Aspergillus. In: Martinelli S.D. , Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp 525-560), Ballance, D.J. 1991 (Transformation systems for Filamentous Fungi and an Overview of Fungal Gene structure. In: Leong,S.A.
- Filamentous fungi have been widely used in industry for production of organic compounds and enzymes. Traditional Japanese koji and soy fermentations have used Aspergillus sp for hundreds of years. In this century Aspergillus niger has been used for production of organic acids particular citric acid and for production of various enzymes for use in industry.
- filamentous fungi There are two major reasons for that filamentous fungi have been so widely used in industry. First filamentous fungi can produce high amounts of extracellular products, for example enzymes and organic compounds such as antibiotics or organic acids. Second filamentous fungi can grow on low cost substrates such as grains, bran, beet pulp etc. The same reasons have made filamentous fungi attractive organisms as hosts for heterologous gene expression according to the present invention.
- expression constructs are prepared by inserting the recombinant nucleotide sequence of the present invention into a construct designed for expression in filamentous fungi.
- the constructs will contain a promoter which is active in fungi.
- promoters include a fungal promoter for a highly expressed extracellular enzyme, such as an glucoamylase promoter or an ⁇ -amylase promoter.
- the recombinant nucleotide sequence of the present invention can be fused to a signal sequence which directs the enzyme encoded by the nucleotide sequence to be secreted. Usually a signal sequence of fungal origin is used.
- a terminator active in fungi ends the expression system.
- Another type of expression system has been developed in fungi where the recombinant nucleotide sequence of the present invention is fused to a smaller or a larger part of a fungal gene encoding a stable protein. This can stabilize the protein encoded by the recombinant nucleotide sequence of the present invention.
- a cleavage site recognized by a specific protease, can be introduced between the fungal protein and the enzyme encoded by the recombinant nucleotide sequence of the present invention, so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the protein or enzyme encoded by die recombinant nucleotide sequence of the present invention.
- a fusion leads to cleavage in vivo resulting in protection of the recombinant enzyme and production of the recombinant enzyme and not a larger fusion protein or enzyme.
- Heterologous expression in Aspergillus has been reported for several genes coding for bacterial, fungal, vertebrate and plant proteins or enzymes.
- the proteins can be deposited intracellularly if the recombinant nucleotide sequence of the present invention is not fused to a signal sequence.
- Such proteins or enzymes will accumulate in the cytoplasm and will usually not be glycosylated which can be an advantage for some bacterial proteins or enzymes. If the recombinant nucleotide sequence of the present invention is equipped with a signal sequence the enzyme will , accumulate extracellulary.
- product stability and host strain modifications some heterologous proteins or enzymes are not very stable when they are secreted into the culture fluid of fungi. Most fungi produce several extracellular proteases which degrade heterologous proteins or enzymes. To avoid this problem special fungal strains with reduced protease production have been used as hosts for heterologous production.
- transformation protocols For die transformation of filamentous fungi, several transformation protocols have been developed for many filamentous fungi. Many of them are based on preparation of protoplasts and introduction of DNA into the protoplasts using PEG and Ca 2+ ions. The transformed protoplasts men regenerate and the transformed fungi are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as argB, trpC, niaD and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance. A very common used transformation marker is the amdS gene of A. nidulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source.
- auxotrophic markers such as argB, trpC, niaD and pyrG
- antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance.
- a very common used transformation marker is the amd
- the transgenic organism can be a yeast.
- yeast have been widely used as a vehicle for heterologous gene expression.
- the species Saccharomyces cerevisiae has a long history of industrial use, including use for heterologous gene expression.
- Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107- 133, Blackie, Glasgow).
- Saccharomyces cerevisiae is well suited for heterologous gene expression.
- An additional advantage is that yeasts are capable of post-translational modifications of proteins and thereby have the potential for glycosylation and/or secretion of heterologous gene products into the growth medium.
- yeasts are known to secrete very few proteins into the culture medium. This makes yeast a very attractive host for expression of heterologous genes, since secretable gene products can easily be recovered and purified.
- yeast vectors include integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
- expression constructs are prepared by inserting the recombinant nucleotide sequence of the present invention into a construct designed for expression in yeast.
- the constructs contain a promoter active in yeast fused to the recombinant nucleotide sequence of the present invention, usually a promoter of yeast origin, such as the GAL1 promoter, is used.
- the recombinant nucleotide sequence of the present invention can be fused to a signal sequence which directs the enzyme encoded by the nucleotide sequence to be secreted.
- a signal sequence of yeast origin such as the sequence encoding the SUC2 signal peptide, is used.
- a terminator active in yeast ends the expression system. For expression in yeast, it may be advantageous to remove some or all of the intron or intron sequences.
- Heterologous expression in yeast has been reported for several genes.
- the gene products can be glycosylated which is advantageous for some enzymes intended for specific application where heat tolerance is desirable.
- the protein or enzyme can be deposited intracellularly if the recombinant nucleotide sequence of the present invention is not fused to a signal sequence, or they can be secreted extracelluarly if the recombinant nucleotide sequence of the present invention is equipped with a signal sequence.
- transgenic Saccharomyces For the transformation of yeast several transformation protocols have been developed.
- the transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
- the transformed yeast cells are selected using various selective markers.
- markers used for transformation are a number of auxo trophic markers such as LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, eg G418.
- the present invention relates to the manufacture of a foodstuff.
- the foodstuff may be based on starch and can be, for example, any one of bread, pasta, noodles etc.
- the present invention provides a method of improving the shelf-life and resistance to staling of bread and other products made from wheat flour dough which comprises incorporating into the dough an ⁇ -amylase according to the first aspect of the present invention.
- the recombinant ⁇ -amylase is incorporated into me dough in an amount sufficient to provide from 50 to 400 units of ⁇ -amylase activity per kg of wheat flour used to form the dough.
- the recombinant ⁇ -amylase can be added to the dough at any convenient time during the mixing process, and may be used in conjunction with other enzymes, such as enzymes added as dough improvers, which may also be added in the conventional manner.
- the recombinant ⁇ -amylase enzyme of the present invention may be added in combination with other enzymes or chemical additives, such as emulsifiers, rheology modifiers, stabilisers, surfactants, preservatives, flavourings, colourings etc.
- other ingredients and procedures used to form the dough and to bake die dough will be conventional and can be found in any relevant textbook.
- plasmids have been prepared in accordance with the present invention by adaptation, including in vivo excision, of ZAPII phage.
- These plasmids which have been called pAMYA-1, pAMYA-2, ⁇ AMYA-3 and pAMYA-5, contain the ⁇ - amylase gene of the present invention.
- pAMYA-1 is 8.48 Kb in size
- pAMYA-2 is 7.98 Kb in size
- pAMYA-3 is 9.98 Kb in size
- pAMYA-5 is 10.48 Kb in size.
- a further plasmid has been prepared in accordance with the present invention.
- This plasmid has been called pPR28.
- This plasmid also contains the ⁇ -amylase gene of the present invention.
- This plasmid has 6573 base pairs.
- Figure 1 is a restriction map of plasmid pAMYA-1
- Figure 2 is a restriction map of plasmid pAMYA-2
- Figure 3 is a restriction map of plasmid pAMYA-3;
- Figure 4 is a restriction map of plasmid pAMYA-5;
- Figure 5 is a restriction map of plasmid pPR28
- Figure 6 is a graph used to determine a pH optimum
- Figure 7 is a graph used to determine a temperature optimum
- Figure 8 contains plots of a starch degradation study
- Figure 9 contains plots of a baking study.
- ⁇ -amylase activity is determined by use of the following protocol. Protocol For Measurement of ⁇ -Amylase Activity ⁇ -amylase activity was analysed by determining me degree of hydrolysis of a standard starch solution (modification of method described by Sandstedt et al. 1939). The reaction mixture consisted of 2.5 ml of a 2% (w/v) solution of soluble wheat starch in 0.1 M sodium acetate buffer, 20 mM CaCl 2 (pH 4.8) and 0.5 ml of an enzyme solution. After 12, 14, 16, 18 and 20 minutes 0.5 ml extracts of the reaction mixture were transferred to 2.5 mis of a I 2 -KI solution (0.7 mM I 2 and 241 mM KI). The absorbance of the blue-coloured complex between starch and I 2 -KI was then measured at 620nm against water. The time needed to reach an absorbance of 0.48 was determined and used to calculate enzyme activity:
- A enzyme dilution (mg enzyme/ml solution)
- T time (min) necessary to reach an absorbance of 0.48.
- FAU Fungal Amylase Unit
- amylase activity was estimated by adding 25-100 ⁇ l of me enzyme solution to 1 ml of the standard starch solution at 50°C and transferring 0.5 ml of the reaction mixture to 2.5 ml I 2 -KI solution after 5-10 minutes. Absorbance at 620 nm of the starch-iodine solution was used as an arbitrary activity unit.
- the fungus Thermomyces lanuginosus was used as the source organism to generate DNA for a genomic library for subsequent studies and procedures.
- the Thermomyces lanuginosus used was strain CBS 224.63 obtained from Centraalbureau voor Schimmelcultures, Delft (CBS). A sample of this strain was grown on YPS agar plates at 40°C (yeast extract 4 g/1, wheat starch 15 g/1, K 2 HPO 4 1 g/l, MgSO 4 0.5 g/l and agar 20 g/l). 1.2. DNA extraction
- 1g of frozen T lanuginosus mycelium was ground in a mortar under liquid nitrogen. Following evaporation of the nitrogen cover, the ground mycelium was extracted with 15ml of an extraction buffer (100mM Tris-HCl,pH8.0, 0.50mM EDTA, 500mM NaCl, 10mM ⁇ -mercaptoethanol) containing 1ml 20% sodium dodecyl sulphate. After incubation at 65°C for 10 min. 5ml 5M KAc. pH 5.0, was added and die mixture further incubated, after mixing, on ice for 20 mins. The mixture was then centrifuged for 20 mins. and the supernatant mixed with 0.6 vol. isopropanol to precipitate the extracted DNA.
- an extraction buffer 100mM Tris-HCl,pH8.0, 0.50mM EDTA, 500mM NaCl, 10mM ⁇ -mercaptoethanol
- DNA pellet was dissolved in 0.7 ml TE (10mM Tris, HCl pH 8.0, 1mM EDTA) and precipitated with 75 ⁇ l 3M NaAc, pH 4.8, and 500 ⁇ l isopropanol.
- the adaptor can be prepared by annealing equal amounts of the nucleotide sequence shown as SEQ. I.D. No. 3 phosphorylated with T4 polynucleotide kinase and the nucleotide sequence shown as SEQ. I.D. No. 4.
- T4 ligase activity was destroyed by heating for 15 min. at 65°C.
- the fragments were then phosphorylated with T4 polynucleotide kinase.
- the two separate DNA preparations were run on a 1.0% preparative agarose gel and the DNA fragments in the size range 5-10kb were recovered and combined.
- 150 ng of the combined 5-10kb fragments were then mixed with l ⁇ g of an EcoRI predigested and dephosphorylated ⁇ ZAPII vector (Stratagene #236211) and ligated.
- the ligated vectors were packed in vitro with Gigapack II Gold packing extracts (Stratagene #200214), and the titre determined.
- the resultant library contained 300,000 independent phages of which about 95 % contained inserts.
- the reaction produced a ca. 800nt fragment which was cloned in pT7-Blue (Novagen #69829-1). Both ends of the fragment were sequenced using a commercial sequencing kit (USB: Sequenase) using a universal primer and a T7 primer.
- USB Sequenase
- the determined DNA sequences and me deduced amino acid sequences are set out herein as SEQ I.D. Nos. 7 and 8, and SEQ I.D. Nos. 9 and 10.
- the T. lanuginosus gene was sequenced using an ALF (Pharmacia) sequencer.
- the complete sequence which is shown as SEQ I.D. No. 1, comprises eight introns and nine exons.
- An Aspergillus niger strain, 3M43 (Danisco Ingredients strain collection) was transformed with a construct containing the promoter and me terminator of the xylan- ase-A gene from A. niger (xlnA) and me structural gene for amylase, amyA, from T. lanuginosus.
- the resultant plasmid is pPR28 is shown in Figure 5.
- the xylA gene has been described by Graaff et al. (de Graaff, L.H. et al. (1994) Molecular Microbiology 12(3), 479-490).
- the xylanase-promoter is a 427 bp BspHI fragment ending immediately before the ATG start codon of xlnA.
- a 1079 bp BamHI-Xbal fragment contains the xlnA terminator. This fragment starts 64 bp downstream of the translation stop codon of the xlnA gene.
- a 2106 bp, ScaI-Ecl136II fragment of the amyA gene contains me protein coding region.
- This fragment starts 21 bp before the amyA start codon and ends 68 bp after the stop codon.
- the fragments were assembled in pBluescript II SK+ (Stratagene) which was transformed into A. niger using a co-transformation procedure (Werners, K. et al. (1987), Molecular and General Genetics 209, 71-77).
- the transformants were grown in shake flasks (250 ml with 100 ml substrate) in 100 mM MES, pH 5.5 with 2% wheat bran, 2% beet pulp and 0.2% KNO 3 . After 6 days' growth a high ⁇ -amylase activity was found in the cultures of the transformants whereas the untransformed control did not produce amylase.
- the amylolytic activity was purified as follows: 20 ml of filtered culture material was desalted on PD10 columns (Pharmacia) equilibrated with 20 mM triethanolamine, pH 7.3. 30 ml of the desalted material was applied to an anion exchange column, Q- Sepharose Fast Flow (21 x 2.5 cm, flow 1.5 ml/min, Pharmacia) equilibrated with 20 mM triethanolamine, pH 7.3. A NaCl gradient was applied by adding triethanolamine, pH 7.3 with 1 M NaCl (buffer B) as follows: 0 - 25% buffer B in 300 min. and thereafter 25 - 50% buffer B in 700 min. Fractions of 7.5 ml were - collected.
- the ⁇ -amylase containing fractions were pooled (total volume 72 ml) and concentrated to a volume of 10 ml. This sample was loaded onto a gel filtration column, Superdex G75 Hiload (60 x 2.5 cm) equilibrated with 50 mM sodium acetate buffer containing 0.1 M NaCl, pH 5.8. Protein was eluted with a flow of 1.66 ml/min and fractions of 3.3 ml were collected. Samples of ⁇ -amylase containing fractions were analysed by SDS-PAGE on a 4-12% Tris-glycine gel. The ⁇ -amylase containing fractions gave a broad band at 54 - 60 kDa. The ⁇ -amylase containing fractions were pooled and frozen for further analysis. 5.3 Characterization of the recombinant ⁇ -amylase
- N-terminal of the purified ⁇ -amylase was determined according to conventional procedures (Matsudaira, P.: A Practical Guide to Protein and Peptide Purification for Microsequencing. Second Edition. Academic Press, San Diego 1993) and was found to have the following sequence: A T P D E W K A Q S
- SEQ I.D. No. 11 The sequence, which is identical to the amino acid sequence (amino acids number 1 to 10) in SEQ I.D. No. 1, is shown as SEQ I.D. No. 11. This confirms that the purified ⁇ -amylase is derived from the introduced Thermomyces lanuginosus ⁇ - amylase gene.
- the MW of the purified ⁇ -amylase on a Tris-glycine gel was determined to be 54 - 60 kDa.
- the MW was 55265 +/- 100 Da when determined by mass spectrometry.
- Specific colouring of the gel showed that the ⁇ -amylase is a glycoprotein which was also confirmed by mass spectrometry.
- the MW is 51.737kDa when calculated from the amino acid sequence and the enzyme probably has two N-glycosylated sites.
- the enzyme solution contained an enzyme activity of 176 ⁇ mol reducing equivalents/min/ml when measured in an acetate buffer, pH 6 (acetic acid) at 50°C.
- pH optimum pH optimum was determined as follows: To 400 ⁇ l 0.1 % starch solution in 100 mM of either MES (pH 4.8 to 7) or MOPS (pH 7 to 8) buffers was added 75 ⁇ l of enzyme solution (0.16 ⁇ mol reducing equivalents/min/ml). After 6 min. the activity was stopped by adding 240 ⁇ l stop solution. Then 360 ⁇ l colour reagent was added, and the samples were boiled for 5 min. Activity was determined as described above.
- the pH optimum of the recombinant ⁇ -amylase of the present invention was measured to be from 5.8 to 6 (see Figure 6).
- Temperature optimum was determined as follows: 75 ⁇ l 0.5% starch solution in 100 mM sodium acetate buffer, pH 6 (acetic acid) was placed in a water bath thermostatted at the measuring temperature. Then 400 ⁇ l ⁇ -amylase (29 nmol reducing equivalents/min/ml) was added. Enzyme activity was stopped after 5 and 8 min. , respectively by adding 240 ⁇ l stop solution. 360 ⁇ l colour reagent was added and activity was determined as described above.
- Results are shown in Figure 7. The same results were obtained when 100 mM MES buffer at pH 6 was used.
- the temperature optimum of the recombinant ⁇ -amylase of the present invention was determined to be between 60 and 70°C.
- Heat stability was determined as follows: 600 ⁇ l 100 mM sodium acetate buffer, pH 6 (acetic acid) was placed in a water bath maintained at die measuring temperature. 50 ⁇ l enzyme solution was added and after appropriate time intervals 100 ⁇ l samples were taken out and immediately added to 750 ⁇ l acetate buffer which was kept on ice. ⁇ - Amylase activity in these samples was determined by incubating 400 ⁇ l of the samples with 75 ⁇ l 0.5% starch solution in 0.1 M acetate buffer, pH 6 at 50°C for 10 min. 240 ⁇ l stop solution and 360 ⁇ l colour reagent were added and activity was determined as described above. Heat inactivation of the enzyme is an exponential function of time and heat stability at different temperatures can be described by the half times of inactivation. The results are presented in Table 1.
- Recombinant ⁇ -amylase of the present invention 100 FAU/kg flour
- Recombinant ⁇ -amylase of the present invention 350 FAU/kg flour Handling properties and stickiness of the dough were evaluated during preparation of the bread and after baking the crumb was evaluated.
- the recombinant ⁇ -amylase of me present invention lowers firmness as compared to a high dosage of TAKA amylase, meaning that it is a better anti-staling enzyme than the TAKA amylase.
- Dough handling properties were similar for doughs prepared with TAKA amylase and wim me recombinant ⁇ -amylase of the present invention.
- the crumb of bread with the recombinant ⁇ -amylase of the present invention was evaluated and was found to be more moist than me crumb of bread baked with TAKA amylase.
- present invention relates to a recombinant ⁇ -amylase and its use in baking.
- Other aspects of the present invention include the deposited microorganisms, and mutants, variants and progeny obtained directly or indirectly therefrom and retaining the ability, when cultured, of producing a recombinant ⁇ -amylase having the activity described herein.
- Other modifications of the present invention will be apparent to those skilled in the art.
- ORGANISM Thermomyces lanuginosus
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Abstract
A recombinant enzyme is described that is useful for preparing bakery products. The enzyme has the following characteristics: (i) α-amylase activity over the range from about 60 °C to about 80 °C; (ii) a molecular weight of about 54-60 kDa when measured by SDS-PAGE, or of about 55265 Da +/- 100 Da when measured by mass spectrometry; (iii) a pI value of about 3.7 (on a Pharmacia IEF gel, 3-9); (iv) a pH optimum of from about 5.8 to about 6; (v) a temperature optimum of from about 60 °C to about 70 °C.
Description
Alpha amylase from thermomyces lanuginosus
The present invention relates to an α-amylase enzyme. The present invention also relates to the use of the α-amylase enzyme in the manufacture of foodstuffs, in particular foodstuff based on starch, and especially a bakery product, such as bread and similar baked products based on wheat flour.
Bread has a limited shelf-life because it physically changes with time. Among other things, the crumb texture becomes firm, this process being generally known as staling. One explanation of the process of staling appears to be that the wheat starch undergoes a process of retrogradation (re-crystallisation) over a period of time, but undoubtedly there are many other factors. It has been shown that the addition of bacterial α-amylases to bread, and to a lesser extent, fungal and cereal α-amylases, can retard staling by modifying the gelatinised starch so that retrogradation (recrystallisation) is delayed. For example, see Eliasson, A. and Larssen, K. in Cereals In Breadmaking - A Molecular Colloidal Approach, (1993), Marcel Dekker Inc. , 351. However, the activity of bacterial amylases is difficult to control during the baking process and in a manner such as to achieve the desired result. α-Amylases are also traditionally added to the wheat flour used in bread making to improve the baking quality of the flour. Amongst the α-amylases used for this purpose is the fungal α-amylase from Aspergillus oryzae, Drapon, R. and Godon, B. in Enzymes And Their Role In Cereal Technology, (1987), Edited by Kinger, J.E. et al, 281-234, and known as the TAKA-amylase.
However, as a means of delaying the staling process in bread, the TAKA-amylase is far too thermolabile. The gelatinisation of starch (i.e. the melting of starch crystals) starts at about 65°C so that by the time gelatinisation begins much of the TAKA amylase added to the wheat flour will have been inactivated. Bacterial α-amylases,
on the other hand, are too thermally stable so that they are not properly inactivated by the baking process and may lead to a gummy product - see Leloup et al [(1993) Bioconversion of Cereal Products, pp 79-127, B.Godon Ed.]. An α-amylase has been isolated from Thermomyces lanuginosus (T. lanuginosus) and was found to have a half-life of 10 minutes at 70°C (Enzyme Microbiol. Technol. (1992), 14, 112-116). This α-amylase was not suggested for use in baking.
EP-A-0579360 and WO 90/11352 disclose α-amylases obtained from Pyrococcus and from the bacterium P. fiiriosus, respectively. These α-amylases were not suggested for use in baking.
For optimal baking properties, an α-amylase should have at least two main criteria. First, the enzyme should be active during the starch gelatinisation stage, which occurs from about 60°C to about 70°C. Second, the enzyme should be inactivated during the baking stage, which occurs from about 80°C to about 100°C. Thus, an α-amylase with intermediate heat stability will be suitable for baking purposes.
The present invention seeks to provide an α-amylase that can be beneficial for baking and which can be prepared easily and in large amounts.
According to a first aspect of the present invention there is provided a recombinant enzyme having the following characteristics: i. α-amylase activity over the range from about 60°C to about 80°C; ii. a molecular weight of about 54 - 60 kDa when measured by SDS- PAGE, or of about 55265 Da +/- 100 Da when measured by mass spectrometry;
iii. a pi value of about 3.7 (on a Pharmacia IEF gel, 3-9);
iv. a pH optimum of from about 5.8 to about 6;
v. a temperature optimum of from about 60°C to about 70°C.
According to a second aspect of the present invention there is provided a recombinant nucleotide sequence comprising the coding nucleotide sequence shown in SEQ. I.D. No. 1. or a variant, homologue or fragment thereof. According to a third aspect of the present invention there is provided a recombinant nucleotide sequence comprising the nucleotide sequence shown as SEQ. I.D. No. 1. or a variant, homologue or fragment thereof.
According to a fourth aspect of the present invention there is provided a construct comprising the recombinant nucleotide sequence according to the present invention.
According to a fifth aspect of the present invention there is provided a vector comprising the recombinant nucleotide sequence according to the present invention or the construct according to the present invention.
According to a sixth aspect of the present invention there is provided a cell, tissue or organism comprising the recombinant nucleotide sequence according to the present invention or the construct according to the present invention or the vector according to the present invention.
According to a seventh aspect of the present invention there is provided a transgenic organism comprising the recombinant nucleotide sequence according to the present invention or the construct according to the present invention or the vector according to the present invention or the cell, tissue or organism according to the present invention.
According to an eighth aspect of the present invention there is provided NCIMB deposit No. 40655, NCIMB deposit No. 40656, NCIMB deposit No. 40657 or NCIMB deposit No. 40658.
According to a ninth aspect of the present invention there is provided a method of preparing a bakery product comprising forming a mixture by contacting a wheat-flour dough with an α-amylase and baking the mixture, wherein the α-amylase is a recombinant α-amylase according to the present invention, or is a recombinant α- amylase expressed by the nucleotide sequence of the present invention, which may be present in any of the afore-mentioned nucleotide aspects of the present invention.
According to a tenth aspect of the present invention there is provided a bakery product obtained by use of the recombinant α-amylase according to the present invention, or a recombinant α-amylase expressed by the nucleotide aspect of the present invention.
According to an eleventh aspect of the present invention there is provided the use of a recombinant α-amylase having α-amylase activity over the range from about 60°C to about 80°C in baking.
According to a twelfth aspect of the present invention there is provided a method of preparing a foodstuff comprising forming a mixture by contacting starch or a starch based ingredient with an α-amylase and optionally processing the mixture, wherein the α-amylase is a recombinant α-amylase as defmed above or is a recombinant α- amylase expressed by the nucleotide aspect of the present invention.
According to a thirteenth aspect of the present invention there is provided a foodstuff obtained by use of an α-amylase enzyme wherein the enzyme is a recombinant α- amylase as defined above or is a recombinant α-amylase expressed by the nucleotide aspect of the present invention.
According to a fourteenth aspect of the present invention there is provided the use of a recombinant α-amylase having α-amylase activity over the range from about 60°C to about 80°C in preparing a foodstuff.
Other aspects of the present invention include inserting the recombinant α-amylase nucleotide sequence of the present invention into a suitable vector and/or transforming a suitable host organism with the recombinant α-amylase nucleotide sequence of the present invention and/or expressing that recombinant α-amylase nucleotide sequence gene within the host and/or collecting the expressed recombinant α-amylase and/or isolating the recombinant α-amylase.
Further aspects of the present invention include: i) other than when forming part of the natural genome, a recombinant nucleotide sequence, which may be either an RNA or DNA sequence, that encodes for a recombinant protein according to the first aspect of the present invention, and in particular the DNA sequence shown in SEQ I.D. No. 1; ii) a nucleotide sequence that is functionally equivalent to the DNA sequence shown in SEQ I.D. No. 1, that is to say a DNA sequence showing substantial (at least 90%) homology (similarity) therewith and coding for a recombinant α-amylase having the same properties of the recombinant α-amylase having the amino acid sequence as set out in SEQ I.D. No. 1, but differing from the DNA sequence of SEQ I.D. No. 1 by virtue only of the degeneracy of the genetic code; iii) a recombinant nucleotide sequence that is complementary to the DNA sequence shown in SEQ I.D. No. 1, and the equivalent sequence mentioned in ii); iv) a vector containing the sequences identified in i), ii), and iii) and especially expression vectors containing that sequence downstream from a suitable promoter sequence capable of expressing the recombinant α-amylase when inserted in a suitable host; v) a transformant when transformed with a vector according to iv), and especially a transformant transformed with an expression vector and capable of expressing the recombinant α-amylase, and especially an Aspergillus niger transformant; and
vi) a method for the production of a recombinant α-amylase according to the present invention which comprises culturing a transformant according to v) and recovering from the culture the recombinant α-amylase expressed in the culture by the transformant.
Preferably the enzyme comprises an N-terminal sequence shown as SEQ. I.D. No. 11.
Preferably the enzyme comprises the sequence shown as SEQ. I.D. No. 1 or SEQ. I.D. No. 2, or a variant, homologue or fragment thereof.
Preferably the enzyme is expressed by a nucleotide sequence comprising the coding sequence shown in SEQ. I.D. No. 1, or a variant, homologue or fragment thereof. Preferably the enzyme is expressed by a nucleotide sequence comprising the nucleotide sequence shown as SEQ. I.D. No. l, or a variant, homologue or fragment thereof.
Preferably the enzyme is obtained or obtainable from T. lanuginosus.
Preferably the enzyme is expressed by a member of the Aspergillus family.
Preferably the enzyme is expressed by Aspergillus niger. Preferably the enzyme is obtainable from any one of NCIMB deposit No. 40655, NCIMB deposit No. 40656, NCIMB deposit No. 40657 and NCIMB deposit No. 40658.
Preferably, the transgenic organism is a filamentous fungi, preferably an Aspergillus fungus, more preferably Aspergillus niger.
Preferably, in the method, the recombinant α-amylase is incorporated into the dough in an amount sufficient to provide from 50 to 400 units of α-amylase activity per kg of wheat flour used to form the dough. Preferably, in the method, the recombinant α-amylase is incorporated into the mixture in an amount sufficient to provide from 50 to 400 units of α-amylase activity per kg of starch or starch based ingredient to form the mixture.
Preferably, in the method, the foodstuff or bakery product is bread.
Preferably, in the use, the recombinant α-amylase is a recombinant α-amylase according to the first aspect of the present invention or is a recombinant α-amylase expressed by the nucleotide aspects of the present invention. In a first highly preferred embodiment, the recombinant α-amylase enzyme of the present invention has a protein sequence as set out in SEQ.I.D. No. 1 and SEQ. I.D.
No. 2.
In a second highly preferred embodiment, the recombinant α-amylase enzyme of the present invention is coded by the nucleotide sequence as set out in SEQ.I.D. No. 1.
In a third highly preferred embodiment, the recombinant nucleotide sequence is the nucleotide sequence as set out in SEQ.I.D. No. 1. One of the advantages of the recombinant enzyme of the present invention is that when it is used in baking it improves the shelf-life of bread and other products made from a wheat-flour dough. In this regard, it improves the resistance of bread, and other products made from a wheat-flour dough, to staling. In particular, the recombinant α-amylase has a beneficial thermal activity profile for use in baking.
In addition, the recombinant enzyme of the present invention can be beneficially used in combination with other enzymes useful in the preparation of bakery products.
Other advantages are that the recombinant enzyme can be prepared easily, reliably and cheaply and in large amounts. For example, expressing the gene in Aspergillus niger has the advantage that purification and recovery procedures of the recombinant α-amylase are simpler. Moreover, expression in Aspergillus niger would obviate the need to remove the intron sequences prior to transformation of that host organism with the recombinant nucleotide sequence of the present invention.
The term "recombinant" is used in its normal sense. For example, the term "recombinant enzyme" does not include an enzyme when prepared by expression of the genomic nucleotide sequence coding for the enzyme when that nucleotide sequence is in its natural environment. Thus, the term covers the enzyme when prepared by expression of a recombinant nucleotide sequence coding for the enzyme when that nucleotide sequence is not in the natural environment for the genomic nucleotide coding sequence. Thus the term "recombinant nucleotide" includes recombinant DNA and recombinant RNA. Preferably it means recombinant DNA, and in some instances it preferably means cDNA.
The recombinant enzyme can be used on its own or in combination with one or more other enzymes.
The terms "variant" or "homologue" or "fragment" include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid or amino acid from or to a respective sequence providing the resultant sequence has the respective ability to code for or act as an enzyme according to the present invention, preferably having at least the same activity of the enzyme comprising the sequence shown as the sequence listing SEQ. I.D. No. 1. In particular, the term "homologue" covers homology with respect to similarity of structure and/or similarity of function providing the resultant nucleotide sequence has the ability to code for an enzyme according to the present invention. With respect to sequence homology, preferably there is more than 80% homology, more preferably at least 85% homology, more preferably at least 90% homology, even more preferably at least 95% homology, more preferably at least 98% homology. The
expressions also cover any codon replacement or substitution with another codon coding for the same amino acid or any addition or removal thereof providing the resultant enzyme has α-amylase activity. Thus, the present invention also covers a modified nucleotide sequence in which at least one nucleotide has been deleted, substituted or modified or in which at least one additional nucleotide has been inserted so as to encode an enzyme having the activity of an α-amylase, preferably having an increased α-amylase activity. The above terms are also synonymous with allelic variations of the sequences. The term "complementary" means that the present invention also covers recombinant nucleotide sequences that can hybridise to the recombinant nucleotide sequences.
The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes the nucleotide sequence according to the present invention directly or indirectly attached to the promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. The terms do not cover the natural combination of the wild type α-amylase gene ordinarily associated with the wild type gene promoter and the wild type promoter and when they are both in their natural environment.
The construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a fungus or a plant cell into which it has been transferred. Various markers exist which may be used in, for example, plants - such as mannose. Other examples of markers include those that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin and gentamycin.
The construct of the present invention preferably comprises a promoter.
The term "promoter" is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression. Examples of suitable promoters are those that can direct efficient expression of the nucleotide sequence of the present invention and/or in a specific type of cell. The promoter could additionally include conserved regions such as a Pribnow Box or a TATA box. The promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention. For example, suitable examples of such sequences include the Shl-intron or an ADH intron. Other sequences include inducible elements - such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5' leader sequence (see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97). The terms "obtained" and "obtainable" from T. lanuginosus include products obtained directly from cultures of transformed T. lanuginosus and products obtained by expression of a recombinant α-amylase gene of the present invention obtainable from T. lanuginosus when the gene is in another cellular organism. Preferably, the terms cover expression of the recombinant α-amylase gene of the present invention in another cellular organism, preferably Aspergillus niger.
The term "vector" includes an expression vector and a transformation vector. The term "expression vector" means a construct capable of in vivo or in vitro expression. The term "transformation vector" means a construct capable of being transferred from one species to another - such as from an E.Coli plasmid to a fungus or a plant cell, or from an Agrobacterium to a plant cell.
The terms "cell", "tissue" and "organ" include cell, tissue and organ per se and when within an organism.
The term "organism" in relation to the present invention includes any organism that could comprise the recombinant nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein the recombinant nucleotide sequence according to the present invention can be expressed when present in the organism. Preferably the organism is an α-amylase producing organism such as any one of a plant, algae, fungi, yeast and bacteria, as well as cell lines thereof. Preferably the organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger. The term "transgenic orgamsm" in relation to the present invention includes any organism that comprises the recombinant nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein the recombinant nucleotide sequence according to the present invention can be expressed within the organism. Preferably the recombinant nucleotide sequence is incorporated in the genome of the organism. Preferably the transgenic organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.
If the organism can not naturally splice intron regions post expression then a pre- transformation step may be necessary wherein any intron sections are removed.
The host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press). If a prokaryotic host is used then the recombinant nucleotide sequence of the present invention may need to be suitably modified before transformation - such as by removal of introns. As mentioned above, a preferred host organism is of the genus Aspergillus, such as Aspergillus niger.
The transgenic Aspergillus according to the present invention can be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-393), Davis R.W. 1994 (Heterologous gene expression and protein secretion in Aspergillus. In: Martinelli S.D. , Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp 525-560), Ballance, D.J. 1991 (Transformation systems for Filamentous Fungi and an Overview of Fungal Gene structure. In: Leong,S.A. , Berka R.M. (Editors) Molecular Industrial Mycology. Systems and Applications for Filamentous Fungi. Marcel Dekker Inc. New York 1991. pp 1-29) and Turner G. 1994 (Vectors for genetic manipulation. In: Martinelli S.D. , Kinghorn J.R.( Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641-666). However, the following commentary provides a summary of those teachings for producing transgenic Aspergillus according to the present invention.
Filamentous fungi have been widely used in industry for production of organic compounds and enzymes. Traditional Japanese koji and soy fermentations have used Aspergillus sp for hundreds of years. In this century Aspergillus niger has been used for production of organic acids particular citric acid and for production of various enzymes for use in industry.
There are two major reasons for that filamentous fungi have been so widely used in industry. First filamentous fungi can produce high amounts of extracellular products, for example enzymes and organic compounds such as antibiotics or organic acids. Second filamentous fungi can grow on low cost substrates such as grains, bran, beet pulp etc. The same reasons have made filamentous fungi attractive organisms as hosts for heterologous gene expression according to the present invention.
In order to prepare the transgenic Aspergillus, expression constructs are prepared by inserting the recombinant nucleotide sequence of the present invention into a construct designed for expression in filamentous fungi. In this regard, several types of constructs used for heterologous gene expression have been developed. The
constructs will contain a promoter which is active in fungi. Examples of promoters include a fungal promoter for a highly expressed extracellular enzyme, such as an glucoamylase promoter or an α-amylase promoter. The recombinant nucleotide sequence of the present invention can be fused to a signal sequence which directs the enzyme encoded by the nucleotide sequence to be secreted. Usually a signal sequence of fungal origin is used. A terminator active in fungi ends the expression system.
Another type of expression system has been developed in fungi where the recombinant nucleotide sequence of the present invention is fused to a smaller or a larger part of a fungal gene encoding a stable protein. This can stabilize the protein encoded by the recombinant nucleotide sequence of the present invention. In such a system a cleavage site, recognized by a specific protease, can be introduced between the fungal protein and the enzyme encoded by the recombinant nucleotide sequence of the present invention, so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the protein or enzyme encoded by die recombinant nucleotide sequence of the present invention. By way of example, one can introduce a site which is recognized by a KEX-2 like peptidase found in at least some Aspergilli. Such a fusion leads to cleavage in vivo resulting in protection of the recombinant enzyme and production of the recombinant enzyme and not a larger fusion protein or enzyme.
Heterologous expression in Aspergillus has been reported for several genes coding for bacterial, fungal, vertebrate and plant proteins or enzymes. The proteins can be deposited intracellularly if the recombinant nucleotide sequence of the present invention is not fused to a signal sequence. Such proteins or enzymes will accumulate in the cytoplasm and will usually not be glycosylated which can be an advantage for some bacterial proteins or enzymes. If the recombinant nucleotide sequence of the present invention is equipped with a signal sequence the enzyme will , accumulate extracellulary.
With regard to product stability and host strain modifications, some heterologous proteins or enzymes are not very stable when they are secreted into the culture fluid of fungi. Most fungi produce several extracellular proteases which degrade heterologous proteins or enzymes. To avoid this problem special fungal strains with reduced protease production have been used as hosts for heterologous production.
For die transformation of filamentous fungi, several transformation protocols have been developed for many filamentous fungi. Many of them are based on preparation of protoplasts and introduction of DNA into the protoplasts using PEG and Ca2+ ions. The transformed protoplasts men regenerate and the transformed fungi are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as argB, trpC, niaD and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance. A very common used transformation marker is the amdS gene of A. nidulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source.
In another embodiment the transgenic organism can be a yeast. In this regard, yeast have been widely used as a vehicle for heterologous gene expression. The species Saccharomyces cerevisiae has a long history of industrial use, including use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107- 133, Blackie, Glasgow).
For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability.
Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large- scale fermentation characteristics of Saccharomyces cerevisiae. An additional advantage is that yeasts are capable of post-translational modifications of proteins and thereby have the potential for glycosylation and/or secretion of heterologous gene products into the growth medium.
A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
Also, yeasts are known to secrete very few proteins into the culture medium. This makes yeast a very attractive host for expression of heterologous genes, since secretable gene products can easily be recovered and purified.
Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting the recombinant nucleotide sequence of the present invention into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed. The constructs contain a promoter active in yeast fused to the recombinant nucleotide sequence of the present invention, usually a promoter of yeast origin, such as the GAL1 promoter, is used. The recombinant nucleotide sequence of the present invention can be fused to a signal sequence which directs the enzyme encoded by the nucleotide sequence to be secreted. Usually a signal sequence of yeast origin, such as the sequence encoding the SUC2 signal peptide, is used. A terminator active in yeast ends the expression system.
For expression in yeast, it may be advantageous to remove some or all of the intron or intron sequences.
Heterologous expression in yeast has been reported for several genes. The gene products can be glycosylated which is advantageous for some enzymes intended for specific application where heat tolerance is desirable. The protein or enzyme can be deposited intracellularly if the recombinant nucleotide sequence of the present invention is not fused to a signal sequence, or they can be secreted extracelluarly if the recombinant nucleotide sequence of the present invention is equipped with a signal sequence.
For the transformation of yeast several transformation protocols have been developed. The transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
The transformed yeast cells are selected using various selective markers. Among the markers used for transformation are a number of auxo trophic markers such as LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, eg G418.
The present invention relates to the manufacture of a foodstuff. The foodstuff may be based on starch and can be, for example, any one of bread, pasta, noodles etc.
However, in a preferred aspect, the present invention provides a method of improving the shelf-life and resistance to staling of bread and other products made from wheat flour dough which comprises incorporating into the dough an α-amylase according to the first aspect of the present invention. Typically, the recombinant α-amylase is incorporated into me dough in an amount sufficient to provide from 50 to 400 units of α-amylase activity per kg of wheat flour used to form the dough.
The recombinant α-amylase can be added to the dough at any convenient time during the mixing process, and may be used in conjunction with other enzymes, such as enzymes added as dough improvers, which may also be added in the conventional manner.
Thus, the recombinant α-amylase enzyme of the present invention may be added in combination with other enzymes or chemical additives, such as emulsifiers, rheology modifiers, stabilisers, surfactants, preservatives, flavourings, colourings etc. Typically, the other ingredients and procedures used to form the dough and to bake die dough will be conventional and can be found in any relevant textbook.
A number of plasmids have been prepared in accordance with the present invention by adaptation, including in vivo excision, of ZAPII phage. These plasmids, which have been called pAMYA-1, pAMYA-2, ρAMYA-3 and pAMYA-5, contain the α- amylase gene of the present invention. pAMYA-1 is 8.48 Kb in size, pAMYA-2 is 7.98 Kb in size, pAMYA-3 is 9.98 Kb in size and pAMYA-5 is 10.48 Kb in size.
E. coli containing these plasmids have been deposited with the National Collections of Industrial and Marine Bacteria (NCIMB), 23 St. Machar Drive, Aberdeen AB2 1RY, United Kingdom, on 23 June 1994 under the deposit numbers: pAMYA-1 NCIMB 40655
pAMYA-2 NCIMB 40656
pAMYA-3 NCIMB 40657
pAMYA-5 NCIMB 40658
A further plasmid has been prepared in accordance with the present invention. This plasmid has been called pPR28. This plasmid also contains the α-amylase gene of the present invention. This plasmid has 6573 base pairs.
The present invention will now be described only by way of examples, in which reference shall be made to the following Figures:
Figure 1 is a restriction map of plasmid pAMYA-1;
Figure 2 is a restriction map of plasmid pAMYA-2;
Figure 3 is a restriction map of plasmid pAMYA-3; Figure 4 is a restriction map of plasmid pAMYA-5;
Figure 5 is a restriction map of plasmid pPR28;
Figure 6 is a graph used to determine a pH optimum;
Figure 7 is a graph used to determine a temperature optimum;
Figure 8 contains plots of a starch degradation study; and Figure 9 contains plots of a baking study.
In the following experiments, α-amylase activity is determined by use of the following protocol. Protocol For Measurement of α-Amylase Activity α-amylase activity was analysed by determining me degree of hydrolysis of a standard starch solution (modification of method described by Sandstedt et al. 1939). The reaction mixture consisted of 2.5 ml of a 2% (w/v) solution of soluble wheat starch in 0.1 M sodium acetate buffer, 20 mM CaCl2 (pH 4.8) and 0.5 ml of an enzyme solution. After 12, 14, 16, 18 and 20 minutes 0.5 ml extracts of the reaction mixture were transferred to 2.5 mis of a I2-KI solution (0.7 mM I2 and 241 mM KI).
The absorbance of the blue-coloured complex between starch and I2-KI was then measured at 620nm against water. The time needed to reach an absorbance of 0.48 was determined and used to calculate enzyme activity:
A = enzyme dilution (mg enzyme/ml solution)
T = time (min) necessary to reach an absorbance of 0.48. 1 Fungal Amylase Unit (FAU) is the quantity of enzyme which will dextrinise the standard starch solution at a rate of 1 g per hour at 40°C.
During purification, amylase activity was estimated by adding 25-100μl of me enzyme solution to 1 ml of the standard starch solution at 50°C and transferring 0.5 ml of the reaction mixture to 2.5 ml I2 -KI solution after 5-10 minutes. Absorbance at 620 nm of the starch-iodine solution was used as an arbitrary activity unit.
1. Construction of T. lanuginosus genomic library 1.1. Source Orgamsm
The fungus Thermomyces lanuginosus was used as the source organism to generate DNA for a genomic library for subsequent studies and procedures. The Thermomyces lanuginosus used was strain CBS 224.63 obtained from Centraalbureau voor Schimmelcultures, Delft (CBS). A sample of this strain was grown on YPS agar plates at 40°C (yeast extract 4 g/1, wheat starch 15 g/1, K2HPO4 1 g/l, MgSO4 0.5 g/l and agar 20 g/l).
1.2. DNA extraction
1g of frozen T lanuginosus mycelium was ground in a mortar under liquid nitrogen. Following evaporation of the nitrogen cover, the ground mycelium was extracted with 15ml of an extraction buffer (100mM Tris-HCl,pH8.0, 0.50mM EDTA, 500mM NaCl, 10mM β-mercaptoethanol) containing 1ml 20% sodium dodecyl sulphate. After incubation at 65°C for 10 min. 5ml 5M KAc. pH 5.0, was added and die mixture further incubated, after mixing, on ice for 20 mins. The mixture was then centrifuged for 20 mins. and the supernatant mixed with 0.6 vol. isopropanol to precipitate the extracted DNA. After further centrifuging for 15 mins. the DNA pellet was dissolved in 0.7 ml TE (10mM Tris, HCl pH 8.0, 1mM EDTA) and precipitated with 75 μl 3M NaAc, pH 4.8, and 500 μl isopropanol.
After centrifuging the pellet was washed with 70% ETOH and dried under vacuum. The DNA was dissolved in 200 μl TE and stored at -20°C.
1.3 DNA digestion and cloning
40 μg of the DNA prepared as in 1.2 was digested with 1 U Sau3A in a 500 μl volume. 125 μl digests were removed after 5, 10, 15 and 20 minutes. Using the 10 and 15 minute digests, the digested DNA was precipitated, dissolved in 20 μl water, mixed with a 50-fold excess of a double stranded DNA adapter having the sequences shown as SEQ I.D. No. 3 and SEQ I.D. No. 4 which has Sau3A and EcoRI overhangs at either end and two internal restriction sites, and ligated with T4 ligase overnight.
The adaptor can be prepared by annealing equal amounts of the nucleotide sequence shown as SEQ. I.D. No. 3 phosphorylated with T4 polynucleotide kinase and the nucleotide sequence shown as SEQ. I.D. No. 4.
After ligation, T4 ligase activity was destroyed by heating for 15 min. at 65°C. The fragments were then phosphorylated with T4 polynucleotide kinase.
The two separate DNA preparations were run on a 1.0% preparative agarose gel and the DNA fragments in the size range 5-10kb were recovered and combined.
150 ng of the combined 5-10kb fragments were then mixed with lμg of an EcoRI predigested and dephosphorylated λZAPII vector (Stratagene #236211) and ligated. The ligated vectors were packed in vitro with Gigapack II Gold packing extracts (Stratagene #200214), and the titre determined.
The resultant library contained 300,000 independent phages of which about 95 % contained inserts.
2. Construction of a T. lanuginosus DNA probe. 2.1 Construction of PCR Primers.
By comparisons of the known and published protein sequences of the α-amylase genes from Schwanniomyces occidentalis, Saccharomycopsis fibuligera and Aspergillus oryzae (Swiss protein release 24 database) two conserved regions of the α-amylase gene were identified, being respectively the sequences:
Met-Glu-Phe-Thr-Ala-Ile-Trp-Ile
M G P T A I W I
and
Tyr-Thr-Cys-Pro-Tyr-Gln-Asn
Y T C P Y Q N
Corresponding oligonucleotides were then synthesised for each of the possible DNA sequences encoding for those two sequences, taking the degeneracy of the genetic code into account, and for use as degenerate primers in the subsequent PCR amplification of genetic DNA from T. lanuginosus. Collectively those degenerate primers are represented by the sequences SEQ I.D. No. 5 (Primer 1) and SEQ I.D. No. 6 (Primer 2).
2.2 PCR Cloning of the T. lanuginosus α-amylase gene
1 μg of T. lanuginosus DNA in a 100 μl volume was amplified by PCR using 100 pmol of each of the Primers 1 and 2 according to the following programme:
The determined DNA sequences and me deduced amino acid sequences are set out herein as SEQ I.D. Nos. 7 and 8, and SEQ I.D. Nos. 9 and 10.
3. Isolation of T. lanuginosus α-amylase gene
3.1 2 x 30,000 independent clones of the T. lanuginosus gene library were plated on 22 x 22 NZY plates (per liter 5g NaCl, 2g MgSO4.7H2O, 5g Yeast extract, 10g casein hydrolysate. 20g agar, pH 7.5 ) and plaques obtained. Two replicas of each plate were made on Hybond N membranes (Amersham) and treated according to manufacturer's instructions. 3.2 The T. lanuginosus α-amylase gene fragment obtained as in 2.1 above was labelled with α-32P-dCTP using a "Prime-It" random primer labelling kit (Stratagene #300385) and used to probe the membranes obtained as in 3.1. After 24 hours
hybridisation period, the membranes were washed according to the manufacturer's instructions, autoradiographed, and the positive plaques isolated. For this purpose, a positive plaque was declared only if it hybridised to the probe on both membranes. Plaques containing the full length gene were identified. Phages were extracted from the isolated plaques and purified by plating on small NYZ plates and repeating the plaque lift and hybridisations. The purified clones were sub-cloned in vivo by the ExAssist system (Stratagene #200253). The resulting plasmids with the full length α-amylase gene have been named pAMYA 1-3 and 5, and their restriction maps are shown in Figures 1 - 4.
4. Sequencing of the T. lanuginosus α-amylase gene
4.1 The T. lanuginosus gene was sequenced using an ALF (Pharmacia) sequencer. The complete sequence, which is shown as SEQ I.D. No. 1, comprises eight introns and nine exons.
5. Expression of recombinant α-amylase 5.1 Transformation
An Aspergillus niger strain, 3M43 (Danisco Ingredients strain collection) was transformed with a construct containing the promoter and me terminator of the xylan- ase-A gene from A. niger (xlnA) and me structural gene for amylase, amyA, from T. lanuginosus. The resultant plasmid is pPR28 is shown in Figure 5.
The xylA gene has been described by Graaff et al. (de Graaff, L.H. et al. (1994) Molecular Microbiology 12(3), 479-490). The xylanase-promoter is a 427 bp BspHI fragment ending immediately before the ATG start codon of xlnA. A 1079 bp BamHI-Xbal fragment contains the xlnA terminator. This fragment starts 64 bp downstream of the translation stop codon of the xlnA gene.
A 2106 bp, ScaI-Ecl136II fragment of the amyA gene contains me protein coding region. This fragment starts 21 bp before the amyA start codon and ends 68 bp after the stop codon. The fragments were assembled in pBluescript II SK+ (Stratagene) which was transformed into A. niger using a co-transformation procedure (Werners, K. et al. (1987), Molecular and General Genetics 209, 71-77).
5.2 Purification of the transgenic α-amylase
The transformants were grown in shake flasks (250 ml with 100 ml substrate) in 100 mM MES, pH 5.5 with 2% wheat bran, 2% beet pulp and 0.2% KNO3. After 6 days' growth a high α-amylase activity was found in the cultures of the transformants whereas the untransformed control did not produce amylase.
The amylolytic activity was purified as follows: 20 ml of filtered culture material was desalted on PD10 columns (Pharmacia) equilibrated with 20 mM triethanolamine, pH 7.3. 30 ml of the desalted material was applied to an anion exchange column, Q- Sepharose Fast Flow (21 x 2.5 cm, flow 1.5 ml/min, Pharmacia) equilibrated with 20 mM triethanolamine, pH 7.3. A NaCl gradient was applied by adding triethanolamine, pH 7.3 with 1 M NaCl (buffer B) as follows: 0 - 25% buffer B in 300 min. and thereafter 25 - 50% buffer B in 700 min. Fractions of 7.5 ml were - collected. The α-amylase containing fractions were pooled (total volume 72 ml) and concentrated to a volume of 10 ml. This sample was loaded onto a gel filtration column, Superdex G75 Hiload (60 x 2.5 cm) equilibrated with 50 mM sodium acetate buffer containing 0.1 M NaCl, pH 5.8. Protein was eluted with a flow of 1.66 ml/min and fractions of 3.3 ml were collected. Samples of α-amylase containing fractions were analysed by SDS-PAGE on a 4-12% Tris-glycine gel. The α-amylase containing fractions gave a broad band at 54 - 60 kDa. The α-amylase containing fractions were pooled and frozen for further analysis.
5.3 Characterization of the recombinant α-amylase
5.3.1 N-terminal Sequence The N-terminal of the purified α-amylase was determined according to conventional procedures (Matsudaira, P.: A Practical Guide to Protein and Peptide Purification for Microsequencing. Second Edition. Academic Press, San Diego 1993) and was found to have the following sequence: A T P D E W K A Q S
The sequence, which is identical to the amino acid sequence (amino acids number 1 to 10) in SEQ I.D. No. 1, is shown as SEQ I.D. No. 11. This confirms that the purified α-amylase is derived from the introduced Thermomyces lanuginosus α- amylase gene.
5.3.2 Molecular weight
As mentioned above, the MW of the purified α-amylase on a Tris-glycine gel was determined to be 54 - 60 kDa. The MW was 55265 +/- 100 Da when determined by mass spectrometry. Specific colouring of the gel showed that the α-amylase is a glycoprotein which was also confirmed by mass spectrometry.
The MW is 51.737kDa when calculated from the amino acid sequence and the enzyme probably has two N-glycosylated sites.
5.3.3 pI pi of the α-amylase was determined by use of a Pharmacia IEF gel, 3-9, and was measured to be approximately 3.7.
5.4 Activity measurements
Activity measurements for determination of pH and temperature optimum were made by following the formation of reducing equivalents in a 0.08% starch solution (described in detail below). The reaction was stopped by adding an alkaline sodium phosphate solution (0.112 M Na2HPO4, 2 H2O; 55 mM NaOH in distilled water). Then a colour reagent (3.5 mM K3(Fe(CN)6; 56 mM Na2HPO4, 2 H2O; 55 mM NaOH in distilled water) was added. The samples were boiled for 5 min. After cooling, 300 μl of the samples were transferred to a microtiter plate and absorbance at 405 nm was measured. Activity was determined from absorbance differences between samples and a blank. Glucose was used as a standard.
The enzyme solution contained an enzyme activity of 176 μmol reducing equivalents/min/ml when measured in an acetate buffer, pH 6 (acetic acid) at 50°C.
5.4.1 pH optimum pH optimum was determined as follows: To 400 μl 0.1 % starch solution in 100 mM of either MES (pH 4.8 to 7) or MOPS (pH 7 to 8) buffers was added 75 μl of enzyme solution (0.16 μmol reducing equivalents/min/ml). After 6 min. the activity was stopped by adding 240 μl stop solution. Then 360 μl colour reagent was added, and the samples were boiled for 5 min. Activity was determined as described above.
The pH optimum of the recombinant α-amylase of the present invention was measured to be from 5.8 to 6 (see Figure 6).
5.4.2 Temperature optimum
Temperature optimum was determined as follows: 75 μl 0.5% starch solution in 100 mM sodium acetate buffer, pH 6 (acetic acid) was placed in a water bath thermostatted at the measuring temperature. Then 400 μl α-amylase (29 nmol reducing equivalents/min/ml) was added. Enzyme activity was stopped after 5 and 8
min. , respectively by adding 240 μl stop solution. 360 μl colour reagent was added and activity was determined as described above.
Results are shown in Figure 7. The same results were obtained when 100 mM MES buffer at pH 6 was used. The temperature optimum of the recombinant α-amylase of the present invention was determined to be between 60 and 70°C.
5.5 Heat stability Heat stability was determined as follows: 600 μl 100 mM sodium acetate buffer, pH 6 (acetic acid) was placed in a water bath maintained at die measuring temperature. 50 μl enzyme solution was added and after appropriate time intervals 100 μl samples were taken out and immediately added to 750 μl acetate buffer which was kept on ice. α- Amylase activity in these samples was determined by incubating 400 μl of the samples with 75 μl 0.5% starch solution in 0.1 M acetate buffer, pH 6 at 50°C for 10 min. 240 μl stop solution and 360 μl colour reagent were added and activity was determined as described above. Heat inactivation of the enzyme is an exponential function of time and heat stability at different temperatures can be described by the half times of inactivation. The results are presented in Table 1.
To 2.5 ml 5 % starch solution in water was added 250 μl enzyme solution (1.5 μmol reducing equivalents/min/ml). The samples were incubated at 50°C and enzyme activity was stopped by boiling die samples after 0.5, 1, 1.5, 2, 3, 4 and 5 hours, respectively. The samples were diluted 20 times in water and analysed on a Dionex HPAEC system on a CarboPac Pa-1 column (4 x 250 mm, flow 1 ml/min) with 0.2 M NaOH, 1.5 M NaOAc and water as eluents in a gradient optimized for separation of glucose oligomers. 0.3 M NaOH was added after the separation column and the dextrins were detected by PAD (pulse amperometric detection). 20 μl samples were applied to the column.
The results are shown in Figure 8, which show the oligomer pattern in starch samples when treated with α-amylase as mentioned above. B0 is me control (i.e. no enzyme, stopped after 5 hrs), whereas the other plots are for the enzyme of the present invention wherein B1 is stoppage at 0.5 hrs, B2 is stoppage at 1 hrs, B3 is stoppage at 1.5 hrs, B4 i stoppage at 2 hrs, B5 is stoppage at 3 hrs, B6 is stoppage at 4 hrs and B7 is stoppage at 5 hrs. In Figure 8, the peak identification is that for glucose, retention time 7.1 min (in B7, glucose peaks at retention time 11.2 which can not be explained); maltose, retention time 15.9 min. ; maltotriose, retention time 17 min. and maltotetraose, retention time 18 min.
The results show that the recombinant α-amylase of the present invention is endospecific, producing only a litte glucose. After 5 hrs. , only 4% of the total peak area is due to glucose, whereas maltose accounts for 39%.
6,. Baking Study
In this example, the beneficial effect of the α-amylase of the present invention in the manufacture of bread is illustrated.
Enzyme addition:
1. control, no addition
2. TAKA amylase, 100 FAU/kg flour
3. Recombinant α-amylase of the present invention, 100 FAU/kg flour
4. TAKA amylase, 350 FAU/kg flour
5. Recombinant α-amylase of the present invention, 350 FAU/kg flour
Handling properties and stickiness of the dough were evaluated during preparation of the bread and after baking the crumb was evaluated.
After baking, the bread was stored in plastic bags at 20°C. Firmness of the bread crumb was measured on day 3 and 7 using an Instron apparatus. 8 slices from each bread loaf were measured and the mean value was calculated. The results are shown in Figure 9.
As can be seen from Figure 9, there is a dosage effect of the recombinant α-amylase of the present invention. Furthermore, the enzyme of the present invention clearly has a beneficial effect on firmness.
Furthermore, the recombinant α-amylase of me present invention lowers firmness as compared to a high dosage of TAKA amylase, meaning that it is a better anti-staling enzyme than the TAKA amylase.
Dough handling properties were similar for doughs prepared with TAKA amylase and wim me recombinant α-amylase of the present invention. The crumb of bread with the recombinant α-amylase of the present invention was evaluated and was found to be more moist than me crumb of bread baked with TAKA amylase.
In summation me present invention relates to a recombinant α-amylase and its use in baking. Other aspects of the present invention include the deposited microorganisms, and mutants, variants and progeny obtained directly or indirectly therefrom and retaining the ability, when cultured, of producing a recombinant α-amylase having the activity described herein. Other modifications of the present invention will be apparent to those skilled in the art.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: DANISCO A/S
(B) STREET: Langebrogade 1. P.O. Box 17
(C) CITY: Copenhagen
(E) COUNTRY: Denmark
(F) POSTAL CODE (ZIP): 1001
(G) TELEPHONE: +4532662200
(H) TELEFAX: +4532662167
(ii) TITLE OF INVENTION: Alpha amylase from Thermomyces lanuginosus
(iii) NUMBER OF SEQUENCES: 11
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patent In Release #1.0. Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3311 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Thermomyces lanuginosus
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join (427..585. 660..698. 755..876. 956..1064,
1142..1370. 1425..1584. 1637..1783. 1846..2086.
2168..2443)
(ix) FEATURE:
(A) NAME/KEY: sig_ peptide
(B) LOCATION: 427. 480
(ix) FEATURE:
(A) NAME/KEY: mat_ peptide
(B) LOCATION :join(481..585. 660..698. 755..876. 956..1064,
1142..1370. 1425..1584 , 1637..1783. 1846..2086.
2168..2440)
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 427..585
(ix) FEATURE:
(A) NAME/KEY: intron
(B) LOCATION: 586..659 ( ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION. -660..698
(ix) FEATURE:
(A) NAME/KEY: intron
(B) LOCATION: 699..754
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 755..876
(ix) FEATURE:
(A) NAME/KEY: intron
(B) LOCATION: 877..955
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 956..1064
(ix) FEATURE:
(A) NAME/KEY: intron
(B) LOCATION: 1065..1141
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 1142..1370
(ix) FEATURE:
(A) NAME/KEY: intron
(B) LOCATION: 1371..1424
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 1425..1584
(ix) FEATURE:
(A) NAME/KEY: intron
(B) LOCATION: 1585..1636
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 1637..1783
(ix) FEATURE:
(A) NAME/KEY: intron
(B) LOCATION: 1784..1845
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 1846..2086
(ix) FEATURE:
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "adaptor upper strand"
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GATCGTTTAA ACGGCGCCG 19
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "adaptor lower strand"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
AATTCGGCGC CGTTTAAAC 19
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
ATGGGNTTYA CNGCNATHTG GAT 23
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
TTYTGRTANG GRCANGTRTA 20
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 245 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iv) ANTI -SENSE: NO
Claims
1. A recombinant enzyme having the following characteristics: i. α-amylase activity over the range from about 60°C to about 80°C; ii. a molecular weight of about 54 - 60 kDa when measured by SDS- PAGE, or of about 55265 Da +/- 100 Da when measured by mass spectrometry; iii. a pI value of about 3.7 (on a Pharmacia IEF gel, 3-9); iv. a pH optimum of from about 5.8 to about 6; v. a temperature optimum of from about 60°C to about 70°C.
2. A recombinant enzyme according to claim 1 wherein the enzyme comprises an N-terminal sequence shown as SEQ. I.D. No. 11.
3. A recombinant enzyme according to claim 1 or claim 2 wherein the enzyme comprises the sequence shown as SEQ. I.D. No. 1 or SEQ. I.D. No. 2, or a variant, homologue or fragment thereof.
4. A recombinant enzyme according to any one of the preceding claims wherein the enzyme is expressed by a nucleotide sequence comprising the coding sequence shown in SEQ. I.D. No. 1, or a variant, homologue or fragment thereof.
5. A recombinant enzyme according to any one of the preceding claims wherein the enzyme is expressed by a nucleotide sequence comprising the nucleotide sequence shown as SEQ. I.D. No.1, or a variant, homologue or fragment thereof.
6. A recombinant enzyme according to any one of the preceding claims wherein the enzyme is obtained or obtainable from T. lanuginosus.
7. A recombinant enzyme according to any one of the preceding claims wherein the enzyme is expressed by a member of the Aspergillus family.
8. A recombinant enzyme according to claim 7 wherein the enzyme is expressed by Aspergillus niger.
9. A recombinant enzyme according to any one of the preceding claims wherein the enzyme is obtainable from any one of NCIMB deposit No. 40655, NCIMB deposit No. 40656, NCIMB deposit No. 40657 and NCIMB deposit No. 40658.
10. A recombinant nucleotide sequence comprising the coding nucleotide sequence shown in SEQ. I.D. No. 1, or a variant, homologue or fragment thereof.
11. A recombinant nucleotide sequence comprising the nucleotide sequence shown as SEQ. I.D. No. 1, or a variant, homologue or fragment thereof.
12. A construct comprising the recombinant nucleotide sequence according to claim 10 or claim 11.
13. A vector comprising the recombinant nucleotide sequence according to claim 10 or claim 11 or the construct according to claim 12.
14. A cell, tissue or organism comprising the recombinant nucleotide sequence according to claim 10 or claim 11 or the construct according to claim 11 or the vector according to claim 13.
15. A transgenic organism comprising the recombinant nucleotide sequence according to claim 10 or claim 11 or the construct according to claim 12 or the vector according to claim 13 or the cell, tissue or organism according to claim 14.
16. A transgenic organism according to claim 15 wherein the organism is a filamentous fungus, preferably an Aspergillus fungus, preferably Aspergillus niger.
17. NCIMB deposit No. 40655, NCIMB deposit No. 40656, NCIMB deposit No. 40657 or NCIMB deposit No. 40658.
18. A method of preparing a foodstuff comprising forming a mixture by contacting starch or a starch based ingredient with an α-amylase and optionally processing the mixture, wherein the α-amylase is a recombinant α-amylase according to any one of claims 1 to 9, or is a recombinant α-amylase expressed by the invention according to any one of claims 10 to 17.
19. A method according to claim 18 wherein the recombinant α-amylase is incorporated into the mixture in an amount sufficient to provide from 50 to 400 units of α-amylase activity per kg of starch or starch based ingredient to form the mixture.
20. A method of preparing a bakery product comprising forming a mixture by contacting a wheat-flour dough with an α-amylase and baking the mixture, wherein the α-amylase is a recombinant α-amylase according to any one of claims 1 to 9, or is a recombinant α-amylase expressed by the invention according to any one of claims 10 to 17.
21. A method according to claim 20 wherein the recombinant α-amylase is incorporated into the dough in an amount sufficient to provide from 50 to 400 units of α-amylase activity per kg of wheat flour used to form the dough.
22. A method according to any one of claims 18 to 21 wherein the foodstuff or bakery product is bread.
23. A foodstuff obtained by use of the recombinant α-amylase according to any one of claims 1 to 9, or a recombinant α-amylase expressed by the invention according to any one of claims 10 to 17.
24. A bakery product obtained by use of the recombinant α-amylase according to any one of claims 1 to 9, or a recombinant α-amylase expressed by the invention according to any one of claims 10 to 17.
25. Use of a recombinant α-amylase having α-amylase activity over the range from about 60°C to about 80°C in preparing a foodstuff.
26. Use of a recombinant α-amylase having α-amylase activity over the range from about 60°C to about 80°C in baking.
27. Use according to claim 25 or claim 26 wherein the recombinant α-amylase is a recombinant α-amylase according to any one of claims 1 to 9, or is a recombinant α-amylase expressed by the invention according to any one of claims 10 to 17.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU30752/95A AU3075295A (en) | 1994-07-04 | 1995-07-03 | Alpha amylase from thermomyces lanuginosus |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9413419A GB9413419D0 (en) | 1994-07-04 | 1994-07-04 | Amylase enzyme |
| GB9413419.4 | 1994-07-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1996001323A1 true WO1996001323A1 (en) | 1996-01-18 |
Family
ID=10757766
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP1995/002607 WO1996001323A1 (en) | 1994-07-04 | 1995-07-03 | Alpha amylase from thermomyces lanuginosus |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU3075295A (en) |
| GB (1) | GB9413419D0 (en) |
| WO (1) | WO1996001323A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6524816B1 (en) | 1997-02-28 | 2003-02-25 | Danisco A/S | Expression element |
| US6607904B2 (en) | 1998-09-30 | 2003-08-19 | Danisco A/S | Peptidyl prolyl cis-trans isomerases |
| WO2004055178A1 (en) * | 2002-12-17 | 2004-07-01 | Novozymes A/S | Thermostable alpha-amylases |
| WO2005066338A1 (en) * | 2004-01-08 | 2005-07-21 | Novozymes A/S | Amylase |
| US7053268B1 (en) | 1999-06-17 | 2006-05-30 | Danisco A/S | Promoter |
| US8252573B2 (en) | 2001-05-15 | 2012-08-28 | Novozymes A/S | Alpha-amylase variant with altered properties |
| US9434932B2 (en) | 2011-06-30 | 2016-09-06 | Novozymes A/S | Alpha-amylase variants |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4416903A (en) * | 1977-12-20 | 1983-11-22 | Cole Morton S | Antistaling baking composition |
| EP0238023A2 (en) * | 1986-03-17 | 1987-09-23 | Novo Nordisk A/S | Process for the production of protein products in Aspergillus oryzae and a promoter for use in Aspergillus |
| EP0273268A2 (en) * | 1986-12-22 | 1988-07-06 | Enzyme Bio-Systems Ltd. | Reduced-stability alpha-amylase and process for its production |
| EP0419907A1 (en) * | 1989-09-13 | 1991-04-03 | Enzyme Bio-Systems Ltd. | Method for retarding staling of baked goods |
-
1994
- 1994-07-04 GB GB9413419A patent/GB9413419D0/en active Pending
-
1995
- 1995-07-03 WO PCT/EP1995/002607 patent/WO1996001323A1/en active Application Filing
- 1995-07-03 AU AU30752/95A patent/AU3075295A/en not_active Abandoned
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4416903A (en) * | 1977-12-20 | 1983-11-22 | Cole Morton S | Antistaling baking composition |
| EP0238023A2 (en) * | 1986-03-17 | 1987-09-23 | Novo Nordisk A/S | Process for the production of protein products in Aspergillus oryzae and a promoter for use in Aspergillus |
| EP0273268A2 (en) * | 1986-12-22 | 1988-07-06 | Enzyme Bio-Systems Ltd. | Reduced-stability alpha-amylase and process for its production |
| EP0419907A1 (en) * | 1989-09-13 | 1991-04-03 | Enzyme Bio-Systems Ltd. | Method for retarding staling of baked goods |
Non-Patent Citations (1)
| Title |
|---|
| JENSEN B ET AL: "PHYSICOCHEMICAL PROPERTIES OF A PURIFIED ALPHA AMYLASE FROM THE THERMOPHILIC FUNGUS THERMOMYCES -LANUGINOSUS.", ENZYME MICROB TECHNOL 14 (2). 1992. 112-116. CODEN: EMTED2 ISSN: 0141-0229 * |
Cited By (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6524816B1 (en) | 1997-02-28 | 2003-02-25 | Danisco A/S | Expression element |
| US6607904B2 (en) | 1998-09-30 | 2003-08-19 | Danisco A/S | Peptidyl prolyl cis-trans isomerases |
| US6638737B1 (en) | 1998-09-30 | 2003-10-28 | Danisco A/S | Peptidyl prolyl cis-trans isomerases |
| US7053268B1 (en) | 1999-06-17 | 2006-05-30 | Danisco A/S | Promoter |
| US8486681B2 (en) | 2001-05-15 | 2013-07-16 | Novozymes A/S | Alpha-amylase variant with altered properties |
| US10612012B2 (en) | 2001-05-15 | 2020-04-07 | Novozymes A/S | Alpha-amylase variant with altered properties |
| US9796968B2 (en) | 2001-05-15 | 2017-10-24 | Novozymes A/S | Alpha-amylase variant with altered properties |
| US9670471B2 (en) | 2001-05-15 | 2017-06-06 | Novozymes A/S | Alpha-amylase variant with altered properties |
| US9080137B2 (en) | 2001-05-15 | 2015-07-14 | Novozymes A/S | Alpha-amylase variant with altered properties |
| US8617837B2 (en) | 2001-05-15 | 2013-12-31 | Novozymes A/S | Method of hydrolyzing soluble starch with an alpha-amylase variant |
| US8252573B2 (en) | 2001-05-15 | 2012-08-28 | Novozymes A/S | Alpha-amylase variant with altered properties |
| US7189552B2 (en) | 2002-12-17 | 2007-03-13 | Novozymes A/S | Thermostable alpha-amylases |
| US8039241B2 (en) | 2002-12-17 | 2011-10-18 | I Novozymes A/S | Thermostable alpha-amylases |
| US9394533B2 (en) | 2002-12-17 | 2016-07-19 | Novozymes A/S | Thermostable alpha-amylases |
| CN100412191C (en) * | 2002-12-17 | 2008-08-20 | 诺和酶股份有限公司 | Thermostable alpha-amylase |
| US9938512B2 (en) | 2002-12-17 | 2018-04-10 | Novozymes A/S | Thermostable alpha-amylases |
| WO2004055178A1 (en) * | 2002-12-17 | 2004-07-01 | Novozymes A/S | Thermostable alpha-amylases |
| US7666650B2 (en) | 2004-01-08 | 2010-02-23 | Novozymes A/S | Amylase |
| WO2005066338A1 (en) * | 2004-01-08 | 2005-07-21 | Novozymes A/S | Amylase |
| US9434932B2 (en) | 2011-06-30 | 2016-09-06 | Novozymes A/S | Alpha-amylase variants |
| US10167458B2 (en) | 2011-06-30 | 2019-01-01 | Novozymes A/S | Alpha-amylase variants |
| US10752889B2 (en) | 2011-06-30 | 2020-08-25 | Novozymes A/S | Alpha-amylase variants |
| US11091748B2 (en) | 2011-06-30 | 2021-08-17 | Novozymes A/S | Alpha-amylase variants |
| US12012623B2 (en) | 2011-06-30 | 2024-06-18 | Novozymes A/S | Alpha-amylase variants |
Also Published As
| Publication number | Publication date |
|---|---|
| GB9413419D0 (en) | 1994-08-24 |
| AU3075295A (en) | 1996-01-25 |
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