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WO1994000578A1 - Cellulases recombinees - Google Patents

Cellulases recombinees Download PDF

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Publication number
WO1994000578A1
WO1994000578A1 PCT/AU1993/000307 AU9300307W WO9400578A1 WO 1994000578 A1 WO1994000578 A1 WO 1994000578A1 AU 9300307 W AU9300307 W AU 9300307W WO 9400578 A1 WO9400578 A1 WO 9400578A1
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Prior art keywords
cellulase
cdna
enzyme
celd
recombinant
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PCT/AU1993/000307
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English (en)
Inventor
Gang Ping Xue
Colin George Orpin
James Harrison Aylward
Kari Steven Gobius
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Commonwealth Scientific And Industrial Research Organisation
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Priority to JP6501883A priority Critical patent/JPH07507928A/ja
Priority to AU42995/93A priority patent/AU4299593A/en
Priority to EP93912457A priority patent/EP0649471A4/fr
Publication of WO1994000578A1 publication Critical patent/WO1994000578A1/fr
Priority to FI945994A priority patent/FI945994L/fi

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    • 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
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

Definitions

  • FIELD OF INVENTION relates to recombinant cellulases derived from anaerobic fungi and a method of production of recombinant cellulases and clones utilised in the method.
  • Cellulose is one of the most abundant polysaccharides in nature and consists of a polymer of glucose linked by ⁇ -1,4-glucosidic bonds. Conversion of cellulose to simple sugars (cellobiose and glucose) involves at least two types of hydrolases: endoglucanases which hydrolyse internal ⁇ -1,4-glucosidic linkages in less ordered regions of cellulose and exoglucanases (mainly cellobiohydrolases) which cleave cellobiosyl units from non-reducing ends of cellulose chains.
  • Xylan similar to the structure of cellulose, consists of a backbone of ⁇ -1,4-linkedxylose units.
  • xylanases endo- ⁇ -1,4-xylanases
  • CM-cellulose carboxymethyl cellulose
  • MUC methylumbelliferyl cellobioside
  • many xylanases exclusively attack ⁇ -1,4-xylosidic linkages.
  • polysaccharide hydrolases have strict substrate specificity. Due to the similarity in the chemical nature of the substrates, cross specificity occurs not only between two types of cellulase, but also between cellulases and xylanases.
  • rumen anaerobic fungi such as Neocallimastix frontalis might produce multi-functional polysaccharide hydrolases (Gomez de Segura & Fevre, 1991; Li & Calza, 1991).
  • Multi-functional polysaccharide hydrolases are of particular interest in genetic manipulation of rumen bacteria to enrich for the lignocellulose-degrading capacity.
  • Simultaneous enhancement of endoglucanase, cellobiohydrolase and xylanase activities would facilitate the disruption of the complex structure of lignocellulose, of which cellulose and xylan are the major components. It may also circumvent the rate-limiting problem which often occurs when only one of a complex of enzymatic reactions is enhanced.
  • Cellulose and hemicellulose are major components of ruminants' diets, consisting of 50-80% by weight of plant tissue. Effective utilisation of plant feeds is therefore largely dependent on the production of cellulolytic and xylanolytic enzymes by microbial populations residing within the rumen. Compared with other components of the diet, degradation of cellulose and hemicellulose in the rumen is relatively slow and incomplete; digestion may be as low as 30% (Dehority, 1991). Thus, there is potential economic value in enhancing the plant fibre-degrading capacity by introducing plant polysaccharide hydrolase gene(s) into rumen micro-organisms using recombinant DNA techniques.
  • Neocallimastix patriciarum isolated from the sheep rumen, has a high capacity for cellulose degradation and can grow on cellulose as the sole carbohydrate source (Orpin & Munn, 1986; Williams & Orpin, 1987).
  • Cloning of cellulase genes from bacteria can be achieved by isolation of enzymatically active clones from genomic libraries established in E. coli .
  • this approach for isolation of cellulase genes from fungal genomic libraries with functional expression of cellulase is usually not possible. This is because fungi are eucaryotic microorganisms. Most eucaryotic genes contain introns and E. coli is unable to perform post-transcriptional modification of mRNAs in order to splice out introns. Therefore, enzymatically functional protein cannot normally be synthesised in clones obtained from a fungal genomic library.
  • the cDNA cloning approach can be used to overcome the post-transcriptional modification problem in E. coli .
  • cellulases in fungi are usually glycosylated and glycosylation is often required for biological activity of many glycosylated enzymes.
  • E. coli lacks a glycosylation mechanism. This problem can be solved if the cloned gene is transferred to an eucaryotic organism, such as yeast.
  • yeast eucaryotic organism
  • coli are (i) that many eucaryotic mRNAs contain translational stop codons upstream of the translational start codon of a gene which prevents the synthesis of the cloned protein from the translational start provided in the vector, and (ii) that synthesis of the cloned protein is based on fusion proteins and the biological function of the cloned protein is often adversely affected by the fused peptide derived from the cloning vector.
  • a further object of the invention is to provide a method of cloning of cellulase cDNAs from an anaerobic rumen fungus which may encode the recombinant cellulase of the invention.
  • a further object of the invention is to provide cellulase clones which may be produced in the abovementioned method.
  • the method of cloning of the invention includes the following steps:
  • step (ii) isolating total RNA from the culture in step (i);
  • step (i) above in relation to preparation of the recombinant cellulase from anaerobic fungi, particularly alimentary tract fungi, may be cultivated as described hereinbelow.
  • These fungi are strict anaerobes and may be exemplified by Neocallimastix patriciarum, Neocallimastix frontalis, Neocallimastix h url eyensi s , Neocallimastix s tan thorpensi s , Sphaeromonas communis, Caecomyces equi , Piromyces communis, Piromyces equi , Piromyces dumbonica, Piromyces lethargicus, Piromyces mai , Ruminomyces elegans, Anaeromyces mucronatus, Orpinomyces bovis and Orpinomyces joyonii .
  • the cultivation may proceed in appropriate culture media containing rumen fluid and also may contain cellulose such as Avicel (ie. a form of microcrystalline cellulose) as a carbon source under anaerobic conditions.
  • cellulose such as Avicel (ie. a form of microcrystalline cellulose)
  • After cultivation of the fungi total RNA may be obtained in any suitable manner.
  • the fungal cells may be harvested by filtration and subsequently lysed in appropriate cell lysis buffer by mechanical disruption.
  • a suitable RNA preserving compound may also be added to the fungal cells to maintain the RNA intact by denaturing RNAses which would otherwise attack the fungal RNA.
  • the total RNA may subsequently be isolated from the homogenate by any suitable technique such as by ultracentrifugation through a CsCl 2 cushion or alternative technique as described by Sambrook et. al.
  • Total fungal RNA in this alternative technique may also be isolated from the above homogenate by extraction with phenol chloroform at pH4 to remove DNA and associated protein. Total RNA obtained was further purified by washing with lithium chloride-urea solution.
  • Poly (A) + mRNA may then be isolated from the total RNA by affinity chromatography on a compound containing multiple thymine residues such as oligo (dT) cellulose. Alternatively a compound containing multiple uracil residues may be used such as poly (U)-Sephadex. The poly (A) + mRNA may then be eluted from the affinity column by a suitable buffer.
  • a cDNA expression library may then be constructed using a standard technique based on conversion of the poly (A) + mRNA to cDNA by the enzyme reverse transcriptase.
  • the first strand of cDNA may be synthesised using reverse transcriptase and the second strand of the cDNA may be synthesised using E. coli DNA polymerase I.
  • the cDNA may subsequently be fractionated to a suitable size and may be ligated to the bacteriophage expression vector, preferably ⁇ ZAP or ⁇ ZAPII.
  • the cDNA library may then be amplified after packaging in vi tro, using any suitable host bacterial cell such as a suitable strain of E. coli .
  • step (v) The choice of the bacteriophage expression vector in step (v) is important in that such expression vector should include the following features:
  • the fusion peptide derived from the vector should be as small as possible, as the biological function of the cloned protein is usually adversely affected by the fused peptide derived from the vector. Therefore the polyclonal sites of the bacteriophage expression vector are suitably located at the N-terminus of lacZ peptides such as in ⁇ ZAPII.
  • vectors of similar properties to ⁇ ZAP or ⁇ ZAPII includes within its scope expression vectors having the abovementioned features (i), (ii), (iii) and (iv).
  • the screening of cellulase positive recombinant clones may be carried out by any suitable technique based on hydrolysis of cellulose.
  • the clones may be grown on culture media incorporating cellulose and hydrolysis may be detected by the presence of cellulase-positive plaques suitably assisted by a suitable colour indicator.
  • Cellulase positive recombinant clones may then be purified and the cDNA insert in the clones may then be excised into pBluescript (SK(-)).
  • E. coli promoter Any suitable E. coli promoter may be used in the expression vector described above. Suitable promoters include lacZ, Tac, Bacteriophage T 7 and lambda-P L .
  • the cloned celA enzyme has high specific activity on crystalline and amorphous cellulase.
  • the optimal pH and temperature for cellulose hydrolysis are pH5 and 40°C, respectively,
  • the cloned celD enzyme is a multi-functional cellulase with a high activity of endoglucanase, cellobiohydrolase and xylanase.
  • celD cDNA can be truncated to code for three catalytically active domains. Each domain has endoglucanase, cellobiohydrolase and xylanase activity and cellulose-binding capacity.
  • the recombinant celA and celD enzymes also have very high activity on lichenan.
  • a combination of celA and celD enzymes can hydrolyse crystalline cellulose more efficiently.
  • Microbial strains Microbial strains, vectors and culture media.
  • Neocallimastix patriciarum type species
  • the culture medium for N. patriciarum was described previously (Kemp et al., 1984).
  • Microcrystalline cellulose (Avicel) was used as the sole carbohydrate source.
  • Host strains for cDNA cloning were E coli PLK-F and XL1-Blue obtained from Stratagene. E coli strains were grown in L-broth (Sambrook et al., 1989).
  • ⁇ ZAPII vector was obtained from Stratagene and the recombinant phage were grown in E coli strains according to the supplier's instructions.
  • Frozen fungal mycelia were ground into fine powder with a mortar and pestle under liquid N 2 .
  • Powdered mycelia were homogenised in guanidinium thiocyanate solution (4M guanidinium thiocyanate, 0.5% (w/v) sodium lauryl sarcosine, 25 mM-sodium citrate, pH7.0, 1mM-EDTA and 0.1 M- ⁇ -mercaptoethanol) using a mortar and pestle for 5 min and then further homogenised with a Polytron at full speed for 2 min. Total cellular RNA was prepared from the homogenate either by.
  • guanidinium thiocyanate solution 4M guanidinium thiocyanate, 0.5% (w/v) sodium lauryl sarcosine, 25 mM-sodium citrate, pH7.0, 1mM-EDTA and 0.1 M- ⁇ -mercaptoethanol
  • RNA pellet obtained after acid guanidinium thiocyanate/phenol/chloroform extraction and the first step of 2-propanol precipitation, was suspended in a LiCl/urea solution (6 M-urea, 3 M-LiCl, 1 mM-EDTA, pH 7.6). The suspension was shaken at 4°C for 1-2 h to remove contaminating protein and DNA.
  • RNA pellet was briefly washed once with the LiCl/urea solution, twice with 75% (v/v) ethanol and then dissolved in 10 mM-Tris/HCl/1 mM-EDTA, pH 8.0.
  • the RNA was further purified by extraction with phenol/chloroform and ethanpol precipitation.
  • Poly(A) + RNA was selected by oligo(dT)-cellulose chromatography (Sambrook et al. 1989).
  • RNA isolation, restriction endonuclease digestion, ligation , transformation and preparation of RNA probes were performed basically according to procedures described by Sambrook et al. (1989).
  • Double-stranded cDNA was synthesised from mRNA isolated from N. patriciarum grown on the medium containing 1% (w/v) Avicel for 48 h and ligated with ⁇ ZAPII using a ZAP-cDNA synthesis kit, according to the manufacturer's instructions (Stratagene).
  • a cDNA library of 10 6 recombinants was obtained.
  • Recombinant phage were screened for cellulolytic activity by plating in 0.7% (w/v) soft agar overlays containing one of the following substrates 0.5% (w/v) carboxymethylcellulose (CM-cellulose), 1 mm MUC or 0.1% xylan.
  • CM-cellulose hydrolysis was detected by the Congo red staining procedure (Teather & Wood, 1982). MUC hydrolysis was examined for fluorescence under UV light.
  • the cDNA inserts in CM-cellulose positive phage were recovered in the form of pBluescript (SK-) by in vivo excision, according to Stratagene's instructions.
  • celD cDNA Deletion of celD cDNA was achieved by either removing a cDNA fragment with restriction enzymes or by exonuclease III digestion (Sambrook et al., 1989). The truncated celD cDNA was checked either by restriction mapping or by partial nucleotide sequencing at the insert terminals.
  • Single-stranded plasmid DNA was prepared basically according to Stratagene's protocol. Sequencing of the resultant DNA was performed using dideoxynucleotide method (Tabor and Richardson, 1987). Southern blot hybridisation.
  • ⁇ DNA from the cellulase-positive clones was purified by a rapid mini-preparation method as follows.
  • One millilitre of phage lysate from liquid culture was incubated with RNAase A ( 10 ⁇ g ml -1 ) and DNasel (1 ⁇ g ml -1 ) at 37°C for 1 h and with proteinase K ( 1 mg ml -1 ) at 37°C for 3 h and then extracted with phenol/chloroform.
  • the DNA was precipitated by ethanol, digested with EcoRI and Xho1 (the cDNA cloning sites), fractionated by electrophoresis on 1 % (w/v) agarose gel and blotted onto Hybond N membrane (Amersham). Procedures for hybridisation and signal detection were as described previously (Xue & Morris, 1992), using digoxigenin-labelled RNA probes prepared from the 3'-region-deleted cDNA. Hybridisation was carried out at 50°C in a hybridisation mixture of 50% (v/v) formamide, 0.8 M-NaCl, 50 mM-sodium phosphate (pH 7.2), 4mM-EDTA, 0.2% (w/v) SDS.
  • Denhardt's solution 5x Denhardt's solution, 0.2 mg yeast RNA ml -1 , 0.2 mg herring sperm DNA ml -1 (1 x Denhardt's solution is 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone). High-stringency washing was performed in 0.1 ⁇ SSC/0.1% (w/v) SDS at 68°C (1 X SSC is 0.15 M-NaCl, 15 mM-sodium citrate).
  • E coli cells harbouring the recombinant plasmids were grown in LB medium to the end of the exponential phase in the presence of 1mM IPTG. Crude cell lysates prepared according to Schwarz et al . (1987) were used as enzyme sources.
  • the enzyme preparations were incubated at 39°C for 30-60 min in 50 mM-sodium citrate (pH 5.7) with the following substrates: 0.5% (w/v) CM-cellulose (low viscosity, Sigma, 1% (w/v) amorphous cellulose (H 3 PO 4 -swollen Avicel), 1% (w/v) Avicel (Merck), 0.05% (w/v) p-nitrophenyl cellobioside (pNPC, Sigma), p-nitrophenyl glucopyranoside (pNPG, Sigma), 0.25% (w/v) oat spelt xylan (Sigma) and 0.4% Lichenan.
  • the reducing sugars released from cellulose, Lichenan or xylan were measured as described by Lever (1972).
  • the p-nitrophenyl groups released from p-nitrophenyl derivatives were measured as described by Deshpande et al. (1988).
  • the cell lysate prepared from E coli strain XL1-Blue harbouring non-recombinant pBluescript was used as control. Protein concentrations were determined by dye-binding assay using the Bio-Rad protein assay kit II according to the supplier's instructions.
  • cell lysates were incubated with 200 ⁇ l of pre-washed 5% (w/v) Avicel in 50 mM-sodium citrate (pH 5.7) at 0°C with continuous shaking for 1h. The unbound protein was removed after centrifugation and the Avicel pellet was washed three times with 50mM- sodium citrate (pH 5.7). The bound cellulase was assayed for enzyme activity as above.
  • a cDNA library was prepared from poly (A) + RNA isolated from N. patriciarum grown on Avicel as the sole carbohydrate source and was constructed in E coli using a ⁇ ZAPII vector. The library was initially screened for expression of endoglucanase activity on CM-cellulose plates. Two hundred CM-cellulose positive plaques were identified after screening 4 ⁇ 10 5 plaques from library.
  • CM-cellulose positive clones were screened for cellobiohydrolase activity first on MUC plates and were further tested for the ability to hydrolyse microcrystalline cellulose, by assaying the reducing sugar released after absorption of cellulase in the supernatant of the recombinant bacteriophage lysates to Avicel followed by incubation at 39°C for 3 hr (see cellulose-binding assay in Method). Eleven bacteriophage clones exhibited large hydrolysis zones on both CM-cellulose and MUC plates, as well as activity towards Avicel. These eleven clones were then tested for xylanolytic activity on xylan plates and all were positive.
  • Fig. 1 A restriction map of the longest cellulase cDNA sequence, designated celD (pCNP4.1) is shown in Fig. 1.
  • the remaining clone possessed an insert of 7.0 Kb designated as celE and also had a similar restriction pattern to celD, but contained two additional 1.15-Kb internal EcoR1-EcoR1 fragments and a 1.7 Kbp cDNA (Fig 1).
  • Cross hybridisation analysis showed that CelD strongly hybridised to CelE using a nucleic acid probe prepared from CelD cDNA in which the 3' region was deleted. Thus it is most likely that the ten clones originate from the same gene and the celE clone is a related cDNA to celD.
  • Fig. 2 Three other classes of cellulase cDNAs were isolated from the pool of CM-cellulose-positive clones by restriction mapping and cross-hybridisation. Restriction maps of three cellulase cDNAs (the longest cDNA insert for each type), designated celA (2.0 Kb), CelB (1.7 Kb) and CelC (1.6 Kb) respectively, are shown in Fig. 2. Southern hybridisation analysis showed these three cDNA inserts did not cross-hybridise to each other (Fig. 3), using nucleic acid probes prepared from CelA and CelC clones with the 3 'regions of the cDNA insert removed by digestion with Xhol and the enzyme at the upstream restriction site (see Fig 2). Similarly, CelD did not hybridise to celA, celB and CelC using high stringency conditions.
  • the substrate specificity of these recombinant cellulases was further characterised by quantitative measurement of the activity on various cellulosic substrates and xylan. As shown in Table 1, the celD enzyme was most active on CM-cellulose, but it also possessed cellobiohydrolase-like properties, as it was highly active on crystalline cellulose, MUC and p-nitrophenyl cellobioside (pNPC) as well as amorphous cellulose. The enzyme showed no activity on methylumbelliferyl glucoside (MUG) and p-nitrophenyl glucoside (pNPG), substrates for ⁇ -glucosidase.
  • MUC methylumbelliferyl glucoside
  • pNPG p-nitrophenyl glucoside
  • celD enzyme had very high activity towards lichenan (Table 1) and produced a large hydrolysis zone on lichenancontaining agarose gel plates, but did not produce a hydrolysis zone on laminarin plates (Fig. 4). This indicates that cleavage on lichenan is at the ⁇ -1 ,4-linkages. Interestingly, a high xylanase activity was also present in the celD enzyme.
  • the celD enzyme was able to hydrolyse cellodextrins (containing 3-5 glucose units) to glucose and cellobiose. Its catalytic mode on these cellulosic substrates is of a typical endoglucanase (ie. it cleaved ⁇ -1,4-glucosidic linkages at random positions, as shown in Fig. 5).
  • the hydrolysis products of microcrystalline cellulose were mainly cellobiose with a trace amount of glucose (Fig.5), indicative of cellobiohydrolase activity. It appears that it is a truly multi-functional plant polysaccharide-degrading enzyme.
  • celE enzyme Although a number of cellulases and xylanases have been shown to have multiple substrate specificity, most of them possess only residual activity (usually ⁇ 1%) towards the secondary substrate (Saarilahti et al., 1990; Yague et al., 1990; Hazlewood et al., 1990; Flint et al., 1991; Taylor et al., 1987).
  • the substrate specificity of celE enzyme is similar to celD enzyme, but its activity was about 4-fold lower.
  • the enzyme encoded by celA possesses cellobiohydrolase properties. It has very high activity in hydrolysis of crystalline and amorphous cellulose, although it also has relatively weak activity on CM-cellulose (Table 1).
  • the cellobiohydrolase-like properties of the celA enzyme was further confirmed by its hydrolysis pattern as cellobiose was the only product released from cellotetrose or Avicel by the celA enzyme (Fig. 6).
  • the celA enzyme also has very high activity on Lichenan and no activity on laminarin.
  • the enzyme properties of celB and celC resembled endo-glucanase (Table 1 and Fig. 6).
  • the pH and temperature profiles of celA and celD enzymes are shown in Fig. 7 and Fig. 8.
  • the celA and celD enzymes were active from pH4.5 to pH8.5 and preferably at pH5-7.
  • the thermostability of these enzymes was tested at temperature from 30°C-60°C.
  • the celA and celD enzymes are active preferably at 30°C-50°C.
  • the recombinant enzymes remain active in hydrolysis of Avicel at 39°C for at least 21 hr (Fig. 9 and Fig. 10).
  • the hydrolysis rates of Avicel by celA or celD enzyme were not proportional to the enzyme levels tested (Fig. 9 and Fig. 10).
  • a combination of celA and celD enzymes performs much better in hydrolysis of Avicel than doubling the concentration of individual enzyme (Fig. 11), suggesting a complementary effect of the celA and celD enzymes.
  • pNX-Tac is a DNA construct as shown in Fig. 16 and has a DNA sequence as shown in FIG 17.
  • a combination of a recombinant xylanase such as pNX-tAC and celA and celD enzyme has demonstrated that co-operativity or synergy may occur in relation to biological activity on crude cellulosic substrates containing lignin and hemicellulose components. This activity is shown in Table 2.
  • the cellulose-binding capacity of the celA and celD enzymes were assessed by a comparative assay of the enzyme activity with or without prior absorption to crystalline cellulose (Avicel).
  • the amount of reducing sugar released from Avicel after absorption of the enzyme to Avicel followed by extensive washing of the enzyme-substrate complex was 23.3 ⁇ g glucose equivalent min ⁇ 1 per mg protein (the crude cell lysate preparation), compared to 24.3 ⁇ g min ⁇ 1 per mg protein for the enzyme added without prior absorption.
  • This high recovery (95%) of the enzyme activity after absorption and washing suggests that the celD enzyme possesses a strong cellulose-binding capacity.
  • the recovery of celA enzyme after adsorption to Avicel and washing was 77%, slightly lower than celD enzyme.
  • the cellulose-binding capacity is important for efficient degradation of cellulose as a result of the close contact of the enzyme with this insoluble substrate.
  • celD cDNA can be truncated to code for three catalytically active domains, when each domain was fused in frame with the vector's lacZ translation initiation codon. These are designated domain I (pCNP4.2), domain II (pCNP4.4) and domain III (pCNP4.8), respectively.
  • domain I pCNP4.2
  • domain II pCNP4.4
  • domain III pCNP4.8
  • Domain II contained sequence from the position 1.15 Kb to 2.3 Kb of celD cDNA and domain III from 2.3 Kb to 3.37 Kb.
  • the subclone construction of domain II (pCNP4.4) was achieved by deletion of a 1.15-Kbp EcoRI-PvuII fragment at the 5' region and exonuclease III digestion at the 3' region of celD cDNA and domain III by exonuclease III digestion from both the 5' region and 3' region of the celD.
  • all three domains possessed the same pattern of substrate specificities as the enzyme produced by the untruncated celD cDNA.
  • all three domains had cellulose-binding capacity. Recovery of the enzyme activity after absorption to Avicel and subsequent washing ranged from 70% to 80%. This is slightly lower than the enzyme from the untruncated celD cDNA.
  • the celD cDNA was sequenced (see Fig. 13) and graphical presentation of celD structure is shown in Fig. 14.
  • the amino acid sequences of three catalytic domains deduced from the nucleotide sequence are presented in Fig. 15.
  • the third catalytic domain is untranslated, because there is a translation stop codon at the end of the second domain.
  • celD enzyme Although some cellulases and xylanases consist of two mono-functional catalytic domains (Saul et al., 1990; Gilbert et al. 1992) or possess a single multi-functional domain (Foong et al., 1991), there is no previous example of a polysaccharide hydrolase cDNA encoding three multifunctional catalytic domains, with each catalytic domain possessing cellulose-binding capacity.
  • a multi-functional enzyme would be beneficial for the rumen fungus in its natural environment where these polysaccharide substrates exist in a complex structure. Usually, several types of polysaccharide hydrolases are required to form a multi-enzyme complex acting co-operatively on these natural substrates.
  • celA cDNA encodes a highly active cellobiohydrolase which efficiently hydrolyses both crystalline and amorphous cellulose.
  • celD cDNA encodes a highly active enzyme with endoglucanase, cellobiohydrolase and xylanase activities, capable of degrading a wide range of cellulosic materials and xylan.
  • the cloned celD enzyme can actively hydrolyse crystalline cellulose, presumably due to the presence of both endoglucanase and cellobiohydrolase activities which act synergistically in cellulolysis.
  • the celD cDNA contains sequences which can encode three functional domains; each domain possesses endoglucanase, cellobiohydrolase and xylanase activities in addition to strong cellulose-binding capacity.
  • the cellulose- binding capacity is important for efficient degradation of cellulose as a result of the close contact of the enzyme with this insoluble substrate.
  • celA and celD enzymes have very high activity in hydrolysis of lichenan.
  • a multi-functional enzyme could more efficiently degrade the polysaccharide complex existing in plant materials. Although a number of cloned cellulases showed multiple substrate specificity, most of them possess only residual activity (usually ⁇ 1%) towards the secondary substrate. There is no previous example of a cellulase or xylanase gene encoding three multi-functional catalytic domains with each possessing strong cellulose-binding capacity. The activity of the cloned celA and celD enzymes in E coli can be further increased by using stronger promoters.
  • Cellulose and hemicellulose represent the most abundant natural resource on earth.
  • Cellulose alone accounts for about 40% total biomass with an annual production of 4 ⁇ 10 10 tons (Coughlan, 1985), which was equivalent to 70 kg of cellulose synthesises per person each day, as calculated in 1983 by Lutzen et al. (1983).
  • Most plant materials consist of 40-60% cellulose and 15-30% hemicellulose (Dekker and Lindner 1979).
  • Efficient utilisation of plant materials by ruminant animals, such as sheep and cattle, are therefore largely dependent on production of cellulolytic and xylanolytic enzymes by microbial populations residing within the rumen (the enlarged forestomach of the ruminants).
  • cellulase cDNAs include transfer into some industrial strains of microorganisms for more efficient conversion of cheap plant material, even lignocellulosic wastes, to commercially valuable products, such as ethanol, butanol, acetic acid, citric acid and antibiotics.
  • the recombinant cellulases may also be used as a cellulase source for industrial applications.
  • Cellulase is one of the sixteen important industrial enzymes.
  • the current world market for these enzymes is >750 million U.S. dollars with an annual growth rate of 5-10% in volume.
  • the potential use of the recombinant celD enzyme is listed below:
  • the enzyme may be added to waste water to remove cellulose residues in waste water recycling processes. It may also be used to facilitate drainage in paper making and the deinking of newsprint.
  • celA and celD as genetic material for modification of some economically important micro-organisms for improvement of cellulose utilisation 1. Modification of rumen bacteria for improvement of plant fibre digestion by sheep and cattle.
  • lactic acid bacteria to stimulate conversion of cellulosic material to microbial protein and increase nutritive value of silage as animal feeds.
  • the invention also includes within its scope the following - (i) DNA sequences derived from celA, celB, celC, celD and celE cDNA clones;
  • celA, celB, celC, celD or celE enzymes in combination or mixtures of these enzymes as a pair, triplet or as a mixture of four enzymes, and these mixtures in combination with xylanase, eg. recombinant xylanase derived from Neocallimastix patriciarum.
  • Plasmid pCNP4.1 in E coli strain XL1-Blue has been deposited at the International Depository Australian Government Analytical Laboratories on June 22, 1992 under accession number N92/27543.
  • Plasmid pCNP1 has been deposited at the
  • hybridise refers to a standard nucleic acid hybridisation technique described by Sambrook et. al. (1989).
  • BAUCHOP T. (1981). The anaerobic fungi in rumen fiber digestion. Agricul ture and Environment 6,
  • FOONG E., HAMAMOTO, T., SHOSEYOV, O. & DOI, R.H.
  • Neocallimasrix patriciarum contains two homologous catalytic domains. Molecular Microbiology 6(15), 2065- 2072.
  • KILBURN D.G. (1988). Precise excision of the cellulose binding domains from two Cellulomonas fimicellulases by a homologous protease and the effect on carlaysis. The Journal of Biological Chemistry. 263, 10401- 10407.
  • MEINKE A., GILKES, N.R., KILBURN, D.G., MILLER,
  • Neocallimastix patriciarum sp. nov. a new member of the Neocallimasticaceae inhabiting the rumen of sheep. Transactions of the Bri tish Mycological Society 86, 178-180.
  • ROBSON L.M. & CHAMBLISS, G.H. (1989). Cellulases of bacterial origin. Enzyme and Microbial Technology 11, 626-644. ROMANTEC, M.P.M., DAVIDSON, K., WHITE, B.A., HAZLEWOOD, G.P. (1989). Cloning of Ruminococcus albus endo- ⁇ -1, 4-glucanase and xylanase genes. Letters in Applied Bacteriology 9, 101-104. SAARILAHTI, H.T., HENRISSAT, B. & PALVA, E.T. (1990).
  • CelS a novel endoglucanase identified from Erwinia carotovora subsp. carotovora . Gene 90, 9-14.
  • CelB a gene coding for a bifunctional cellulase from the extreme thermophile 'Caldocellum saccharolyticum '. Applied and Environmental Microbiology 56, 3117-3124.
  • TOMME P., VAN TILBEURGH, H., PETTERSSON, G., VAN
  • the rate of plant fibre hydrolysis was measured by assaying reducing sugar production
  • the hydrolysis reaction was performed at 40 C for 2 days using Setaria stem with in vivo digestibility of 64.7%.
  • FIG. 3 Cross-hybridization of three cellulase cDNA inserts by Southern blot analysis. Plasmids containing celA (A), celB (B) and celC (C) were cut with EcoRI and XhoI (the cDNA cloning sites) and fractionated on 1% (w/v) agarose gel. Digoxigenin-labelled RNA probes generated from 3 '-region-deleted cDNA clones were used for hybridization: celA ' probe, left blot; celC' probe, right blot. Large arrows indicate the cDNA inserts being hybridized.
  • the bands indicated by small arrows are the cloning vector being hybridized, as the RNA probes contain part of the sequence from the vector. Numbers on the margins indicate the sizes, in kb, of molecular markers (BstEII fragments of ⁇ DNA).
  • Figure 4 Congo-red staining assay of the celD enzyme activity on lichenan and laminarin. Two microlitres of crude enzyme extract were placed onto wells cut in the agarose plates containing lichenan or laminarin as described in Methods. After incubation at 39°C for 2 hr and staining with Congo red, hydrolysis of substrates is indicated by presence of a yellow halo in the red background around the well.
  • FIG. 5 Analysis of products of cellulosic compounds hydrolysed by the celD enzyme.
  • Crude cell lysate was prepared from E. coli harbouring plasmid pCNP4.1 and low-molecular-mass compounds were removed by spindialysis using a Centricon-10 tube (Amicon). The enzyme preparation was incubated with cellodextrins: [cellotriose (G 3 ), cellotetraose (G 4 ) and cellopentaose (G 5 ), each 2 mg ml -1 ] or with 1% (w/v) Avicel (C) as described in Methods. Partial hydrolysis of cellodextrins is shown to illustrate the intermediate products.
  • FIG. 6 Analysis of hydrolysis products of the celA, celB and CelC enzymes on cellulosic compounds.
  • Crude cell lysates were prepared from E. coli harbouring recombinant plasmids ( celA, celB and celC) and the small molecules were removed by spin-dialysis using Centricon-10 tubes (Amicon). The enzyme preparations were incubated with cellodextrins (2 mg ml -1 ); cellotriose (G 3 ), cellotetraose (G 4 ) and cellopentaose (G 5 ) or with 1% (w/v) Avicel (C) as described in Methods. Products were identified by TLC.
  • Figure 7 Effect of pH on the activity of the recombinant cellulases.
  • Cellulase assays were performed at 40°C in 50 mM Na-citrate (pH4-7) or 25mM Tris-Cl/50mM NaCl (pH7.5-9.5) containing 1% Avicel for 22 hours.
  • Figure 8 Effect of incubation temperature on the activity of the recombinant cellulases.
  • Cellulase assays were performed in 50 mM Na-citrate (pH 5 or 6) containing 1% Avicel for 22 hours.
  • Figure 10 Time course of cellulose hydrolysis by the cloned celD enzyme.
  • Cellulose hydrolysis was performed at 39°C in 50mM Na-citrate (pH6.0) containing 1% Avicel.
  • the celD enzyme (1-12 ⁇ L) was added to the reaction of a final volume of 500 ⁇ L.
  • FIG 11 Effect of celA and celD enzymes in combination on the rate of crystalline cellulose hydrolysis.
  • Cellulose hydrolysis was performed at 39°C in 50mM Na-citrate (pH6) containing 1% Avicel for 4 hr.
  • FIG 12 Restriction map of celD cDNA and its deletion mutants.
  • the positions of the cleavage sites of EcoRI (E ) , Bg/II (B ) , KpnI (K ), PauII(P) and -XhoI(X) are shown.
  • the positions of deletion mutants of celD are indicated by solid bars and numbers in kbp corresponding to the positions in pCNP4.1.
  • the enzyme activity of the clones was determined on substratecontaining agarose gel plates and cellulose-binding capacity was determined with Avicel: +, active, -, inactive; ND, not determined, CMC, CM-cellulose; Xyn, xylan; Av, Avicel: CB, cellulose-binding.
  • Catalytic domains present in triplicate.
  • Predicted amino acid identity of each catalytic domain is >95%.
  • A4 comprises only endoglucanases from anaerobic bacteria, including
  • Neocallimastix patriciarum xynA cDNA encoded by Neocallimastix patriciarum xynA cDNA.
  • SEQ ID NO:1 refers to nucleotide sequence of
  • Neocallimastix patriciarum celD cDNA The sequence underlined is derived from pBluescript SK-vector and the EcoRI adaptor used for cDNA cloning.
  • SEQ ID NO: 2 refers to translated sequence of domains I and II of Neocallimastix patriciarum celD cDNA.
  • Translated polypeptide includes the N-terminus of the ⁇ -galactosidase ⁇ -peptide (derived from nucleotides 1-111) and amino acids derived from the 5' oligonucleotide linker (nucleotides 112-124) used in cDNA library construction.
  • SEQ ID NO: 3 refers to translated sequence of domain III of Neocallimastix patriciarum celD cDNA.
  • SEQ ID NO: 4 refers to the sequence of the modified xylanase cDNA in pNX-Tac.

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Abstract

Procédé de clonage d'ADNc de cellulase à activité enzymatique à partir d'un champignon de rumen anaérobie, consistant à: (ii) cultiver un champignon de rumen anaérobie; (ii) isoler l'ARN complet de la culture de l'étape (i); (iii) isoler l'ARNm à région poly A+ de l'ARN complet obtenu dans l'étape (ii); (iv) construire une bibliothèque d'expression d'ADNc; (v) ligaturer l'ADNc à un vecteur d'expression bactériophage choisi entre μZAP, μZAPII ou des vecteurs présentant des caractéristiques analogues; (vi) détecter, par détection d'hydrolyse de cellulose, les clones recombinés positifs par rapport à la cellulase dans un milieu de culture contenant la cellulose; et (vii) purifier les clones recombinés positifs par rapport à la cellulase. L'invention se rapporte également à des clones recombinés d'ADNc fongique de cellulase obtenus selon le procédé ci-dessus, ainsi qu'aux clones recombinés d'ADNc de cellulase dérivés de N. patriciarum dont une caractéristique est de produire des cellulases à fonctionnalité biologique dans des cellules de E. Coli. Différentes molécules d'ADNc pouvant être utilisées selon le procédé ci-dessus sont également décrites.
PCT/AU1993/000307 1992-06-24 1993-06-24 Cellulases recombinees WO1994000578A1 (fr)

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JP6501883A JPH07507928A (ja) 1992-06-24 1993-06-24 組換えセルラーゼ
AU42995/93A AU4299593A (en) 1992-06-24 1993-06-24 Recombinant cellulases
EP93912457A EP0649471A4 (fr) 1992-06-24 1993-06-24 Cellulases recombinees.
FI945994A FI945994L (fi) 1992-06-24 1994-12-21 Rekombinoitu sellulaasi

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5824533A (en) * 1995-05-19 1998-10-20 University Of Georgia Research Foundation, Inc. Orpinomyces xylanase proteins and coding sequences
WO2000061760A1 (fr) * 1999-04-09 2000-10-19 Babraham Institute Domaines de fixation glucidiques de piromyces equi
WO2000061625A1 (fr) * 1999-04-09 2000-10-19 Human Genome Sciences, Inc. 48 proteines secretees humaines
DE10038969A1 (de) * 2000-08-10 2002-03-14 Infineon Technologies Ag Halbleiterbauelement mit Diagnoseeinrichtung, Diagnoseelement und Schaltungsanordnung zur Diagnose
EP1433844A3 (fr) * 2002-09-17 2005-02-09 Geneway Biotechnology Corporation Nouvelles xylanases recombinantes dérivées de champignons aerobies, et les séquences, vecteurs d'expression et hôtes associés
EP1612267A1 (fr) * 2004-07-02 2006-01-04 GBF Gesellschaft für Biotechnologische Forschung mbH Cellulases de rumen
FR3000500A1 (fr) * 2013-01-02 2014-07-04 Julien Sylvestre Procede d'obtention de microorganismes et enzymes capables de degrader de la cellulose et de l'hemicellulose
CN109750015A (zh) * 2019-03-27 2019-05-14 云南师范大学 一种热稳性提高的木聚糖酶突变体及其应用

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DE68911131T2 (de) * 1988-03-24 1994-03-31 Novonordisk As Cellulosezubereitung.
DK16490D0 (da) * 1990-01-19 1990-01-19 Novo Nordisk As Enzym

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8th Int. Biotechnol. Symp.; (1988) Pt. 2, pages 1015-29, "Recent Aspects in the Biochemistry and Genetics of Cellulose Degradation". *
Applied and Environmental Microbiology; June 1987, pages 1216-1223, "Cellulases and Xylanases of an Anaerobic Rumen Fungus Grown on Wheat Straw, Wheat Straw Holocellulose, Cellulose and Xylan". *
Australian Microbiologist; (1992) 13, (3), A22, "Expression of a Multi-Functional Cellulolytic cDNA from the Rumen Fungus Neocallimastix Patriciarum in E. coli" (published July 1992). *
FEMS Microbiology Letters; (1986) 34, (1), pages 37-40, "A Highly Active Extracellular Cellulase from the Anaerobic Rumen Fungus Neocallimastix Frontalis". *
Journal of General Microbiology; (1992), 138, (7), pages 1413-20, "Cloning and Expression of Multiple Cellulase cDNAs from the Anaerobic Rumen Fungus Neocallimastix Patriciarum in E. coli" (published July 1992). *
See also references of EP0649471A4 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5824533A (en) * 1995-05-19 1998-10-20 University Of Georgia Research Foundation, Inc. Orpinomyces xylanase proteins and coding sequences
WO2000061760A1 (fr) * 1999-04-09 2000-10-19 Babraham Institute Domaines de fixation glucidiques de piromyces equi
WO2000061625A1 (fr) * 1999-04-09 2000-10-19 Human Genome Sciences, Inc. 48 proteines secretees humaines
DE10038969A1 (de) * 2000-08-10 2002-03-14 Infineon Technologies Ag Halbleiterbauelement mit Diagnoseeinrichtung, Diagnoseelement und Schaltungsanordnung zur Diagnose
EP1433844A3 (fr) * 2002-09-17 2005-02-09 Geneway Biotechnology Corporation Nouvelles xylanases recombinantes dérivées de champignons aerobies, et les séquences, vecteurs d'expression et hôtes associés
EP1612267A1 (fr) * 2004-07-02 2006-01-04 GBF Gesellschaft für Biotechnologische Forschung mbH Cellulases de rumen
WO2006003175A1 (fr) * 2004-07-02 2006-01-12 Helmholtz-Zentrum für Infektionsforschung GmbH Cellulases du rumen
FR3000500A1 (fr) * 2013-01-02 2014-07-04 Julien Sylvestre Procede d'obtention de microorganismes et enzymes capables de degrader de la cellulose et de l'hemicellulose
CN109750015A (zh) * 2019-03-27 2019-05-14 云南师范大学 一种热稳性提高的木聚糖酶突变体及其应用
CN109750015B (zh) * 2019-03-27 2023-05-23 云南师范大学 一种热稳性提高的木聚糖酶突变体及其应用

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FI945994A0 (fi) 1994-12-21
CA2139099A1 (fr) 1994-01-06
JPH07507928A (ja) 1995-09-07

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