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WO2013166993A1 - 一种固定化环脂肽酰基转移酶及其制备方法和用途 - Google Patents

一种固定化环脂肽酰基转移酶及其制备方法和用途 Download PDF

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Publication number
WO2013166993A1
WO2013166993A1 PCT/CN2013/075508 CN2013075508W WO2013166993A1 WO 2013166993 A1 WO2013166993 A1 WO 2013166993A1 CN 2013075508 W CN2013075508 W CN 2013075508W WO 2013166993 A1 WO2013166993 A1 WO 2013166993A1
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Prior art keywords
acyltransferase
cyclolipopeptide
immobilized
carrier
enzyme
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PCT/CN2013/075508
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English (en)
French (fr)
Inventor
刘石东
张兆利
季晓铭
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上海天伟生物制药有限公司
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Application filed by 上海天伟生物制药有限公司 filed Critical 上海天伟生物制药有限公司
Priority to US14/400,473 priority Critical patent/US20150140604A1/en
Priority to JP2015510631A priority patent/JP2015519049A/ja
Priority to CA2873090A priority patent/CA2873090C/en
Priority to RU2014149304A priority patent/RU2014149304A/ru
Priority to KR1020147034431A priority patent/KR20150013726A/ko
Priority to EP13788659.4A priority patent/EP2848685B1/en
Priority to AU2013258683A priority patent/AU2013258683A1/en
Publication of WO2013166993A1 publication Critical patent/WO2013166993A1/zh
Priority to US16/741,460 priority patent/US11518990B2/en

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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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/087—Acrylic polymers
    • 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)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • 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)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/814—Enzyme separation or purification
    • Y10S435/815—Enzyme separation or purification by sorption

Definitions

  • the present invention relates to an enzyme immobilization technique, and more particularly to an immobilized cyclolipopeptide acyltransferase, a preparation method and use thereof. Background technique
  • Echinoglobin is a new class of antifungal agents that works well for treating infections caused by Candida or Aspergillus. These drugs are represented by caspofungin, micafungin, and anifenin. Echinoglobin inhibits fungi by inhibiting the formation of 1, 3- ⁇ glycosidic bonds, thereby reducing harm to the human body, reducing side effects as much as possible while efficient, so they are in use Safer than traditional antifungal drugs.
  • FK463 (micafungin) is a compound of the formula [Ie] which is obtained by the synthesis of the compound of the formula [Ic] FR179642 by cleavage of the side chain with the compound of the formula la FR901379 as a precursor.
  • Anidulafungin is a compound of the formula [If], which is obtained by a synthetic method by using a compound of the formula lb echinocandin B as a precursor to obtain a compound of the formula [Id] by cleavage of a side chain.
  • the enzyme for deacylating the acyl side chain of a cyclic lipopeptide substance such as FR901379, echinocandin B (echinomycin B), arachidin A, and the like, has been reported to belong to the Utah swimming line.
  • the acyltransferase can be found in cultured hyphae and in the filtrate.
  • the method for recovering the biologically active substance is usually that the hyphae and the filtrate obtained by filtering or centrifuging the broth are separated and purified by a conventional method, such as extraction with a high-concentration salt solution, solvent extraction, concentration under reduced pressure,
  • the acyltransferase can be obtained by freeze drying, pH adjustment, anion exchange resin or cation exchange resin, crystallization, recrystallization, and the like.
  • a deacylation method of the enzyme which converts a cyclic lipopeptide substance (such as FR901379, echinocandin B, etc.) with a culture solution, which has the disadvantage that an organic solvent is present in the conversion system, and the product is The treatment is difficult, the conversion speed is slow, the acyltransferase is not reusable, the purity of the conversion product is low, and the purification of the product after deacylation is increased.
  • a cyclic lipopeptide substance such as FR901379, echinocandin B, etc.
  • the present invention aims to provide an immobilized cyclolipopeptide acyltransferase.
  • the present invention also provides a method for producing the above immobilized cyclolipopeptide acyltransferase.
  • Still another object of the present invention is to provide the use of the above immobilized cyclolipopeptide acyltransferase.
  • an immobilized cyclolipopeptide acyltransferase immobilized on a carrier the cyclolipopeptide acyltransferase is derived from a natural or artificial mutant, Alternatively, the variant is obtained by transformation after introduction of a foreign cyclic acyltransferase gene selected from an inorganic carrier or a porous hydrophilic enzyme carrier.
  • the immobilized cyclolipopeptide acyltransferase is used for catalyzing the acyl deacylation of R1 in a compound of formula I to give a compound of formula II,
  • R 1 is an acyl group
  • R 2 is a hydroxy group or an acyloxy group
  • R 3 is hydrogen or a hydroxyl group
  • R 4 is hydrogen or a hydroxyl group
  • R 5 is hydrogen or a hydroxysulfonyloxy group
  • R 6 is hydrogen or a carbamoyl group.
  • the compound capable of catalyzing an acyl deacylation reaction, or a pharmaceutically acceptable salt thereof is represented by formula la or formula lb;
  • the support material is selected from the group consisting of: the total weight of the inorganic support, wherein the SiO2 content is greater than 50% by weight, and the A12O3 content is greater than 1% by weight; preferably from: catalyst carrier CELITE, expanded perlite, Diatomaceous earth, kaolin, or porous glass.
  • the carrier material is selected from the group consisting of a porous hydrophilic enzyme carrier: a polymethacrylate as a matrix-bonding epoxide or an amino-functional group-containing porous hydrophilic enzyme carrier; : Relizyme EP403, SEPABEADS EC-EP, Relizyme HA403 or SEPABEADS EC-HA.
  • the immobilized cyclolipopeptide acyltransferase is immobilized with 10-1000 units (u) of cyclolipopeptide acyltransferase per gram of the carrier; preferably 20-600 units (u).
  • the immobilized cyclolipopeptide acyltransferase is immobilized on an inorganic carrier, and 10 to 100 units of (u) cyclolipopeptide acyltransferase is immobilized per gram of the carrier; preferably 20 to 80 units (u ).
  • the immobilized cyclolipopeptide acylase is immobilized on a porous hydrophilic enzyme carrier, such as polymethacrylate as a matrix-bonded epoxide or an amino-functional group-containing enzyme carrier.
  • a porous hydrophilic enzyme carrier such as polymethacrylate as a matrix-bonded epoxide or an amino-functional group-containing enzyme carrier.
  • a method for producing an immobilized cyclolipopeptide acyltransferase provided by the present invention as described above comprising the steps of: The solution containing the free cyclolipopeptide acyltransferase is mixed with a carrier, adsorbed, and immobilized to obtain the immobilized cyclolipopeptide acyltransferase provided by the present invention as described above.
  • the cyclolipopeptide acyltransferase is derived from a natural or artificial mutant, or a variant, and is obtained by transformation after introduction of a foreign cyclic acyltransferase gene.
  • the strain is of the genus Actinomyces or Streptomyces.
  • the solution containing the free cyclolipopeptide acyltransferase is obtained by: culturing the strain to obtain a solution containing the free cyclolipopeptide acyltransferase; or the hyphae obtained after the strain is cultured. The body was broken and a solution containing free cyclolipopeptide acyltransferase was obtained.
  • the solution containing the free cyclolipopeptide acyltransferase can be purified to remove the heteroprotein to obtain a more pure solution of the cyclolipopeptide acyltransferase.
  • the carrier material is selected from an inorganic carrier or a porous hydrophilic enzyme carrier.
  • the inorganic carrier is selected from the group consisting of: the SiO2 content is greater than 50% by weight, and the A12O3 content is greater than 1% by weight based on the total weight of the inorganic carrier; preferably from: catalyst carrier CELITE, expanded perlite, diatomaceous earth , kaolin, or porous glass.
  • the porous hydrophilic enzyme carrier is selected from the group consisting of: an epoxide-bonded epoxide or an amino-functional group-containing enzyme carrier; preferably from: Relizyme EP403, SEPABEADS EC-EP , elizyme HA403 or SEPABEADS EC-HA.
  • the ratio of the free cyclolipopeptide acyltransferase to the carrier is 10-1000 units (u) per gram of the carrier; preferably 20-600 units (u) per gram of the carrier.
  • the mixing ratio of the free cyclolipopeptide acyltransferase to the inorganic carrier is 10-100 units (u) per gram of the carrier; preferably 20-80 units (u) per gram of the carrier.
  • the mixing ratio of the free cyclolipopeptide acyltransferase to the porous hydrophilic enzyme carrier is 100-1000 units (u) per gram of the carrier; preferably 120-600 units per gram of the carrier. (u).
  • the solution containing the free cyclolipopeptide acyltransferase has a pH of 4 to 9; preferably 6 to 7.
  • the solution containing the free cyclolipopeptide acyltransferase and the carrier are mixed at a temperature of 0 to 80 ° C; preferably 20 to 35 ° C; most preferably 20 to 25 ° C.
  • R 1 is an acyl group
  • R 2 is a hydroxy group or an acyloxy group
  • R 3 is hydrogen or a hydroxyl group
  • R 4 is hydrogen or a hydroxyl group
  • R 5 is hydrogen or a hydroxysulfonyloxy group
  • R 6 is hydrogen or a carbamoyl group.
  • the ratio of the immobilized cyclolipopeptide acyltransferase to the compound of formula I is from 0.01 to 10 u/g.
  • the pH of the buffer in the step (a) is 4-9; preferably 6-7; wherein the buffer in the step (a) is selected from one or more of the following solutions; : Sodium citrate buffer, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer and Tris-HCl buffer.
  • the temperature of the deacylation reaction in the step (b) is from 20 to 70 ° C; preferably from 40 to 50 ° C.
  • the immobilized cyclolipopeptide acyltransferase is separated from the reaction solution containing the product to obtain a compound of the formula II; preferably, The method of separating the immobilized cyclolipopeptide acylase from the reaction solution containing the product includes filtration or centrifugation. Accordingly, the present invention provides a method for immobilizing a cyclolipopeptide acyltransferase, thereby recovering and purifying the same The acyltransferase increases the utilization of the enzyme.
  • Fig. 1 is a HPCL analysis chart of the deacylation product of the immobilized enzyme (vi) of Example 10.
  • Figure 2 is a HPCL analysis of the deacylated product of the free enzyme solution of Comparative Example 1. detailed description
  • the present invention relates to FR901379 substance (Patent No. CN1051757A) produced by the microorganism Colephoma sp. F-11899 (FERM BP-2635), and echinocandin B (Echinocandin B) produced by the microorganism Aspergillus nidulans Nrrl 11440 strain.
  • Patent No. US 4288549 and methods for immobilizing an acyltransferase of an acyl side chain deacylated by an analog thereof, and a method of deacylation using the immobilized enzyme.
  • the structural formulas relating to the background art and the compounds of the present invention are listed in the following table:
  • R' is acyl
  • R 2 is hydroxy or acyloxy
  • R j is hydrogen or hydroxy
  • R 4 is hydrogen or hydroxy
  • R 6 is hydrogen or carbamoyl.
  • the cyclic lipopeptide acyltransferase of the present invention is immobilized, and the preparation process comprises the following steps: a. preparing a cyclic lipopeptide acyltransferase solution;
  • the cyclic lipopeptide acyltransferase used in the present invention is not particularly limited in its source, and is derived from a natural or artificial mutant, or a variant, and the enzyme obtained by transformation after introduction of a foreign cyclic acyltransferase gene. included.
  • the carrier material is selected from an inorganic carrier or a porous hydrophilic enzyme carrier.
  • the advantage of the inorganic carrier is that the enzyme is not inactivated, and the enzyme adsorbed on the carrier is active, but the disadvantage is that the specific activity is lower, and the amount of the enzyme adsorbed by the unit carrier is much lower than that of the porous hydrophilic enzyme carrier; While the porous hydrophilic enzyme carrier can adsorb more enzymes, it will inactivate some of the enzymes, that is, the enzyme adsorbed to the carrier is only partially active, and the recovery rate is low.
  • the inorganic carrier may be a hydrophobic carrier such as a catalyst carrier CELITE commonly used for immobilization [chemical composition: 87% SiO2, 0.9% CaO, 6.1% A12O3, 1.6% Fe2O3, 1.6% Na2O+K2O], expanded perlite [chemical composition : SiO2 (70-75%), CaO (0.1-2.0%), A12O3 (12-16%), Na2O (1.0-5.0%), Fe2O3 (0.1-1.5%), K2O (1.0-5.0%)], a carrier such as diatomaceous earth, kaolin, or porous glass, whose chemical composition is based on the total weight of the inorganic carrier, wherein the SiO2 content is more than 50% by weight, and the A12O3 content is more than 1% by weight; preferably from: catalyst carrier CELITE, expanded perlite, diatomaceous earth , kaolin, or porous glass; most preferred are catalyst supports CELITE, expanded perlite.
  • the porous hydrophilic enzyme carrier may be an epoxide-bonded
  • the functional group is an already diamined enzymatic carrier comprising Relizyme HA403.
  • Functional basis The enzyme is an enzyme carrier of hexamethyleneimine containing SEPABEADS EC-HA.
  • the ratio of the enzyme to the carrier ranges from 10 to 100 u per gram of the carrier, and preferably 20 to 80 u / g of the carrier.
  • the amount of the enzyme is less than 10 u/g, the immobilized enzyme activity is too low, and the enzymatic reaction is difficult to exert.
  • the enzyme activity is greater than 100 u/g, the immobilization efficiency of the enzyme is too low, and some enzymes are in a free state. , will be lost when used once.
  • the ratio of the enzyme to the carrier ranges from 100 to 1000 u per gram of the carrier, and preferably 120 to 600 u/g of the enzyme.
  • the enzyme solution used is preferably purified, and the smaller the proportion of the heteroprotein in the enzyme solution, the more advantageous it is for immobilization.
  • the immobilization conditions using the inorganic carrier are as follows:
  • the cyclic lipopeptide acyltransferase in the crude enzyme solution obtained above is subjected to high performance liquid chromatography (HPLC) to measure the enzyme activity, and a solid carrier is added at a predetermined enzyme/carrier ratio. pH 4-9, temperature 0-80 ° C, stirring for more than half an hour, washing thoroughly, filtering, and drying at low temperature, the immobilized cyclic lipopeptide acyltransferase can be obtained and stored at 0-5 °C.
  • HPLC high performance liquid chromatography
  • the immobilization condition using a porous hydrophilic enzyme carrier having a functional group as an epoxide is: measuring the enzyme activity by high performance liquid chromatography (HPLC) using the cyclolipopeptide acyltransferase in the relatively pure enzyme solution obtained above. According to a certain enzyme ⁇ carrier ratio, an enzyme carrier having a functional group as an epoxide is added. pH 4-9, temperature 0-80 ° C, stirred for more than 24 hours, washed thoroughly, filtered, and dried at low temperature to obtain an immobilized cyclic lipopeptide acyltransferase, which was stored at 0-5 ° C.
  • HPLC high performance liquid chromatography
  • the immobilization condition of the porous hydrophilic enzyme carrier having a functional group of amino group is: the enzyme carrier is activated with glutaraldehyde in advance, and after the activation is completed, the residual glutaraldehyde is sufficiently washed to obtain the pure enzyme solution obtained above.
  • the enzyme activity is determined by high performance liquid chromatography (HPLC), and the enzyme carrier activated by glutaraldehyde is added at a certain enzyme/carrier ratio, pH 4-9, temperature 0-80 ° C, After sufficiently stirring for 24 hours or more, filtration, and drying at a low temperature, an immobilized cyclic lipopeptide acyltransferase can be obtained and stored at 0 to 5 °C.
  • HPLC high performance liquid chromatography
  • the above unit of enzyme activity is defined as: the amount of enzyme required to produce 1 micromole of product in one hour at 40 ° C, which is defined as l u .
  • the procedure was as follows: 17.5 ml of a crude enzyme solution containing 5 ml of a buffer solution of FR901379 (0.25 mol/L) pH 6.0, 2.5 ml of methanol was weighed. The reaction was carried out for one hour under a water bath at 40 °C. The deionized water was appropriately diluted, filtered through a 0.22 um nylon membrane, and the product concentration was determined by HPLC. Or
  • the above unit of enzyme activity is defined as: the amount of enzyme required to produce 1 micromole of product in one hour at 40 ° C, which is defined as l u .
  • the steps are as follows: 17.5 ml of crude enzyme solution is separately weighed, 2.5 ml of echinocandin B dimethyl sulfoxide solution (100 mg/ml) is added, and 1.2 M potassium chloride, 0.5 M potassium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is added. 5.0ml. The reaction was carried out for one hour under a water bath at 40 °C. The methanol was appropriately diluted, filtered through a 0.22 um nylon membrane, and the product concentration was determined by HPLC.
  • the method for producing a cyclic lipopeptide acyltransferase in the above step a comprises the steps of: strain culture, and the obtained mycelium is disrupted to obtain a cyclolipopeptide acyltransferase solution.
  • the strain refers to an excellent strain capable of secreting a cyclic lipopeptide acyltransferase, which is an actinomycete or a genus Streptomyces.
  • a cyclic lipopeptide acyltransferase which is an actinomycete or a genus Streptomyces.
  • bacteria belonging to the genus Streptomyces for example, Streptomyces anulatus 481 1 strain, Streptomyces cerevisiae 8703 strain, Streptomyces sp. 6907 are reported. The enzyme produced by the enzyme produced by the strain).
  • Oidiodendron tenuissimum IFO 6797 strain Oidiodendron echinulatum IFO 31963 strain
  • Oidiodendron truncatum IFO 9951 strain Oidiodendron truncatum IFO 31812 strain
  • Oidiodendron sp. 30084 strain An enzyme produced by a strain of Verticillium sp. 30085.
  • Actinoplanes utahensis IFO-13244 Preferably, Actinoplanes utahensis IFO-13244,
  • the medium used for strain culture includes the following substances: sucrose 1-10%, soy peptone 0.1-0.1%,
  • the culture conditions are 25-36 ° C, preferably 30 ° C, aeration of l-2 vvm, and a stirring speed of 200-800 r/min. After 3-5 days of cultivation, a large amount of mycelium was obtained.
  • the mycelium obtained above can be disrupted first, and the intracellular enzymes of the cells are collected, so that a better cyclic lipopeptide acyltransferase can be obtained, and the cell wall can be broken by a known method, such as : High-concentration salt solution extraction, ultrasonic disruption, mechanical disruption, lysozyme, etc. Transfer the cyclic lipopeptide acyltransferase to the extracellular domain of the cell, and then use the filtration method or centrifugation to transfer the enzyme solution to the mycelium Separation to obtain a crude enzyme solution.
  • the cyclic lipopeptide acyltransferase solution may be first purified: the crude enzyme solution obtained above is added to the inorganic carrier used to adsorb the free enzyme in a certain ratio.
  • the inorganic carrier to which the enzyme is adsorbed is separated from the enzyme solution, and after thorough washing, the cyclic lipopeptide acyltransferase is desorbed from the inorganic carrier with a high concentration of a salt solution to obtain a relatively pure enzyme solution.
  • Echinocandin B deacylase of Actinoplanes utahensis purification, characterization, heterologous cloning and enzymatic deacylation reaction. Purification of cyclic lipopeptide acyltransferase by ultrafiltration, ion exchange tree method, this method can obtain a more pure enzyme solution, but this method The disadvantage is that the recovery of the enzyme is very low.
  • the present invention uses an immobilized enzyme for the deacylation process, which comprises the following steps:
  • the ratio of the immobilized acyltransferase to the cyclic lipopeptide is 0.01 to 10 u/g, preferably 0.1 to 5 u/g.
  • the pH of the buffer in step A is controlled to be 4-9, preferably pH 5-7, more preferably pH 6.0.
  • the type of buffer is one of 0.5 M sodium citrate buffer, 0.5 M potassium dihydrogen phosphate-disodium hydrogen phosphate buffer and Tris-HCl buffer or a mixture thereof, preferably 0.5 M potassium dihydrogen phosphate-phosphoric acid. Hydrogen disodium buffer.
  • the temperature of the deacylation reaction in step B is controlled to be about 20-70 ° C, and the preferred temperature range is about 30-50 ° C.
  • the method for separating the immobilized enzyme from the reaction solution containing the product described in the step C includes a filtration method or a centrifugation method.
  • the deacylation of the present invention can be carried out smoothly in a continuous stirred reactor instead of batchwise.
  • cyclic lipopeptide or "cyclolipopeptide” as used in the present invention means a substance containing a polypeptide ring, and the side chain of the ring contains an "acylamino group", and the substance may have other side chains.
  • cyclic lipopeptide is FR901379, echinocandin B (echinomycin B), which is a known substance having antifungal activity.
  • the immobilized cyclic lipopeptide acyltransferase of the invention can deacylate the "amide group" of the side chain of the cyclic lipopeptide to form an "amino group", specifically, the palmitoyl side of the substance FR901379 or a salt thereof
  • the chain or an acyl side chain comprising FR901379, a compound of formula I is deacylated to form a cyclic peptide material of formula II.
  • salts preferably include: metal salts such as alkali metal salts (e.g., sodium salts, potassium salts), alkaline earth metal salts (e.g., calcium salts, magnesium salts, etc.), ammonium salts, and organic bases.
  • metal salts such as alkali metal salts (e.g., sodium salts, potassium salts), alkaline earth metal salts (e.g., calcium salts, magnesium salts, etc.), ammonium salts, and organic bases.
  • Salt such as trimethylamine salt, triethylamine salt, pyridinium salt, methylpyridine salt, dicyclohexylammonium salt, N, N,-dibenzylethylenediamine salt, diisopropylethylamine salt, etc.
  • organic acid addition salts such as formate, acetate, trifluoroacetate, maleate, tartrate, methanesulfonate, besylate, tosylate, etc.
  • inorganic acid plus a salt such as a hydrochloride, a hydrobromide, a hydroiodide, a sulfate, a phosphate, etc.
  • a salt formed with an amino acid such as arginine, aspartic acid, glutamic acid, etc.
  • the lipopeptide acyltransferase immobilization method provided by the present invention can repeatedly use the enzyme, improve the utilization ratio of the cyclic lipopeptide acyltransferase, reduce the production cost, and is advantageous for industrial production and cost reduction.
  • the unit in the weight percent by volume in the present invention is well known to those skilled in the art and, for example, refers to the weight of the solute in a 100 ml solution.
  • the strain of Actinobacillus mirabilis IFO-13244 was used for fermentation culture according to the fermentation method of US Pat. No. 5,376,634 to obtain 150 L of mycelial culture solution. 0.01 M potassium dihydrogen phosphate buffer salt was added, the pH was adjusted to 6.0, 1.0 M potassium chloride was added, and the mixture was stirred at a low temperature for more than 20 hours. Then, it was filtered through a Buchner funnel coated with filter paper, and 1 16 L of the filtrate containing the acyltransferase, that is, the free enzyme solution (a) was collected, and the filtrate was collected for HPLC activity of 2.3 ⁇ 10 5 u.
  • acyltransferase b produced by A. utahensis NRRL-12052 strain was carried out by fermentation of U. serrata NRRL-12052 strain according to the fermentation method of US4320053.
  • the strain of Streptomyces sp. 6907 was subjected to fermentation culture according to the fermentation method of WO97/32975, and 160 L of mycelial culture liquid was obtained. 0.01 M potassium phosphate monobasic buffer salt was added, the pH was adjusted to 6.0, 1.0 M potassium chloride was added, and the mixture was stirred at a low temperature for 20 hours or more. Then, it was filtered through a Buchner funnel coated with filter paper, and 124 L of the filtrate containing the acyltransferase, that is, the free enzyme solution (c), was collected, and the filtrate was collected and detected by HPLC to be 2.9 ⁇ 10 5 u.
  • Example 5 Reference Membrane-associate echinocandin B deacylase of Actinoplanes Utahensis: purification, characterization, heterologous cloning and enzymatic deacylation reaction method The crude enzyme solution obtained in Example 2 is purified to finally obtain 3.0 L of pure acyltransferase solution, ie, free enzyme solution (d), and the collected enzyme solution is detected by HPLC. The enzyme activity is 0.5x l0 5 u.
  • Example 5 The enzyme activity is 0.5x l0 5 u.
  • immobilization 50 liters of free enzyme solution (a), (b), and (c) were respectively taken, and then 1.5 kg of expanded pearl salt was added, and the mixture was stirred and adsorbed at 25 ° C for 1 hour or more.
  • the immobilized acyltransferase was collected by filtration, i.e., immobilized enzymes (i), (ii), (iii) were washed three times with 0.01 M potassium phosphate monobasic buffer, pH 6.0 buffer, and dried at room temperature for several hours. The immobilized enzyme is stored at 4 before use.
  • the carrier is first activated: 8 g of Relizyme HA403 and SEPABEADS EC-HA 8 g, respectively, 90 ml of 2% glutaraldehyde solution, 0.02 mol/L of dipotassium hydrogen phosphate buffer, pH 8.0, After stirring at 20-25 ° C for 1 hour, the supernatant was removed, and the purified water was used to wash the resin. 0.4 liters of the pure acyltransferase solution (d) prepared in the above Example 4 was separately taken, and two activated enzyme carriers were added to the enzyme solution, and stirred at 20-25 ° C for 20 hours or more, and filtered.
  • FR901379 substance 100 mg/ml, HPLC purity 79.2% 100 ml (FR901379 10 g, 38.35 mmol), was added to a buffer (0.2 M potassium dihydrogen phosphate-hydrogen phosphate). Disodium buffer; pH 6.0) 100ml, methanol 100ml, then add free enzyme solution (c) 700ml, 40 ° C reaction ⁇ hours, conversion rate of 71.1%, the purity of the resulting FR17964 substance, the purity by HPLC is 75.7% , The HPLC analysis chromatogram is shown in Figure 2 and Table 10.
  • the converted liquid was measured for enzyme activity, and substantially no enzyme activity was detected by the measurement.
  • the enzyme is almost completely inactivated during the conversion process.
  • the continuous deacylation of FR901379 to FR179642 was carried out in a 50 liter continuous stirred tank reactor at 45 ° C, and an immobilized enzyme (vi) 0.3 kg, an aqueous solution of FR901379 (20 mg/ml, HPLC purity 79.2%) was added. 20 liters, buffer (0.2 M potassium dihydrogen phosphate - disodium hydrogen phosphate buffer; pH 6.0) 20 liters. The substrate conversion rate reached 95% after 3 hours, and the purity of F 179642 substance was 95.99% by HPLC.
  • the HPLC analysis chromatogram is shown in Figure 1 and Table 6.
  • the immobilized acyltransferase maintains stability and activity for at least 30 hours, so it can be continuously used more than 5 times in the production process.
  • Example 11 Comparison of Immobilization of Free Enzymes and Deacylation of Immobilized Enzymes by Different Vectors
  • immobilization For immobilization, take 5 liters of free enzyme solution (c), and then add 0.15 kg of expanded pearl salt, celite (CELITE), and activated carbon (SiO2 content less than 1%), molecular sieve, and porous glass. At 30 ° C, the adsorption was carried out for more than 1 hour.
  • the immobilized acyltransferase was collected by filtration, ie, immobilized enzyme (viii), (ix), (x), (xi), (xii) was washed three times with 0.01 M potassium dihydrogen phosphate buffer salt, pH 6.0 buffer. And drying at room temperature for several hours, the immobilized enzyme was stored at 4 before use.
  • FR901379 substance 20 g, 16.7 mmol a buffer (0.2 M potassium dihydrogen phosphate-disodium hydrogen phosphate buffer; pH 6.0) was added. 2 liters and 15 g (viii), (ix), (x), (xi), (xii), reacted at 40 ° C for 4 hours, and the yield and purity of the FR179642 substance were measured by HPLC.
  • FR901379 10g, 8.35mmol aqueous solution of FR901379 substance (20mg/ml, HPLC purity 79.2%), buffer (0.2M potassium dihydrogen phosphate-disodium hydrogen phosphate buffer; pH6. 0) 1 liter and 75 g of the immobilized enzyme obtained in the above preparation, reacted at 40 ° C for 4 hours, and the production amount and purity of FR179642 substance were measured by HPLC.
  • FR901379 10g, 8.35mmol aqueous solution of FR901379 substance (20mg/ml, HPLC purity 79.2%), buffer (0.2M potassium dihydrogen phosphate-disodium hydrogen phosphate buffer; pH6. 0) 1 liter and 75 g of the immobilized enzyme obtained in the above preparation, reacted at 40 ° C for 4 hours, and the production amount of FR179642 substance was measured by HPLC.

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Abstract

本发明公开了一种固定化环脂肽酰基转移酶及其制备方法和用途。所述环脂肽酰基转移酶固定于载体上；所述环脂肽酰基转移酶来源于天然或人工突变体、或者变种及由通过导入外来的环状酰基转移酶基因后转化得到的，所述载体材料选自无机载体、或聚丙烯树脂载体。本发明还公开了所述固定化环脂肽酰基转移酶的制备方法和用途。

Description

一种固定化环脂肽酰基转移酶及其制备方法和用途
技术领域
本发明涉及到酶固定化技术，尤其涉及一种固定化环脂肽酰基转移酶及其制备方 法和用途。 背景技术
棘球白素作为一类新的抗真菌药物，在治疗由念珠菌或曲霉引起的感染方面效果 良好。 这类药物又以卡泊芬净、 米卡芬净、 阿尼芬净为代表。 棘球白素类药物通过抑 制 1， 3-β糖苷键的形成来抑制真菌， 从而更好地减小了对人体的伤害， 在高效的同 时尽可能的降低了副作用， 因此它们在使用过程中比传统抗真菌药更安全。
FK463(米卡芬净)是如式 〔Ie〕 所示的化合物， 它是以式 la化合物 FR901379为 前体通过切侧链得到式 〔Ic〕 化合物 FR179642, 然后经过合成方法得到的。
Anidulafungin (阿尼芬净） 是如式 〔If〕 所示的化合物， 它是以式 lb化合物 echinocandin B为前体通过切侧链得到式 〔Id〕 化合物， 然后经过合成方法得到的。
关于使环状脂肽物质例如 FR901379、 echinocandin B (棘白菌素 B)、 棘孢曲菌素 A等物质及其类似物的酰基侧链脱酰的酶， 已经报道了属于犹他游动放线菌
(Actinoplanes utahensis)IFO- 13244 , (A.utahensis )N L- 12052生产的酶。 在
WO97/32975号专利中， 报道了属于链霉菌属 (Streptomyces)的细菌 (例如环圈链霉菌 (Streptomyces anulatus)481 1号菌株、 环圈链霉菌 8703号菌株、 链霉菌 (Streptomyces sp.)6907号菌株)生产的酶。 另外， 在 WO97/47738号专利中， 报道了 Oidiodendron tenuissimum IFO 6797菌株、 Oidiodendron echinulatum IFO 31963菌株、 Oidiodendron truncatum IFO 9951菌株、 Oidiodendron truncatum IFO 31812菌株、 树粉孢
(Oidiodendron sp.)30084号菌株、 轮枝孢 (Verticillium sp.)30085号菌株生产的酶。
所述酰基转移酶在培养菌丝中及滤液中可以找到。通常回收这种生物活性物质的 方法为， 培养肉汤经过过滤或离心所得的菌丝及滤液经传统的方法进行分离、 纯化， 如高浓度盐溶液抽提， 常用溶剂抽提， 减压浓缩， 冷冻干燥， PH调整， 阴离子交换 树脂或阳离子交换树脂、结晶、再结晶等，可得到该酰基转移酶。在专利 CN 1 161462C 中， 公开了所述酶的脱酰化方法， 该方法用培养液转化环状脂肽物质 (如 FR901379、 echinocandin B等)，其缺点为转化体系中有有机溶剂，产物后处理困难， 转化速度慢， 酰基转移酶不可重复使用， 转化产物纯度低， 增加脱酰后产物的纯化难度。
因此人们一直在寻求一种固定所述酶的方法， 来回收、纯化所述酰基转移酶， 提 高该酶的利用率。 发明内容
本发明旨在提供一种固定化环脂肽酰基转移酶。 本发明还提供上述固定化环脂肽酰基转移酶的制备方法。
本发明的再一个目的是提供上述固定化环脂肽酰基转移酶的用途。 在本发明的第一方面，提供了一种固定化环脂肽酰基转移酶，所述环脂肽酰基转 移酶固定于载体上； 所述环脂肽酰基转移酶来源于天然或人工突变体、 或者变种及由 通过导入外来的环状酰基转移酶基因后转化得到的， 所述载体材料选自无机载体、 或 多孔亲水性酶载体。
所述固定化环脂肽酰基转移酶， 是用于催化如式 I的化合物中的 R1的酰基脱酰 化， 产生如式 II所示的化合物，
Figure imgf000003_0001
式中， R1为酰基， R2为羟基或酰氧基， R3为氢或羟基， R4为氢或羟基， R5为氢 或羟基磺酰氧基， R6为氢或氨基甲酰基。
在另一优选例中，所述能催化进行酰基脱酰化反应的化合物或其药学上可接受的 盐， 为如式 l a或式 l b所示；
Figure imgf000004_0001
在一优选例中， 所述载体材料选自以下无机载体： 以所述无机载体的总重量计， 其中 SiO2含量大于 50wt%， A12O3含量大于 lwt%； 优选自： 催化剂载体 CELITE、 膨化珍珠岩、 硅藻土、 高岭土、 或多孔玻璃。
在另一优选例中，所述载体材料选自以下多孔亲水性酶载体： 以聚甲基丙烯酸酯 为基体键合环氧化物或者含氨基功能基团的多孔亲水性酶载体； 优选自： Relizyme EP403、 SEPABEADS EC-EP、 Relizyme HA403或 SEPABEADS EC-HA。
在一优选例中，所述固定化环脂肽酰基转移酶，每克载体固定有 10-1000单位 (u) 环脂肽酰基转移酶； 优选 20-600单位 (u)。
在另一优选例中， 所述固定化环脂肽酰基转移酶， 固定在无机载体上， 每克载体 固定有 10— 100单位 (u)环脂肽酰基转移酶； 优选 20-80单位 (u)。
在另一优选例中， 所述固定化环脂肽酰基转移酶， 固定在多孔亲水性酶载体， 例 如聚甲基丙烯酸酯为基体键合环氧化物或者含氨基功能基团的酶载体，每克载体可固 定 100-1000单位 (u)环脂肽酰基转移酶； 优选 120-600单位 (u)。 在本发明的第二方面，提供了一种如上所述的本发明提供的固定化环脂肽酰基转 移酶的制备方法， 所述的方法包括步骤： 将含有游离的环脂肽酰基转移酶的溶液与载体混合， 进行吸附、 固定化， 得到如 上所述的本发明提供的固定化环脂肽酰基转移酶。
在上述制备方法中，所述环脂肽酰基转移酶来源于天然或人工突变体、或者变种 及由通过导入外来的环状酰基转移酶基因后转化得到的。
在上述制备方法中， 所述菌株是放线菌属或链霉菌属的。
在上述制备方法中， 所述含有游离的环脂肽酰基转移酶的溶液通过下述方式得 到： 菌株培养， 得到含有游离的环脂肽酰基转移酶的溶液； 或菌株培养后将得到的菌 丝体进行破壁， 得到含有游离的环脂肽酰基转移酶的溶液。或将含有游离的环脂肽酰 基转移酶的溶液纯化， 除去杂蛋白， 得到更加纯净的环脂肽酰基转移酶的溶液。
在上述制备方法中， 所述载体材料选自无机载体、 或多孔亲水性酶载体。
在上述制备方法中，所述无机载体选自： 以所述无机载体的总重量计，其中 SiO2 含量大于 50wt%， A12O3含量大于 lwt%； 优选自： 催化剂载体 CELITE、 膨化珍珠 岩、 硅藻土、 高岭土、 或多孔玻璃。
在上述制备方法中，所述多孔亲水性酶载体选自： 以聚甲基丙烯酸酯为基体键合 环氧化物或者含氨基功能基团的酶载体； 优选自： Relizyme EP403、 SEPABEADS EC-EP、 elizyme HA403或 SEPABEADS EC-HA。
在上述制备方法中，所述游离的环脂肽酰基转移酶与载体的混合比例为每克载体 需酶 10-1000单位 (u); 优选每克载体需酶 20-600单位 (u)。
在上述制备方法中，所述游离的环脂肽酰基转移酶与无机载体的混合比例为每克 载体需酶 10-100单位 (u); 优选每克载体需酶 20-80单位 (u)。
在上述制备方法中，所述游离的环脂肽酰基转移酶与多孔亲水性酶载体的混合比 例为每克载体需酶 100-1000单位 (u); 优选每克载体需酶 120-600单位 (u)。
在上述制备方法中， 所述含有游离的环脂肽酰基转移酶的溶液的 pH值为 4-9; 优选 6-7。
在上述制备方法中，所述含有游离的环脂肽酰基转移酶的溶液和载体混合的温度 为 0-80°C ; 优选 20— 35°C ; 最优选 20-25°C。
在上述制备方法中，将含有游离的环脂肽酰基转移酶的溶液与载体混合后，将游 离的环脂肽酰基转移酶和结合于载体的环脂肽酰基转移酶分离，得到固定化环脂肽酰 基转移酶。 在本发明的第三方面，提供了一种如式 II所示的化合物的制备方法，所述的方法 包括步骤：
(a)将如式 I的化合物和缓冲溶液混合， 得到溶液 1 ;
(b)将溶液 1和如上所述的本发明提供的固定化环脂肽酰基转移酶混合， 进行脱 酰化反应， 得到如式 II所示的化合物；
Figure imgf000006_0001
式中， R1为酰基， R2为羟基或酰氧基， R3为氢或羟基， R4为氢或羟基， R5 为氢或羟基磺酰氧基， R6为氢或氨基甲酰基。
在另一优选例中， 所述固定化环脂肽酰基转移酶和式 I的化合物的比例在 0.01-10 u/g。
在另一优选例中， 所述步骤 (a)中缓冲液的 pH在 4-9; 优选 6-7; 所述步骤 (a) 中缓冲液选自下述溶液中的一种或一种以上： 柠檬酸钠缓冲液、 磷酸二氢钾 -磷酸 氢二钠缓冲液和 Tris-HCl缓冲液。
在另一优选例中，所述步骤 (b)中脱酰化反应的温度为 20-70°C ;优选 40-50°C。 在另一优选例中， 所述步骤 (b)中脱酰化反应后， 将固定化环脂肽酰基转移酶 与含产物的反应液分离， 得到如式 II所示的化合物； 较佳地， 所述将固定化环脂 肽酰基转移酶与含产物的反应液分离的方法包括过滤或离心。 据此， 本发明提供了一种固定环脂肽酰基转移酶的方法， 从而回收、 纯化所 述酰基转移酶， 提高了该酶的利用率。 附图说明
图 1为实施例 10固定化酶 (vi)脱酰化产物的 HPCL分析图谱 。
图 2为对比例 1游离酶液脱酰化产物的 HPCL分析图谱 。 具体实施方式
发明人经过广泛而深入的研究，找到了一种固定化环状脂肽酰基转移酶的工业化 生产方法， 以及利用该固定化酶制剂使环状脂肽物侧链的"酰胺基"脱酰化形成"氨基" 的生产方法， 该方法技术先进， 工艺简单， 操作方便， 脱酰化产物纯度高。
进一步讲， 本发明涉及由微生物 Colephoma sp.F- 11899株 (FERM BP-2635)所产 生的 FR901379物质 (专利号 CN1051757A)、 由微生物 Aspergillus nidulans Nrrl 11440 株所生产的 echinocandin B (棘白菌素 B)(专利号 US4288549)及其它们的类似物的酰基 侧链脱酰化的酰基转移酶的固定化方法， 以及用该固定化酶进行脱酰化的方法。 关于背景技术和本发明涉及的化合物的结构式列于下表:
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0001
式中， R'为酰基， R2为羟基或酰氧基， Rj为氢或羟基， R4为氢或羟基， 为氢或羟基磺酰氧基， 及 R6为氢或氨基甲酰基。 本发明的环状脂肽酰基转移酶是固定化的， 其制备过程包括以下步骤： a.制备环状脂肽酰基转移酶液；
b.将环状脂肽酰基转移酶液与载体按一定的比例混合，将环脂肽酰基转移酶固定 在载体材料上；
c.将环脂肽酰基转移酶液与酶载体分离， 得到固定化的环脂肽酰基转移酶。 本发明所用的环状脂肽酰基转移酶对于其来源没有特别的限制，来源于天然或人 工突变体、或者变种及由通过导入外来的环状酰基转移酶基因后转化得到的所述酶也 全都包括在内。
所述载体材料选自无机载体或多孔亲水性酶载体。无机载体的优点是不会使酶失 活， 吸附到载体上的酶都是有活性的， 但缺点是比活较低， 单位载体可吸附的酶量远 远低于多孔亲水性酶载体； 而多孔亲水性酶载体虽然能吸附更多的酶， 但会使部分酶 失活， 即吸附到载体上的酶只有部分是有活性的， 回收率较低。
无机载体可以是疏水性载体例如常用于固定化作用的催化剂载体 CELITE [化学 组成： 87%SiO2， 0.9%CaO， 6.1 %A12O3, 1.6%Fe2O3, 1.6%Na2O+K2O] ,膨化珍珠岩 [化 学组成： SiO2(70-75%)、 CaO(0.1-2.0%)、 A12O3(12-16%)、 Na2O (1.0-5.0%)、 Fe2O3(0.1-1.5%), K2O( 1.0-5.0%)], 硅藻土， 高岭土， 或多孔玻璃等载体， 其化学组 成以无机载体的总重量计， 其中 SiO2含量大于 50wt%， A12O3含量大于 lwt%； 优选 自： 催化剂载体 CELITE、 膨化珍珠岩、 硅藻土、 高岭土、 或多孔玻璃； 最优选的为 催化剂载体 CELITE、 膨化珍珠岩。 多孔亲水性酶载体可以是以聚甲基丙烯酸酯为基体键合环氧化物或者含氨基功 能基团的酶载体。 例如功能基团为环氧化物的酶载体， 包含有 Relizyme EP403、
SEPABEADS EC-EP。 功能基团为已二胺的酶载体， 包含有 Relizyme HA403。 功能基 团为六亚甲基亚胺的酶载体， 包含有 SEPABEADS EC-HA。 使用无机载体在固定化过程中， 酶和载体的比例范围是每克载体需酶 10-100 u， 优选的酶 20-80u/g载体。 当酶用量小于 10 u/g载体时， 固定化的酶活太低， 酶促反应 很难发挥出来， 但酶活大于 100u/g载体时， 酶的固定化效率太低， 部分酶呈游离状 态， 使用一次就会流失。
使用多孔亲水性酶载体在固定化过程中， 酶和载体的比例范围是每克载体需酶 100-lOOOu，优选的酶 120-600u/g载体。特别地，使用多孔亲水性酶载体固定化过程中， 使用的酶液优选纯化的， 酶液中杂蛋白的比例越小越有利于固定化。
使用无机载体的固定化条件是： 将上述得到的粗酶液中的环状脂肽酰基转移酶， 用高效液相色谱 (HPLC)测定酶活力， 按一定的酶 \载体比例，加入固态载体。 pH4-9，温 度 0-80°C， 搅拌半小时以上， 充分洗涤， 过滤， 低温干燥， 可得固定化环状脂肽酰基 转移酶， 于 0-5 °C保存。
使用功能基团为环氧化物的多孔亲水性酶载体的固定化条件是：将上述得到的较 为纯净的酶液中环状脂肽酰基转移酶，用高效液相色谱 (HPLC)测定酶活力，按一定的 酶\载体比例， 加入功能基团为环氧化物的酶载体。 pH4-9，温度 0-80°C， 搅拌 24小时 以上充分洗涤， 过滤， 低温干燥， 可得固定化环状脂肽酰基转移酶， 于 0-5°C保存。
使用功能基团为氨基的多孔亲水性酶载体的固定化条件是:预先将酶载体用戊二 醛活化， 活化完成后充分洗涤去除残余的戊二醛， 将上述得到的较为纯净的酶液中环 状脂肽酰基转移酶， 用高效液相色谱 (HPLC)测定酶活力， 按一定的酶 \载体比例， 加 入戊二醛活化好的酶载体， pH4-9，温度 0-80°C， 搅拌 24小时以上充分洗涤， 过滤， 低温干燥， 可得固定化环状脂肽酰基转移酶， 于 0-5°C保存。
在本发明中， 上述酶活力单位定义为： 40°C时， 在一小时内生成 1微摩尔产物所 需的酶量， 即定义为 l u。 步骤为: 分别量取 17.5ml粗酶液， 含有 FR901379的磷酸 二氢钾缓冲盐 (0.25mol/L)pH6.0的缓冲液 5ml，2.5ml甲醇。 于 40°C水浴条件下， 反应 一小时。 去离子水适当稀释， 0.22um尼龙膜过滤， HPLC测定产物浓度。 或者
在本发明中， 上述酶活力单位定义为： 40°C时， 在一小时内生成 1微摩尔产物所 需的酶量， 即定义为 l u。 步骤为: 分别量取 17.5ml粗酶液， 将 echinocandin B 的二 甲基亚砜溶液 (100mg/ml)2.5ml， 加入 1.2M氯化钾、 0.5M磷酸二氢钾-磷酸氢二钠缓 冲液 5.0ml。于 40°C水浴条件下， 反应一小时。去甲醇适当稀释， 0.22um尼龙膜过滤， HPLC测定产物浓度。 在本发明的一个优选例中在上述步骤 a中环状脂肽酰基转移酶的制备方法包括 下述步骤： 菌株培养， 将得到的菌丝体进行破壁， 获得环脂肽酰基转移酶液。
所述的菌株是指具有能分泌环状脂肽酰基转移酶的优良菌株，是放线菌属或链霉 菌属的。 主要有以下几种： 犹他游动放线菌 (Actinoplanes utahensis)IFO-13244、 (A.utahensis)NRRL-12052生产的酶。在 WO97/32975号专利中， 报道了属于链霉菌属 (Streptomyces)的细菌 (例如环圈链霉菌 (Streptomyces anulatus)481 1号菌株、 环圈链霉 菌 8703号菌株、 链霉菌 (Streptomyces sp.)6907号菌株)生产的酶生产的酶。 另外， 在 WO97/47738号专利中，报道了 Oidiodendron tenuissimum IFO 6797菌株、 Oidiodendron echinulatum IFO 31963菌株、 Oidiodendron truncatum IFO 9951菌株、 Oidiodendron truncatum IFO 31812菌株、树粉孢 (Oidiodendron sp.)30084号菌株、轮枝孢 (Verticillium sp.)30085号菌株生产的酶。
优选犹他游动放线菌 (Actinoplanes utahensis)IFO-13244、
(A.utahensis )N L- 12052, 链霉菌 (Streptomyces sp.)6907号菌株生产的酶。
菌株培养所用的培养基包括以下几种物质： 蔗糖 1-10%， 大豆蛋白胨 0.1-0.1%，
K2HPO4 0.1-0.2%,KH2PO4 0.01-0.1%, MgSO4.7H2O 0.01-0.05%。 培养条件是在 25-36°C , 优选 30°C， 通气量 l-2vvm， 搅拌速度 200-800r/min。 经过 3-5天培养， 获 得大量菌丝体。
上述得到的菌丝体可以先进行破壁，收集这些菌体细胞的胞内酶，这样可以获得 较好的环状脂肽酰基转移酶， 所述的菌体破壁可用已知的方法， 如： 高浓度盐溶液抽 提法， 超声波破碎法， 机械破碎法， 溶菌酶法等将环状脂肽酰基转移酶转移到细胞的 胞外， 再用过滤法或离心法将酶液与菌丝体分离， 得到粗酶液。
为了获得更好的固定化酶，还可以先将环状脂肽酰基转移酶液进行纯化：将上述 所得的粗酶液， 按照一定的比例加入所用的无机载体吸附游离酶。将吸附有酶的无机 载体与酶液分离， 经过充分洗涤后， 用高浓度的盐溶液将环状脂肽酰基转移酶从无机 载体上解吸下来， 得到较为纯净的酶液。
环状脂肽酰基转移酶的纯化过程还可以参照文献 Membrane-associate
echinocandin B deacylase of Actinoplanes utahensis: purification, characterization, heterologous cloning and enzymatic deacylation reaction使用超滤、 离子交换树月旨的方 法纯化环状脂肽酰基转移酶， 此方法能够得到更加纯净的酶液， 但此方法的缺点是酶 的回收率很低。 本发明使用固定化酶进行脱酰化过程， 包括以下步骤：
A.加入缓冲溶液， 制备含有环状脂肽物的溶液 1 ;
B.在溶液 1中加入固定化酰基转移酶， 进行脱酰化反应；
C.将固定化酰基转移酶与含产物的反应液分离。
其中， 固定化酰基转移酶与环状脂肽物的比例在 0.01-10u/g， 优选 0.1-5u/g。 步骤 A中所述缓冲液的 pH控制在 4-9， 优选 pH 5-7， 更佳地 pH约 6.0。 缓冲 液的种类为 0.5M柠檬酸钠缓冲液、 0.5M磷酸二氢钾-磷酸氢二钠缓冲液和 Tris-HCl 缓冲液的的一种或它们的混合物， 优选 0.5M磷酸二氢钾-磷酸氢二钠缓冲液。
步骤 B中所述脱酰化反应的温度控制在约为 20-70°C， 优选的温度范围是约 30-50°C。
步骤 C中所述的将固定化酶与含产物的反应液分离的方法包括过滤法或离心法。 当将酶固定到合适的载体上时，本发明的脱酰化作用可顺利地在连续搅拌反应釜 器中进行而不是分批地进行。
本发明中所讲 "环状脂肽物 "或"环脂肽 "指含有多肽环， 该环上侧链含有"酰基氨 基"的物质， 该物质也可有其它的侧链。
该"环状脂肽物"的代表为 FR901379、 echinocandin B (棘白菌素 B)， 该类物质是 具有抗真菌活性的已知物。
该发明的固定化环状脂肽酰基转移酶，能使环状脂肽物侧链的"酰胺基 "脱酰化形 成"氨基"， 具体的讲它能使物质 FR901379或其盐的棕榈酰侧链或含有 FR901379、式 I中所示的化合物的酰基侧链脱酰化， 形成式 II所示的环状肽物质。 如本文所用， "药学上可接受的盐"优选： 包括金属盐例如碱金属盐 (如钠盐、 钾 盐)、 碱土金属盐 (如钙盐、 镁盐等)、 铵盐、 与有机碱形成的盐 (如三甲胺盐、 三乙胺 盐、 吡啶盐、 甲基吡啶盐、 二环己铵盐、 N，N，-二苄基乙二胺盐、 二异丙基乙胺盐等) 等、 有机酸加成盐 (如甲酸盐、 乙酸盐、 三氟乙酸盐、 马来酸盐、 酒石酸盐、 甲磺酸 盐、 苯磺酸盐、 甲苯磺酸盐等)、 无机酸加成盐 (如盐酸盐、 氢溴酸盐、 氢碘酸盐、 硫 酸盐、 磷酸盐等)、 与氨基酸 (如精氨酸、 天冬氨酸、 谷氨酸等)形成的盐等。 本发明提到的上述特征，或实施例提到的特征可以任意组合。本案说明书所揭示 的所有特征可与任何组合物形式并用， 说明书中所揭示的各个特征， 可以任何可提供 相同、 均等或相似目的的替代性特征取代。 因此除有特别说明， 所揭示的特征仅为均 等或相似特征的一般性例子。 本发明的主要优点在于：
1、 本发明提供的脂肽酰基转移酶固定化方法， 可反复使用该酶， 提高环状脂肽 酰基转移酶的利用率、 降低生产成本， 有利于工业化生产和降低成本。
2、 本发明的脱酰化产物纯度得到特别显著提高。 下面结合具体实施例， 进一步阐述本发明。应理解， 这些实施例仅用于说明本发 明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照 常规条件或按照制造厂商所建议的条件。 除非另外说明， 否则所有的百分数、 比率、 比例、 或份数按重量计。
本发明中的重量体积百分比中的单位是本领域技术人员所熟知的， 例如是指在 100毫升的溶液中溶质的重量。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意 义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。 文中所述的较佳实施方法与材料仅作示范之用。 下述实施例中化合物 〔II〕 HPLC检测所用的方法：
样本在 Waters 分析性 HPLC体系上进行分析。 反相 HPLC分析用于测定
FR179642、棘白菌素 B核物质及其它类似物。反相分析采用 PLATISIL ODS色谱柱 (粒 径 5μιη， 4.6mmi.dx250cm), 并且保持在 30°C。 以 3%乙腈 /0.5%磷酸二氢钠作流动相， 流速为 1 ml/分钟， 并在 21 Onm下 UV检测。 实施例 1
犹他游动放线菌 (Actinoplanes utahensis)IFO-13244株生产的酰基转移酶 a 的制备
采用犹他游动放线菌 IFO-13244菌株， 按照 US5376634专利上的发酵方法， 进行发酵培养， 得到菌丝体培养液 150 L。 加入 0.01 M磷酸二氢钾缓冲盐， pH调 至 6.0， 加入 1.0 M氯化钾， 低温下搅拌抽提 20小时以上。 然后于铺有滤纸的布 氏漏斗过滤，收集含有酰基转移酶的滤液 1 16 L,即游离酶液 (a)，收集滤液经 HPLC 检测酶活 2.3x l05 u。 实施例 2
犹他游动放线菌 (A.utahensis )NRRL-12052株生产的酰基转移酶 b的制备 采用犹他游动放线菌 NRRL-12052菌株， 按照 US4320053 专利上的发酵方法， 进行发酵培养，得到菌丝体培养液 160L。加入 0.01M磷酸二氢钾缓冲盐， pH调至 6.0， 加入 1.0M氯化钾， 低温下搅拌抽提 20小时以上。 然后于铺有滤纸的布氏漏斗过滤， 收集含有酰基转移酶的滤液 121L， 即游离酶液 (b)，收集滤液经 HPLC检测酶活
2.1 x l05u。 实施例 3
链霉菌 (Streptomyces sp.)6907号菌株生产的酰基转移酶 c的制备
采用链霉菌 6907号菌株， 按照 WO97/32975专利上的发酵方法，进行发酵培养， 得到菌丝体培养液 160L。 加入 0.01M磷酸二氢钾缓冲盐， pH调至 6.0， 加入 1.0M氯 化钾， 低温下搅拌抽提 20小时以上。 然后于铺有滤纸的布氏漏斗过滤， 收集含有酰 基转移酶的滤液 124L, 即游离酶液 (c)，收集滤液经 HPLC检测酶活 2.9x l05u。 实施例 4
酰基转移酶 b的纯化
参照文献 Membrane-associate echinocandin B deacylase of Actinoplanes utahensis: purification, characterization, heterologous cloning and enzymatic deacylation reaction的方法纯化实施例 2中得到的粗酶液，最终得到纯净的酰基转移酶液 3.0L, 即 游离酶液 (d)， 收集酶液经 HPLC检测酶活 0.5x l05 u。 实施例 5
无机载体固定酰基转移酶
为了固定化， 分别取 50升游离酶液 (a)、 （b)、 (c), 然后分别加入 1.5公斤的膨化 珍珠盐， 25°C下，搅拌吸附 1小时以上。过滤收集固定化酰基转移酶，即固定化酶（ i )、 ( ii)、（iii)用 0.01M磷酸二氢钾缓冲盐， pH6.0的缓冲液洗涤三次， 并在室温下干燥几 小时。 该固定化酶用前储存在 4 。
Figure imgf000015_0001
实施例 6
功能基团为环氧化物的多孔亲水性酶载体固定酰基转移酶
为了固定化， 分别取 0.8升上述实施例 4制备的纯净的酰基转移酶液 (d)两份， 分 别加入 59.2g氯化钾，和 0.01mol/L的磷酸二氢钾缓冲盐， pH7.0，分别加入 30g elizyme EP403和 SEPABEADS EC-EP 30g, 25°C下，搅拌 24小时以上， 过滤收集固定化酰基转 移酶， 即固定化酶 (iv)、 （V )用 0.01M磷酸二氢钾缓冲盐， pH7.0的缓冲液洗涤三次， 并在室温下干燥几小时。 该固定化酶用前储存在 4 。
表 2 用环氧化物的多孔亲水性酶载体对游离酶液的固定情况
Figure imgf000015_0002
实施例 7
功能基团为氨基的多孔亲水性酶载体固定酰基转移酶
为了固定化， 先对载体进行活化： 分别取 8g Relizyme HA403和 SEPABEADS EC-HA 8g，加入 90毫升 2%的戊二醛溶液， 0.02mol/L的磷酸氢二钾缓冲盐， pH8.0，在 20-25 °C下搅拌 1小时， 去除上清， 纯化水清洗树脂。 分别取 0.4升上述实施例 4制备 的纯净的酰基转移酶液 (d)两份，将两份活化好的酶载体添加到酶液中，在 20-25 °C下， 搅拌 20小时以上， 过滤收集固定化酰基转移酶， 即固定化酶 (vi)、（vii)用 0.01M磷酸 二氢钾缓冲盐， pH7.0的缓冲液洗涤三次，并在室温下干燥几小时。该固定化酶用前储 存在 4°C。
表 3 用氨基型的多孔亲水性酶载体对游离酶液的固定情况
Figure imgf000016_0001
实施例 8
用固定化酶进行分批脱酰化反应
分别向 FR901379物质的水溶液 (20mg/ml、 HPLC纯度 79.2%)2升 (FR901379物 20g，16.7mmol)中加入缓冲液 (0.2M磷酸二氢钾-磷酸氢二钠缓冲液； pH6.0)2升禾卩 15g 固定化酶（ i )、 固定化酶（ϋ )、 固定化酶 (iii)， 40°C反应 4小时， 通过 HPLC测得 F 179642物质的产量和纯度。
表 4 固定化酶 4小时脱酰化后 FR901379物质的转化率及 FR179642物质的纯度
Figure imgf000016_0002
实施例 9
用固定化酶进行分批脱酰化反应
分别向 FR901379物质的水溶液 (20mg/ml、 HPLC纯度 79.2%)2升 (FR901379物 20g, 16.7mmol)中加入缓冲液 (0.2M磷酸二氢钾-磷酸氢二钠缓冲液； pH6.0)2升禾卩 15g 固定化酶 (iv)、 固定化酶 ( v )、 固定化酶 (vi)、 固定化酶 (vii)， 40°C反应 1小时， 通过 HPLC测得 FR 179642物质的产量和纯度。
表 5 固定化酶 1小时脱
Figure imgf000016_0003
对比例 1
用游离酶液进行分批试验
按照 WO97/32975专利上的脱酰化方法， 将 FR901379物质的水溶液 (100mg/ml、 HPLC纯度 79.2%) 100ml(FR901379物 10g，38.35mmol)、加入缓冲液 (0.2M磷酸二氢钾 -磷酸氢二钠缓冲液； pH6.0)100ml， 甲醇 100ml, 然后加入游离酶液 (c)700ml，40°C反 应 Ί小时，转化率 71.1%，生成的 FR17964物质的纯度，经 HPLC检测纯度为 75.7%， 其 HPLC分析色谱图见图 2和表 10。
转化后的液体， 测定酶活， 经过测定基本上检测不到酶活。酶在转化过程中几乎 全部失活。
表 10
Figure imgf000017_0001
从上述实施例和对比例 1可以得出，利用固定酶进行脱酰基化与游离酶液脱酰基 相比较具有显著地优势。 转化率和产物的纯度都得到极大地提高。 实施例 10
在连续搅拌釜式反应器中的连续脱酰化反应
在 50升连续搅拌釜式反应器中于 45°C进行 FR901379到 FR179642物质的连续 脱酰化反应， 加入固定化酶 (vi)0.3Kg， FR901379物质的水溶液 (20mg/ml、 HPLC纯 度 79.2%)20升， 缓冲液 (0.2M磷酸二氢钾-磷酸氢二钠缓冲液； pH6.0)20升。 底物在 3小时后转化率达到 95%以上， F 179642物质经 HPLC检测纯度为 95.99%，其 HPLC 分析色谱图见图 1和表 6。
表 6
Figure imgf000017_0002
固定化的酰基转移酶至少可保持 30小时的稳定性和活性， 因此在生产过程中可 连续重复使用 5次以上。
从实施例 10与对比例 1相比可以得出，固定化酶的另一大优势是可以重复利用。 极大地提高使用效率， 减少对环境的破坏污染。 实施例 11 不同载体对游离酶的固定化及固定化酶脱酰化比较
为了固定化， 分别取 5升游离酶液 (c)， 然后分别加入 0.15公斤的膨化珍珠盐， 硅藻土 (CELITE), 和活性碳 (SiO2含量小于 1%)， 分子筛、 多孔玻璃。 30°C下， 搅拌 吸附 1小时以上。 过滤收集固定化酰基转移酶， 即固定化酶 (viii)、 （ix)、 （x )、 （xi)、 (xii)用 0.01M磷酸二氢钾缓冲盐， pH6.0的缓冲液洗涤三次， 并在室温下干燥几小时， 该固定化酶用前储存在 4 。
然后，分别向 FR901379物质的水溶液 (20mg/ml、HPLC纯度 79.2%)2升 (FR901379 物质 20g，16.7mmol)中加入缓冲液 (0.2M磷酸二氢钾-磷酸氢二钠缓冲液； pH6.0)2升和 15g(viii)、 (ix)、 ( x )、 （xi)、 （xii)， 40°C反应 4小时， 通过 HPLC测得 FR179642物质 的产量和纯度。
Figure imgf000018_0001
实施例 12
不同比例载体对游离酶的固定化及固定化酶脱酰化比较
为了固定化， 分别取 5升游离酶液 (c)， HPLC检测酶活 1.17xl04U， 然后分别加 入 0.05公斤、 0.12公斤、 0.15公斤、 0.24公斤、 0.58公斤、 1.17公斤、 1.75公斤的 硅藻土 (CELITE), 30°C下， 搅拌吸附 1小时以上。 过滤收集固定化酰基转移酶， 用 0.01M磷酸二氢钾缓冲盐， pH6.0的缓冲液洗涤三次， HPLC检测滤液中的酶活。 固定 化酶在室温下干燥几小时， 该固定化酶用前储存在 4°C。
然后，分别向 FR901379物质的水溶液 (20mg/ml、 HPLC纯度 79.2%) 1升 (FR901379 物 10g， 8.35mmol)中， 加入缓冲液 (0.2M磷酸二氢钾-磷酸氢二钠缓冲液； pH6.0)l升 和 75g上述制备获得的固定化酶， 40°C反应 4小时， 通过 HPLC测得 FR179642物质 的生产量和纯度。
表 8 不同比例载体对游离酶的吸附率及固定化酶脱酰化比较
Figure imgf000018_0002
实施例 13
不同 pH对载体固定化游离酶的比较
为了固定化， 分别取 5升游离酶液 (a)， HPLC检测酶活 1.0xl04U， 然后分别加入 0.15公斤的硅藻土 (CELITE)，用 2mol/L的盐酸或 2mol/L氢氧化钠溶液调节 pH至 3.5， 4.0， 6.0， 9.0， 9.5。 25°C下， 搅拌吸附 1小时以上。 过滤收集固定化酰基转移酶。 固 定化酶在室温下干燥几小时， 该固定化酶用前储存在 4°C。
然后，分别向 FR901379物质的水溶液 (20mg/ml、 HPLC纯度 79.2%) 1升 (FR901379 物 10g， 8.35mmol)中， 加入缓冲液 (0.2M磷酸二氢钾-磷酸氢二钠缓冲液； pH6.0)l升 和 75g上述制备获得的固定化酶， 40°C反应 4小时， 通过 HPLC测得 FR179642物质 的生产量。
表 9 不同 pH对载体固定化游离酶的比较
Figure imgf000019_0001
实施例 14
将 echinocandin B的二甲基亚砜溶液（100mg/ml)100ml (棘白菌素 B为 10g; 9.43mmol), 加入 1.2M氯化钾、0.5M磷酸二氢钾 -磷酸氢二钠缓冲液 500ml， pH7.0 然后加入固定化酶（ii )25g，于 50°C转化 2小时， 转化率 85.6%。 以上所述仅为本发明的较佳实施例而已， 并非用以限定本发明的实质技术内 容范围， 本发明的实质技术内容是广义地定义于申请的权利要求范围中， 任何他 人完成的技术实体或方法， 若是与申请的权利要求范围所定义的完全相同， 也或 是一种等效的变更， 均将被视为涵盖于该权利要求范围之中。

Claims

权 利 要 求
1. 一种固定化环脂肽酰基转移酶。
2. 如权利要求 1所述的固定化环脂肽酰基转移酶， 其特征在于， 所述环脂肽 酰基转移酶固定于载体上； 所述载体材料为无机载体、 或多孔亲水性酶载体。
3. 如权利要求 2所述的固定化环脂肽酰基转移酶， 其特征在于， 所述无机载 体以总重量计， 其中 SiO2含量大于 50wt%， A12O3含量大于 lwt%。
4. 如权利要求 2所述的制备方法，其特征在于，所述多孔亲水性酶载体选自： 以聚甲基丙烯酸酯为基体键合环氧化物、 或者含氨基功能基团的酶载体。
5. 如权利要求 1所述的固定化环脂肽酰基转移酶， 其特征在于， 所述的固定 化环脂肽酰基转移酶用于催化如式 I的化合物中的 R1的酰基脱酰化， 产生如式 II所示的化合物，
Figure imgf000020_0001
式中， R1为酰基， R2为羟基或酰氧基， R3为氢或羟基， R4为氢或羟基， R: 为氢或羟基璜酰氧基， R6为氢或氨基甲酰基。
6. 如权利要求 5所述的固定化环脂肽酰基转移酶， 其特征在于， 所述式 I的化 合物为式 I a或式 I b所示化合物或其药学上可接受的盐；
Figure imgf000021_0001
Figure imgf000021_0002
lb
7.一种如权利要求 1-6任一所述的固定化环脂肽酰基转移酶的制备方法， 其 特征在于， 所述的方法包括步骤：
将含有游离的环脂肽酰基转移酶的溶液与载体混合， 得到如权利要求 1-6任 一所述的固定化环脂肽酰基转移酶。
8. 如权利要求 7所述的制备方法， 其特征在于， 所述环脂肽酰基转移酶来源 于天然或人工突变体、 或者变种及由通过导入外来的环状酰基转移酶基因后转化 得到的。
9. 如权利要求 7所述的制备方法， 其特征在于， 所述含有游离的环脂肽酰基 转移酶的溶液通过下述方式得到： 菌株培养， 得到含有游离的环脂肽酰基转移酶 的溶液； 或菌株培养后将得到的菌丝体进行破壁， 得到含有游离的环脂肽酰基转 移酶的溶液。
10. 如权利要求 9所述的制备方法， 其特征在于， 所述菌株是放线菌属或链 霉菌属的。
11. 如权利要求 7所述的制备方法， 其特征在于， 所述载体材料选自无机载 体、 或多孔亲水性酶载体。
12. 如权利要求 7所述的制备方法， 其特征在于， 所述游离的环脂肽酰基转 移酶与载体的混合比例为每克载体需酶 10-1000单位 (u); 优选每克载体需酶 20-600单位 (u)。
13. 如权利要求 7所述的制备方法， 其特征在于， 所述含有游离的环脂肽酰 基转移酶的溶液的 pH值为 4-9。
14. 如权利要求 7所述的制备方法， 其特征在于， 所述含有游离的环脂肽酰 基转移酶的溶液和载体混合的温度为 0-80°C ; 优选 20-35°C。
15. 如权利要求 7-14任一所述的制备方法， 其特征在于， 将含有游离的环脂 肽酰基转移酶的溶液与载体混合后， 将游离的环脂肽酰基转移酶和结合于载体的 环脂肽酰基转移酶分离， 得到固定化环脂肽酰基转移酶。
16. 一种如式 II所示的化合物的制备方法， 其特征在于， 所述的方法包括步 骤：
(a)将如式 I所示的化合物和缓冲溶液混合， 得到溶液 1 ;
(b)将溶液 1和如权利要求 1-6任一所述的固定化环脂肽酰基转移酶混合， 进 行脱酰化反应， 得到如式 II所示的化合物；
Figure imgf000022_0001
II
Figure imgf000023_0001
式中， R1为酰基， R2为羟基或酰氧基， R3为氢或羟基， R4为氢或羟基， R5 为氢或羟基璜酰氧基， R6为氢或氨基甲酰基。
17. 如权利要求 16所述的制备方法， 其特征在于， 所述固定化环脂肽酰基转 移酶和式 I的化合物的比例在 0.01-10 u/g。
18. 如权利要求 16所述的制备方法， 其特征在于， 所述步骤 (a)中缓冲液的 pH在 4-9。
19. 如权利要求 16所述的制备方法， 其特征在于， 所述步骤 (a)中缓冲液选 自下述溶液中的一种或一种以上： 柠檬酸钠缓冲液、 磷酸二氢钾-磷酸氢二钠缓冲 液和 Tris-HCl缓冲液。
20. 如权利要求 16所述的制备方法， 其特征在于， 所述步骤 (b)中脱酰化反 应的温度为 20-70°C。
21. 如权利要求 16-20任一所述的制备方法， 其特征在于， 所述步骤 (b)中脱 酰化反应后， 将固定化环脂肽酰基转移酶与含产物的反应液分离， 得到如式 II所 示的化合物。
22. 如权利要求 21所述的制备方法， 其特征在于， 所述将固定化环脂肽酰基 转移酶与含产物的反应液分离的方法包括过滤或离心。
PCT/CN2013/075508 2012-05-11 2013-05-10 一种固定化环脂肽酰基转移酶及其制备方法和用途 WO2013166993A1 (zh)

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