WO2017143945A1 - 一种头孢菌素c酰化酶突变体 - Google Patents

一种头孢菌素c酰化酶突变体 Download PDF

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WO2017143945A1
WO2017143945A1 PCT/CN2017/074029 CN2017074029W WO2017143945A1 WO 2017143945 A1 WO2017143945 A1 WO 2017143945A1 CN 2017074029 W CN2017074029 W CN 2017074029W WO 2017143945 A1 WO2017143945 A1 WO 2017143945A1
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seq
acylase
mutant
replaced
cephalosporin
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French (fr)
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王金刚
梁岩
陈舒明
任亮
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上海星维生物技术有限公司
山西新宝源制药有限公司
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Definitions

  • the present invention belongs to the field of genetic engineering technology, and in particular to a cephalosporin C acylase for the one-step enzymatic production of 7-ACA (7-aminocephalosporanic acid) constructed by a point mutation method.
  • Cephalosporins are the most widely used ⁇ -lactam antibiotics. Most of these antibiotics are 7-ACA derivatives synthesized by 7-aminocephalosporanic acid (7-ACA). Antibiotics account for 40% of the global antibiotic market.
  • 7-ACA is generally obtained by chemically or biologically enzymatically cleavage of Cephalosporin C (CPC) and removal of molecular side chains. Due to the complicated chemical process, high energy consumption and serious pollution, in recent years, industrial production of 7-ACA has basically been replaced by biological enzymatic preparation.
  • the currently used biological enzymatic method is further divided into two-step enzymatic method and one-step enzymatic method.
  • the two-step enzymatic method was used earlier, mainly using D-Amino Acid Oxidase (hereinafter referred to as DAAO) and Glutaryl-7-Amidocephalospranic Acid (hereinafter referred to as Glutaryl-7-Amidocephalospranic Acid).
  • CPC produces GL-7-ACA under the action of DAAO, and then the side chain is removed by the action of GL-7-ACA acylase to form 7-ACA.
  • the method has basically replaced the chemical method due to the characteristics of environmental protection, low energy consumption and high yield
  • the H 2 O 2 as a by-product of the DAAO catalytic reaction has a degradation effect on CPC, and is a two-step catalytic reaction. complex. Therefore, a one-step enzymatic preparation of 7-ACA has been developed, in which the side chain is catalyzed by CPC acylating enzyme to form 7-ACA.
  • CPC acylase cephalosporin C acylase
  • Pseudomonas sp. SE83 Pseudomonas diminuta N176, Pseudomonas sp. P130, Pseudomonas sp. GK16, etc.
  • these enzymes are strictly GL-7-ACA acylases, and their CPC acylase activities are relatively low, only 2-4% of the GL-7-ACA acylase activity.
  • no wild bacterium of the CPC acylase producing high catalytic activity has been found in nature.
  • Wild-type CPC acylase can not meet the requirements of industrial production of CPC, so one-step enzymatic method can not completely replace the two-step enzymatic method to produce 7-ACA on a large scale.
  • CPC acylation enzymes derived from Pseudomonas sp. SE83.
  • the screening activity of CPC acylase is several times higher than that of wild enzymes, but this type of CPC acylase has a strong 7-ACA product inhibition Systematic.
  • the 45th position of the ⁇ subunit of the derived CPC acylase was replaced by I to the V and ⁇ subunits.
  • the 58th position of the 153th position replaced by F with the V and ⁇ subunits was replaced by Y with the T, ⁇ subunit.
  • the 177th position was replaced by F to the L, and the 382th position of the ⁇ subunit was replaced by V to L.
  • the body's CPC acylase activity was increased by 25.3 times, but the enzyme activity still could not meet the requirements of industrial production.
  • the present invention utilizes genetic engineering technology to microbial-derived wild-type CPC acyl group.
  • the enzyme was engineered and screened to construct a highly enzymatically active CPC acylase mutant, thereby realizing the industrialization of 7-ACA by one-step enzymatic production.
  • the present invention modifies Pseudomonas sp. GK16-derived GL-7-ACA acylase (SEQ ID NO: 1) by random mutation, semi-rational design and the like to obtain a high enzyme using CPC as a specific substrate. Live CPC acylase mutants to efficiently catalyze the formation of 7-ACA by CPC.
  • a first object of the present invention is to provide a high accommodating CPC acylase mutant for producing 7-ACA.
  • a second object of the present invention is to provide a gene encoding the above-described CPC acylase mutant.
  • a third object of the present invention is to provide a plasmid comprising the above gene.
  • a fourth object of the present invention is to provide a microorganism transformed with the above plasmid.
  • a fifth object of the present invention is to provide the use of the above-described CPC acylase mutant or microorganism in the production of 7-ACA.
  • the present invention provides the following cephalosporin C acylase:
  • cephalosporin C acylase (CPC acylase) whose amino acid sequence is:
  • SEQ ID NO: 3 which is a mutant in which the V at position 240 of SEQ ID NO: 1 is replaced by F, the amino acid sequence of which is:
  • SEQ ID NO: 4 which is a mutant in which A of position 306 of SEQ ID NO: 1 is replaced by T, and the amino acid sequence thereof is:
  • SEQ ID NO: 5 which is a mutant in which R of position 553 of SEQ ID NO: 1 is replaced by L, and the amino acid sequence thereof is:
  • SEQ ID NO: 6 which is a mutant in which the 623th position of SEQ ID NO: 1 is replaced by N, and the amino acid sequence thereof is:
  • SEQ ID NO:7 which is a mutant in which V at position 240 of SEQ ID NO: 1 is replaced by F, A at position 306 is replaced by T, and H at position 623 is replaced with T, and the amino acid sequence thereof is:
  • SEQ ID NO:8 which is the substitution of V at position 240 of SEQ ID NO: 1 with F, replacement of A at position 306 with T, mutation at position 553 with R replaced by L, and replacement of H at position 623 with The mutant of T has an amino acid sequence of:
  • SEQ ID NO: 9 which is the substitution of the 215th position of SEQ ID NO: 1 with V, the 228th F for V, the 240th V for F, the 306 for A for T, Y at position 323 is replaced by T, F at position 347 is replaced by L, mutant at position 552 is replaced by L, mutant at position 553 is replaced by L, and H at position 623 is replaced by T.
  • the mutant has an amino acid sequence of:
  • amino acid sequence of the above cephalosporin C acylase is SEQ ID NO: 9.
  • the gene encoding the above cephalosporin C acylase SEQ ID NO: 9 has the following base sequence:
  • a plasmid comprising the above gene.
  • the plasmid contains a vector for expressing the above gene, and preferably the vector is a PET series, for example, the vector is pET24a(+), but is not limited thereto.
  • a microorganism transformed with the above plasmid which can be used as a host for expressing the above cephalosporin C acylase.
  • the microorganism is selected from the group consisting of Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).
  • cephalosporin C acylase or microorganism can be used to produce 7-ACA, especially one-step enzymatic production of 7-ACA.
  • cephalosporin C is used as a substrate raw material, and the above cephalosporin C acylating enzyme or microorganism is used as a catalyst to catalyze the reaction.
  • the concentration of cephalosporin C (CPC) can be selected from 1 to 3 wt%, preferably 2.5% by weight; and the reaction temperature is selected from 10 to 37 ° C, preferably 15 ° C.
  • the CPC acylase mutant of the present invention hydrolyzes CPC to produce 7-ACA, which has an activity of 20.5-150 times higher than that of wild-type enzyme, higher substrate specificity and lower product inhibition, when applied to one-step production 7- At ACA, the 7-ACA generation rate exceeds 98%, which is highly industrialized.
  • amino acid abbreviations herein may be either English three-letter or English single-letter, which are well known to those skilled in the art and are listed in the following table:
  • the amino acid sequence of the wild type GL-7-ACA acylase derived from Pseudomonas sp. GK16 is SEQ ID NO: 1 in the Sequence Listing. Its coding gene is SEQ ID NO: 2 in the Sequence Listing.
  • the present invention performs point mutation of the wild-type CPC acylase SEQ ID NO: 1 gene sequence SEQ ID NO: 2.
  • the above sites were then randomly combined and mutated by site-directed mutagenesis to obtain mutants having the amino acid sequence of SEQ ID NOS: 7-8 of the present invention.
  • site-directed mutagenesis was carried out on the basis of SEQ ID NO: 8, to obtain a mutant having the amino acid sequence of SEQ ID NO: 9 of the present invention.
  • SEQ ID NO: 1 is a common sequence of these amino acid sequences of SEQ ID NOs: 3-9, and these amino acid sequences are each one, or two, up to nine amino acid substitutions based on SEQ ID NO:
  • the mutants obtained, the amino acid sequences of these mutants maintained a homology of more than 98%.
  • CPC acylase mutant means the same meaning, and both refer to cephalosporin C acylation.
  • a mutant of the enzyme means the same meaning, and both refer to cephalosporin C acylation.
  • wild (type) means the same meaning, and all refer to wild type GL-7-ACA acylase or CPC acylase (SEQ ID NO: 1).
  • the CPC acylase mutant of the present invention has only 692 amino acid numbers and has a well-defined structure, and thus a gene encoding the same, an expression cassette and plasmid containing the same, and a transformant comprising the same are easily obtained by those skilled in the art.
  • genes, expression cassettes, plasmids, transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.
  • the above transformant host can be any microorganism suitable for expressing the CPC acylase, including bacteria and fungi.
  • the microorganism is Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, preferably Escherichia coli, more preferably E. coli BL21 (DE3) was selected.
  • the CPC acylase of the present invention may take the form of an enzyme or a form of a microbial cell.
  • the form of the enzyme includes a free enzyme, an immobilized enzyme, and includes a purified enzyme, a crude enzyme, a fermentation broth, a carrier-immobilized enzyme, and the like; the form of the bacterial body includes a living cell and a dead cell.
  • CPC acylase wild-type gene SEQ ID NO: 2 Using the CPC acylase wild-type gene SEQ ID NO: 2 as a template, a random mutant library was constructed using error-prone PCR technology.
  • the forward primer CPC-Nde-F is 5'- CATATG GAGCCGACCTCGAC-3'
  • the reverse primer CPC-Xho-R is 5'- CTCGAG TGGCTTGAAGTTGAAG-3'
  • the 50 ⁇ L error-prone PCR reaction system includes: 50 ng plasmid template pET24a-wt-CPC, 30 pmol pair of primers CPC-Nde-F and CPC-Xho-R, 1X Taq buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP , 7 mM MgCl 2 , (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM) MnCl 2 , 2.5 units of Taq enzyme (fermentas).
  • the PCR reaction conditions were: 95 ° C for 5 min; 94 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 2 min / kbp; 30 cycles; 72 ° C for 10 min.
  • the 2.0 kb random mutant fragment was recovered as a large primer and subjected to MegaPrimer PCR using KOD-plus DNA polymerase: 94 ° C for 5 min, 98 ° C for 10 s, 60 ° C for 30 s, 68 ° C for 2 min/kbp, 25 cycles, and 68 ° C for 10 min.
  • DpnI digestion of the template plasmid electroporated into E. E.coli BL21 (DE3), to obtain more than 10 4 clones of the library of random mutations.
  • the transformants in the mutant library were selected and inoculated into 96-well deep-well culture plates containing 700 ⁇ L of LB medium containing 100 ⁇ g/mL kanamycin and 0.1 mM IPTG. After incubation at 37 ° C for 6 h, the temperature was lowered to 25 ° C. , culture overnight. After centrifugation at 5000 rpm for 10 min, the supernatant was discarded, frozen at -70 ° C for 1 h, and thawed at room temperature for 30 min.
  • Substrate reaction solution 0.1 M potassium phosphate buffer (pH 8.0) containing 2% by weight of CPC sodium salt,
  • Enzyme Activity Definition The amount of enzyme required to catalyze the production of 1 micromolar ( ⁇ mol) of 7-ACA per minute by CPC is defined as 1 unit (U).
  • *wtCPC refers to an expression strain of wild-type cephalosporin C acylase.
  • Example 3 directed evolution by site-directed mutagenesis
  • N A/G/C/T.
  • the pET24a-wtCPC plasmid was used as a template, and the PCR primers were amplified by 240-F1 and 306-R1, 306-F2 and 553-R2, 553-F3 and 623-R3, respectively, and amplified by over-lapping PCR. A large fragment was then subjected to MegaPrimer PCR using a large fragment as a primer to construct a site-directed mutant library.
  • the 50 ⁇ L PCR reaction system included: 10 ng of plasmid template, 10 pmol of primer pair, 1 x KOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO 4 , and 5 units of KOD-plus DNA polymerase.
  • the PCR reaction conditions were: 95 ° C for 1 min; 98 ° C for 10 s, 57 ° C for 30 s, 68 ° C for 1 min / kbp; 30 cycles; 68 ° C for 10 min.
  • the gel recovers three segments P1, P2, and P3.
  • the second round of PCR was carried out using P1, P2, and P3 as templates, and 240-F1 and 623-R3 were used as primers to obtain fragment P, and the gel was recovered.
  • the PCR reaction conditions were: 95 ° C for 3 min; 98 ° C for 10 s, 60 ° C for 30 s, 68 ° C for 1 min / kbp; 25 cycles; 68 ° C for 10 min.
  • Fragment P was used as a large primer, and MegaPrimer PCR was performed with KOD-plus DNA polymerase: 94 ° C for 5 min, 98 ° C for 10 s, 60 ° C for 30 s, 68 ° C for 2 min/kbp, 25 cycles, and 68 ° C for 10 min.
  • the plasmid template was digested with DpnI and electrotransformed into E. coli BL21 (DE3) to obtain a random mutation library of more than 3 ⁇ 10 4 clones.
  • the method is the same as step 2.2 of the embodiment 2. After screening, the ED2 strain with relatively high viability was obtained, and the strain was confirmed to contain mutations of four sites of V240F, A306T, R553L and H623T by sequencing.
  • the plasmid extracted from the ED2 strain was used as a template, and 45/58-F and 45/58-R, 153-F and 153-R, 177-F and 177-R, 382-F and 382-R were used as primers.
  • the final product was digested with Dpn I and transformed into E. coli BL21 (DE3).
  • the 50 ⁇ L PCR reaction system included: 10 ng of plasmid template, 10 pmol of primer pair, 1 x KOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO 4 , and 5 units of KOD-plus DNA polymerase.
  • the PCR reaction conditions were: 95 ° C for 1 min; 98 ° C for 10 s, 57 ° C for 30 s, 68 ° C for 1 min / kbp; 20 cycles; 68 ° C for 10 min.
  • Substrate reaction solution 0.1 M potassium phosphate buffer (pH 8.0) containing 2% by weight of CPC sodium salt,
  • Enzyme Activity Definition The amount of enzyme required to catalyze the production of 1 micromolar ( ⁇ mol) of 7-ACA per minute by CPC is defined as 1 unit (U).
  • the cells were resuspended in 50 mL of equilibration buffer (50 mM potassium phosphate buffer, 200 mM NaCl, pH 8.0), then sonicated, and the disrupted cells were centrifuged at 4 ° C, 12000 rpm for 20 min, and the supernatant was collected.
  • the supernatant was added to an affinity chromatography column containing 10 mL of Ni-NAT matrix at a rate of 1 mL/min, and then the column was washed with an equilibration buffer containing 30 mM imidazole to elute impurities. Finally, the deproteinized protein was washed with an equilibration buffer containing 500 mM imidazole, and the peak eluate was collected.
  • the eluate was subjected to desalting treatment through an ultrafiltration tube having a molecular weight cutoff of 10 kDa to obtain a pure enzyme.
  • the solution used in this step was the same as the reagent used in step 2.3 of Example 2.
  • the protein concentration of the pure enzyme was determined by using the Thermo Scientific BCA Protein Assay Kit to obtain the specific activity of the pure enzyme.
  • *wtCPC refers to an expression strain of wild-type cephalosporin C acylase.
  • the enzyme activity of the cephalosporin C acylase mutant of the present invention SEQ ID NO: 7-9 was increased by 20.5 times compared to the wild-type cephalosporin C acylase SEQ ID NO: 1. Up to 150-fold, wherein the mutant SEQ ID NO: 9 has the highest enzyme activity.
  • the present invention constructs a CPC acylase mutant, which increases the specific activity of the wild-type CPC acylase by 20.5-150 times, and uses a mutant pure enzyme to carry out one-step enzymatic production of 7-ACA for 40 minutes.
  • the conversion rate of CPC is over 98%, which has broad industrial prospects.

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Abstract

提供一种通过点突变法构建的头孢菌素C酰化酶、编码该头孢菌素C酰化酶的基因、包含该基因的质粒、转化了该质粒的微生物以及该头孢菌素C酰化酶或该微生物在生产7-氨基头孢烷酸（即7-ACA）中的用途。

Description

一种头孢菌素C酰化酶突变体 技术领域
本发明属于基因工程技术领域，具体地说，涉及通过点突变法构建的用于一步酶法生产7-ACA(7-氨基头孢烷酸)的头孢菌素C酰化酶。
背景技术
头孢类抗生素是现在应用最广泛的β-内酰胺类抗生素，该类抗生素大部分是通过7-氨基头孢烷酸(7-aminocephalosporanic acid，简称为7-ACA)合成的7-ACA衍生物，这类抗生素占到了全球抗生素市场40％的份额。
7-ACA一般通过化学法或生物酶法裂解头孢菌素C(Cephalosporin C，简称为CPC)，脱去分子侧链而获得。因化学法工艺复杂、能耗高，污染严重，近几年来，工业生产7-ACA基本已替换为生物酶法制备。目前使用的生物酶法又分为两步酶法和一步酶法。两步酶法采用得较早，主要用到D-氨基酸氧化酶(D-Amino Acid Oxidase，以下简称为DAAO)和戊二酰基-7-氨基头孢烷酸(Glutaryl-7-Amidocephalospranic Acid，以下简称为GL-7-ACA)酰化酶，CPC在DAAO的作用下生成GL-7-ACA，然后再在GL-7-ACA酰化酶的作用下脱去侧链，生成7-ACA。虽然该方法因环保、低能耗、高收率等特点已经基本取代了化学法，但该方法中DAAO催化反应的副产物H2O2对CPC有降解作用，且为两步催化反应，步骤较为复杂。因此，人们开发出了一步酶法制备7-ACA的技术，即利用CPC酰化酶催化CPC脱去侧链，生成7-ACA。
自20世纪80年代以来，人们从自然界中发现了产CPC酰化酶(头孢菌素C酰化酶)的菌株，如Pseudomonas sp.SE83、Pseudomonas diminuta N176、Pseudomonas sp.P130、Pseudomonas sp.GK16等，但这些酶严格来说是GL-7-ACA酰化酶，它们的CPC酰化酶活力均比较低，只有GL-7-ACA酰化酶活力的2-4％。迄今为止，自然界尚未发现产高催化活力的CPC酰化酶野生菌。野生型的CPC酰化酶还不能满足工业化生产CPC的要求，因此一步酶法至今不能完全取代两步酶法来大规模生产7-ACA。现在对Pseudomonas sp.SE83来源的CPC酰化酶改造的研究相对比较多，通过改造筛选，CPC酰化酶活性较野生酶提高了几十倍，但该类型的CPC酰化酶都有很强的7-ACA产物抑 制性。近年来，对Pseudomonas sp.GK16菌株来源的CPC酰化酶改造出现了较大的进展，将该来源的CPC酰化酶的β亚基的第45位由I替换成V、β亚基的第58位由F替换成V、β亚基的第153位由Y替换成T、β亚基的第177位由F替换成L、β亚基的第382位由V替换成L，获得的突变体的CPC酰化酶酶活提高了25.3倍，但该酶活仍然无法满足工业生产的要求。
发明内容
为了克服现有一步酶法生产7-ACA技术中的上述缺陷，得到酶活性更高、底物特异性更高的CPC酰化酶，本发明利用基因工程技术来对微生物来源的野生型CPC酰化酶进行改造和筛选，构建高酶活性的CPC酰化酶突变体，从而实现一步酶法生产7-ACA的工业化。
为此，本发明通过随机突变、半理性设计等技术对Pseudomonas sp.GK16来源的GL-7-ACA酰化酶(SEQ ID NO：1)进行改造，获得以CPC作为特异性底物的高酶活的CPC酰化酶突变体，以便高效地将CPC催化生成7-ACA。
因此，本发明的第一个目的在于提供一种用于生产7-ACA的高酶活力的CPC酰化酶突变体。
本发明的第二个目的在于提供编码上述CPC酰化酶突变体的基因。
本发明的第三个目的在于提供包含上述基因的质粒。
本发明的第四个目的在于提供转化了上述质粒的微生物。
本发明的第五个目的在于提供上述CPC酰化酶突变体或微生物在生产7-ACA中的用途。
为了达到上述目的，本发明提供如下头孢菌素C酰化酶：
一种头孢菌素C酰化酶(CPC酰化酶)，其氨基酸序列为：
SEQ ID NO：3，其为SEQ ID NO：1第240位的V替换为F的突变体，其氨基酸序列为：
Figure PCTCN2017074029-appb-000001
Figure PCTCN2017074029-appb-000002
SEQ ID NO：4，其为SEQ ID NO：1第306位的A替换为T的突变体，其氨基酸序列为：
Figure PCTCN2017074029-appb-000003
SEQ ID NO：5，其为SEQ ID NO：1第553位的R替换为L的突变体，其氨基酸序列为：
Figure PCTCN2017074029-appb-000004
Figure PCTCN2017074029-appb-000005
SEQ ID NO：6，其为SEQ ID NO：1第623位的H替换为N的突变体，其氨基酸序列为：
Figure PCTCN2017074029-appb-000006
SEQ ID NO：7，其为SEQ ID NO：1第240位的V替换为F、第306位的A替换为T、第623位的H替换为T的突变体，其氨基酸序列为：
Figure PCTCN2017074029-appb-000007
Figure PCTCN2017074029-appb-000008
SEQ ID NO：8，其为SEQ ID NO：1第240位的V替换为F、第306位的A替换为T、第553位的R替换为L的突变体、第623位的H替换为T的突变体，其氨基酸序列为：
Figure PCTCN2017074029-appb-000009
SEQ ID NO：9，其为SEQ ID NO：1第215位的I替换为V、第228位的F替换为V、第240位的V替换为F、第306位的A替换为T、第323位的Y替换为T、第347位的F替换为L、第552位的V替换为L的突变体、第553位的R替换为L的突变体、第623位的H替换为T的突变体，其氨基酸序列为：
Figure PCTCN2017074029-appb-000010
Figure PCTCN2017074029-appb-000011
优选上述头孢菌素C酰化酶的氨基酸序列为SEQ ID NO：9。
一种编码上述头孢菌素C酰化酶的基因。
优选地，编码上述头孢菌素C酰化酶SEQ ID NO：9的基因具有下述碱基序列：
Figure PCTCN2017074029-appb-000012
一种包含上述基因的质粒。该质粒包含用于表达上述基因的载体，优选载体是PET系列，比如载体是pET24a(+)，但并不受限于此。
一种转化了上述质粒的微生物，该微生物可作为宿主用于表达上述头孢菌素C酰化酶。
优选地，上述微生物选自枯草芽孢杆菌、毕赤酵母、酿酒酵母、大肠杆菌，优选大肠杆菌，更优选大肠杆菌BL21(DE3)。
上述头孢菌素C酰化酶或者微生物可以用于生产7-ACA、尤其是一步酶法生产7-ACA。
在生产7-ACA中，以头孢菌素C为底物原料，用上述头孢菌素C酰化酶或者微生物作为催化剂来催化反应。
生产7-ACA可采用常规的工艺条件，比如，头孢菌素C(CPC)的浓度可选择1～3wt％，优选2.5wt％；反应温度选择10～37℃，优选15℃。
本发明的CPC酰化酶突变体水解CPC产生7-ACA的活性相较野生酶提高了20.5倍至150倍，底物特异性更高，产物抑制性更低，当应用于一步法生产7-ACA时，7-ACA生成率超过98％，极具工业化前景。
具体实施方式
以下结合具体实施例对本发明做进一步详细说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。
为简要起见，本文中的氨基酸缩写既可以使用英文三字母、也可以采用英文单字母，这是本领域技术人员熟知的，这些缩写列于下表中：
表1氨基酸中英文对照及缩写
丙氨酸 Alanine A或Ala 脂肪族类
精氨酸 Arginine R或Arg 碱性氨基酸类
天冬酰胺 Asparagine N或Asn 酰胺类
天冬氨酸 Aspartic acid D或Asp 酸性氨基酸类
半胱氨酸 Cysteine C或Cys 含硫类
谷氨酰胺 Glutamine Q或Gln 酰胺类
谷氨酸 Glutamic acid E或Glu 酸性氨基酸类
甘氨酸 Glycine G或Gly 脂肪族类
组氨酸 Histidine H或His 碱性氨基酸类
异亮氨酸 Isoleucine I或Ile 脂肪族类
亮氨酸 Leucine L或Leu 脂肪族类
赖氨酸 Lysine K或Lys 碱性氨基酸类
蛋氨酸 Methionine M或Met 含硫类
 苯丙氨酸 Phenylalanine F或Phe 芳香族类
脯氨酸 Proline P或Pro 亚氨基酸
丝氨酸 Serine S或Ser 羟基类
苏氨酸 Threonine T或Thr 羟基类
色氨酸 Tryptophan W或Trp 芳香族类
酪氨酸 Tyrosine Y或Tyr 芳香族类
缬氨酸 Valine V或Val 脂肪族类
作为构建头孢菌素C酰化酶突变体的基础模板，Pseudomonas sp.GK16来源的野生型GL-7-ACA酰化酶的氨基酸序列是序列表中的SEQ ID NO：1。其编码基因是序列表中的SEQ ID NO：2。
为了获得酶活性更高的CPC酰化酶突变体，本发明对野生型CPC酰化酶SEQ ID NO：1的基因序列SEQ ID NO：2进行点突变。通过易错PCR技术获得一个或多个氨基酸位点取代的突变体氨基酸序列，筛选出多个可提高CPC酰化酶的酶活的位点，包括240位缬氨酸(β肽第70位)、306位丙氨酸(β肽第136位)、553位精氨酸(β肽第383位)和623位组氨酸(β肽第453位)。然后以定点突变技术对上述位点进行随机组合突变，获得本发明中具有氨基酸序列SEQ ID NO：7-8的突变体。最后再在SEQ ID NO：8的基础上进行定点突变，获得本发明中具有氨基酸序列SEQ ID NO：9的突变体。
其中，SEQ ID NO：1是这些氨基酸序列SEQ ID NO：3-9的共同序列，这些氨基酸序列都是在SEQ ID NO：1的基础上进行1个、或2个、最多9个氨基酸的替换而获得的突变体，这些突变体氨基酸序列保持了98％以上的同源性。
在本发明中，术语“CPC酰化酶突变体”、“突变体CPC酰化酶”、“突变CPC酰化酶”和“突变酶”表示相同的意义，都是指头孢菌素C酰化酶的突变体。
在本发明中，术语“野生(型)”、“野生酶”、“野生型酶”表示相同的意义，都是指野生型的GL-7-ACA酰化酶或称CPC酰化酶(SEQ ID NO：1)。
本发明的CPC酰化酶突变体的氨基酸数量只有692个，且结构明确，因此本领域技术人员很容易获得其编码基因、包含这些基因的表达盒和质粒、以及包含该质粒的转化体。
这些基因、表达盒、质粒、转化体可以通过本领域技术人员所熟知的基因工程构建方式获得。
上述转化体宿主可以使任何适合表达CPC酰化酶的微生物，包括细菌和真菌。优选微生物是枯草芽孢杆菌、毕赤酵母、酿酒酵母、或者大肠杆菌，优选大肠杆菌，更优 选大肠杆菌BL21(DE3)。
当作为生物催化剂用于生产7-ACA时，本发明的CPC酰化酶可以呈现酶的形式或者菌体的形式。所述酶的形式包括游离酶、固定化酶，包括纯化酶、粗酶、发酵液、载体固定的酶等；所述菌体的形式包括存活菌体和死亡菌体。
本发明的CPC酰化酶分离纯化、包括固定化酶制备技术也是本领域技术人员所熟知的。
实施例
本文中的全基因合成、引物合成及测序委托苏州金唯智公司完成。
实施例1野生型CPC酰化酶基因重组大肠杆菌的构建
对于Pseudomonas sp.GK16来源的CPC酰化酶，以其已经公布的基因序列SEQ ID NO：2为基础(Matsuda et.al.，J.Bacteriol.163：1222-1228，1985)，全基因合成基因序列，并在基因两端设计限制性内切酶位点NdeI和XhoI，亚克隆到载体pET24a(Novagen)的相应位点，获得重组质粒pET24a-wt-CPC，转化表达宿主大肠杆菌BL21(DE3)，得到野生型CPC酰化酶的重组大肠杆菌。
实施例2易错PCR法构建随机突变点库及筛选
2.1易错PCR法构建随机突变点库
以CPC酰化酶野生型基因SEQ ID NO：2为模板，应用易错PCR技术构建随机突变体库。正向引物CPC-Nde-F为5’-CATATGGAGCCGACCTCGAC-3’，反向引物CPC-Xho-R为5’-CTCGAGTGGCTTGAAGTTGAAG-3’
50μL易错PCR反应体系包括：50ng质粒模板pET24a-wt-CPC，30pmol一对引物CPC-Nde-F和CPC-Xho-R，1X Taq buffer，0.2mM dGTP，0.2mM dATP，1mM dCTP，1mM dTTP，7mM MgCl2，(0mM、0.05mM、0.1mM、0.15mM、0.2mM)MnCl2，2.5个单位的Taq酶(fermentas)。PCR反应条件为：95℃ 5min；94℃ 30s，55℃ 30s，72℃ 2min/kbp；30个循环；72℃ 10min。胶回收2.0kb随机突变片段作为大引物，用KOD-plus DNA聚合酶做MegaPrimer PCR：94℃ 5min，；98℃ 10s，60℃ 30s，68℃2min/kbp，25个循环；68℃ 10min。DpnI消化质粒模板，电转化大肠杆菌E.coli BL21(DE3)，得到超过104个克隆的随机突变库。
2.2突变体库的高通量筛选
选取突变体库中的转化子接种到含700μL LB培养基的96孔深孔培养板中，培养基中含100μg/mL卡那霉素和0.1mM IPTG，37℃培养6h后，降温至25℃，培养过夜。5000rpm离心10min，弃上清，置于-70℃冷冻1h，室温融化30min。加入200μL含1mg/mL溶菌酶的0.1M磷酸钾盐缓冲液(pH8.0)，重悬菌体，37℃孵育1h，4℃，5000rpm离心20min，取20μL上清，用于CPC活力测定。
2.3高通量CPC酰化酶活力测定
底物反应液：含2wt％CPC钠盐的0.1M磷酸钾盐缓冲液(pH8.0)，
终止反应液：0.05M NaOH，20v/v％冰醋酸，
显色剂：含0.5wt％的PDAB(p-二甲氨基苯甲醛，p-Dimethyl Aminobenzaldehyde)甲醇溶液。
酶活力定义：每分钟催化CPC产生1微摩尔(μmol)7-ACA所需要的酶量定义为1个单位(U)。
将上述步骤2.2中的上清20μL加入20μL底物反应液，在37℃的条件下反应过夜，加入200μL终止反应液，然后5000rpm离心10min。取200μL离心上清，加入40μL显色液，室温反应10min后，检测415nm下的吸光度。
在随机突变库，通过对约30000个突变体克隆筛选，结果显示V240F、A306T、R553L、H623N这4个突变点能提高CPC酰化酶的酶活。
表2随机突变菌在37℃下的发酵液相对比活结果
菌种编号 突变位点 氨基酸序列号 发酵液相对比活
wtCPC —— 1 1.0
EP 1 V240F 3 2.8
EP 2 A306T 4 2.2
EP 3 R553L 5 4.0
EP 4 H623N 6 3.1
*wtCPC是指野生型头孢菌素C酰化酶的表达菌株。
实施例3通过定点突变技术进行定向进化
根据实例2中筛选出的V240F、A306T、R553L、H623N这4个位点，设计简并引物，以pET24a-wtCPC质粒为模板，构建定点组合突变库。然后通过定点突变技术，以组合突变库中筛选出的高活力菌株质粒为模板，加入I215V、F228V、Y323T、F347L、V552L 即β亚基的I45βV、F58βV、Y153βT、F177βL、V382βL五个突变点。构建过程中所用的引物见表3。
表3定向进化引物列表
Figure PCTCN2017074029-appb-000013
*其中：N＝A/G/C/T。
3.1通过定点突变技术构建定向进化突变库
以pET24a-wtCPC质粒为模板，以240-F1和306-R1、306-F2和553-R2、553-F3和623-R3三组引物对分别进行PCR扩增，通过over-lapping PCR扩增出大片段，然后以大片段为引物进行MegaPrimer PCR，构建定点组合突变库。
50μL PCR反应体系包括：10ng质粒模板，10pmol的引物对，1xKOD plus buffer，0.2mM dNTP，1.5mM MgSO4，5个单位的KOD-plus DNA聚合酶。
PCR反应条件为：95℃ 1min；98℃ 10s，57℃ 30s，68℃ 1min/kbp；30个循环； 68℃ 10min。胶回收三个片段P1、P2、P3。
以P1、P2、P3为模板，以240-F1和623-R3为引物进行第二轮PCR，获得片段P，切胶回收。
PCR反应条件为：95℃ 3min；98℃ 10s，60℃ 30s，68℃ 1min/kbp；25个循环；68℃ 10min。
以片段P作为大引物，用KOD-plus DNA聚合酶做MegaPrimer PCR：94℃ 5min，；98℃ 10s，60℃ 30s，68℃ 2min/kbp，25个循环；68℃ 10min。DpnI消化质粒模板，电转化大肠杆菌E.coli BL21(DE3)，得到超过3×104个克隆的随机突变库。
3.2突变体库的高通量筛选
方法同实施例2的步骤2.2。经筛选获得活力相对较高的ED2菌株，经测序确定该菌株含有V240F，A306T，R553L，H623T四个位点的突变。
3.3定点突变
以ED2菌株提取的质粒为模板，以45/58-F和45/58-R、153-F和153-R、177-F和177-R、382-F和382-R为引物，获得的最终产物经Dpn I消化后转化大肠杆菌E.coli BL21(DE3)。
50μL PCR反应体系包括：10ng质粒模板，10pmol的引物对，1xKOD plus buffer，0.2mM dNTP，1.5mM MgSO4，5个单位的KOD-plus DNA聚合酶。
PCR反应条件为：95℃ 1min；98℃ 10s，57℃ 30s，68℃ 1min/kbp；20个循环；68℃ 10min。
3.4高通量CPC酰化酶活力测定
底物反应液：含2wt％CPC钠盐的0.1M磷酸钾盐缓冲液(pH8.0)，
终止反应液：0.05M NaOH，20v/v％冰醋酸，
显色剂：含0.5wt％的PDAB(p-二甲氨基苯甲醛，p-Dimethyl Aminobenzaldehyde)甲醇溶液。
酶活力定义：每分钟催化CPC产生1微摩尔(μmol)7-ACA所需要的酶量定义为1个单位(U)。
将上述步骤2.2中的上清20μL加入20μL底物反应液，在37℃的条件下反应10min后，加入200μL终止反应液，然后5000rpm离心10min。取200μL离心上清，加入40μL显色液，室温反应10min后，检测415nm下的吸光度，与7-ACA定量标准曲线进行比较定量。
3.5摇瓶发酵
挑取单菌落，接种至5mL含50μg/mL硫酸卡那霉素的LB液体培养基中，37℃，250rpm 培养过夜。取2mL过夜培养物接种至200mL TB培养基中，37℃，250rpm培养2-3h，至OD6000.6-0.8时，加入0.1mM IPTG，28℃，200rpm培养过夜。4℃，10000rpm，离心10min，收集菌体。
3.6酶的提取
菌体用50mL平衡缓冲液(50mM磷酸钾盐缓冲液，200mM NaCl，pH8.0)重悬，然后超声破碎，破碎后的菌体4℃，12000rpm，离心20min，收集上清。上清以1mL/min的速率加入含10mL Ni-NAT基质的亲和层析柱中，然后用含有30mM咪唑的平衡缓冲液冲洗柱料，洗脱杂质。最后用含有500mM咪唑的平衡缓冲液冲洗脱目的蛋白，收集峰值洗脱液。
洗脱液经截留分子量为10kDa的超滤管进行脱盐处理，得纯酶。
3.7纯酶比活力测定
该步骤所用溶液与实施例2中步骤2.3所用试剂相同。
将步骤3.6中的脱盐溶液20μL加入20μL底物反应液，在37℃的条件下反应5min后，加入200μL终止反应液，然后5000rpm离心10min。取200μL离心上清，加入40μL显色液，室温反应10min后，检测415nm下的吸光度，与7-ACA定量标准曲线进行比较定量。
同时采用Thermo Scientific公司的BCA Protein Assay Kit试剂盒测定纯酶的蛋白浓度，从而获得纯酶的比活力。
表4定向进化的纯酶在37℃下的酶活力对比结果
Figure PCTCN2017074029-appb-000014
*wtCPC是指野生型头孢菌素C酰化酶的表达菌株。
由表4可以看出，相比野生型头孢菌素C酰化酶SEQ ID NO：1，本发明的头孢菌素C酰化酶突变体SEQ ID NO：7-9的酶活力提高了20.5倍至150倍，其中突变体SEQ ID NO：9的酶活力最高。
实施例4 7-ACA的生产
称取2.5g CPC钠盐，加水溶解，降温至15℃，调整pH至8.2，加入实施例3中步骤3.5制备的500U的突变体SEQ ID NO：9纯酶，搅拌反应。反应过程中控制温度15±0.5℃，pH8.0±0.2，反应40min，检测反应样品。
精确量取100μL反应40min后的样品，用水定容至10mL，进行HPLC分析，分析条件：C18 200mm×4.6柱，波长262nm，流动相为0.02M醋酸钠PH5.5∶乙腈(93∶7)，温度25℃。结果显示，反应中CPC钠盐的转化率超过98％。
综上所述，本发明构建了CPC酰化酶突变体，将野生型CPC酰化酶的比活提高了20.5-150倍，用突变体纯酶进行一步酶法生产7-ACA，反应40min，使CPC的转化率超过98％，具有广阔的工业化前景。

Claims (10)

  1. 一种头孢菌素C酰化酶，其氨基酸序列为：
    SEQ ID NO：3，其为SEQ ID NO：1第240位的V替换为F的突变体；
    SEQ ID NO：4，其为SEQ ID NO：1第306位的A替换为T的突变体；
    SEQ ID NO：5，其为SEQ ID NO：1第553位的R替换为L的突变体；
    SEQ ID NO：6，其为SEQ ID NO：1第623位的H替换为N的突变体；
    SEQ ID NO：7，其为SEQ ID NO：1第240位的V替换为F、第306位的A替换为T、第623位的H替换为T的突变体；
    SEQ ID NO：8，其为SEQ ID NO：1第240位的V替换为F、第306位的A替换为T、第553位的R替换为L、第623位的H替换为T的突变体；或者
    SEQ ID NO：9，其为SEQ ID NO：1第215位的I替换为V、第228位的F替换为V、第240位的V替换为F、第306位的A替换为T、第323位的Y替换为T、第347位的F替换为L、第552位的V替换为L、第553位的R替换为L、第623位的H替换为T的突变体。
  2. 如权利要求1所述头孢菌素C酰化酶，其特征在于，所述氨基酸序列为SEQ ID NO：9。
  3. 编码如权利要求1或2所述头孢菌素C酰化酶的基因。
  4. 编码如权利要求2所述头孢菌素C酰化酶的基因，其序列为SEQ ID NO：10。
  5. 包含如权利要求3或4所述基因的质粒。
  6. 转化了如权利要求5所述质粒的微生物。
  7. 如权利要求6所述的微生物，其特征在于，是所述微生物选自枯草芽孢杆菌、毕赤酵母、酿酒酵母、大肠杆菌。
  8. 如权利要求7所述的微生物，其特征在于，是所述微生物是大肠杆菌BL21(DE3)。
  9. 如权利要求1所述头孢菌素C酰化酶或者如权利要求7所述微生物在生产7-ACA中的用途。
  10. 如权利要求9所述的用途，其特征在于，以头孢菌素C为底物、用权利要求1所述头孢菌素C酰化酶或者如权利要求7所述微生物催化生产7-ACA。
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