GL-7ACA酰化酶表达检测系统的建立

戊二酰－7－氨基头孢烷酸（GL－7ACA）酰化酶能够催化GL－7ACA分解生成7－ACA,后者是工业半合成生产头孢类抗菌素所需的重要前体。为了准确地检测GL－7ACA酰化酶及其突变体的表达,本研究通过构建一系列质粒载体,建立了两个简便有效地测定GL－7ACA酰化酶基因acy表达量的系统,从而可对酶的比活力进行定量。我们将两个报告基因,即儿茶酚双加氧酶基因（xylE)和β－半乳糖苷酶基因（lacZ)分别置于acy基因的下游,使之与acy基因共用一个启动子,进行串联表达,各自构成一个多顺反子系统。实验证明,基因融合后的儿茶酚双加氧酶或β－半乳糖苷酶的活力可以间接反映acy的表达量。     

In vivo post-translational processing and subunit reconstitution of cephalosporin acylase from Pseudomonas sp. 130.

Cephalosporin acylases are a group of enzymes that hydrolyze cephalosporin C (CPC) and/or glutaryl 7-amino cephalosporanic acid (GL-7ACA) to produce 7-amino cephalosporanic acid (7-ACA). The acylase from Pseudomonas sp. 130 (CA-130) is highly active on GL-7ACA and glutaryl 7-aminodesacetoxycephalosporanic acid (GL-7ADCA), but much less active on CPC and penicillin G. The gene encoding the enzyme is expressed as a precursor polypeptide consisting of a signal peptide followed by alpha- and beta-subunits, which are separated by a spacer peptide. Removing the signal peptide has little effect on precursor processing or enzyme activity. Substitution of the first residue of the beta-subunit, Ser, results in a complete loss of enzyme activity, and substitution of the last residue of the spacer, Gly, leads to an inactive and unprocessed precursor. The precursor is supposed to be processed autocatalytically, probably intramolecularly. The two subunits of the acylase, which separately are inactive, can generate enzyme activity when coexpressed in Escherichia coli. Data on this and other related acylases indicate that the cephalosporin acylases may belong to a novel class of enzymes (N-terminal nucleophile hydrolases) described recently.     

Thermal and Operational Characteristics of Glutaryl-7-aminocephalosporanic acid Acylase Immobilized on Silica Gel Modified by Epoxide Silanization

Summary In this study, an investigation was performed into the thermal and operational characteristics of glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase (EC 3.5.1.-) immobilized on silica gel that had been modified by epoxide silanization. The pH values for the optimum activity of free and immobilized GL-7-ACA acylase were almost the same. However, the pH-dependent activity profile for the immobilized GL-7-ACA acylase is considerably expanded. Both free and immobilized enzymes generally had the highest activity at 50 °C. In thermodynamic studies, it was found that immobilization using epoxide silanization made GL-7-ACA acylase thermodynamically stable. In the results of repeated batch production of 7-ACA, 89.0 and 83.5% of the 7-ACA produced at the initial cycle were maintained after 20 times of recycle at 25 °C and 30 °C, respectively. Hence it was suggested that mass production of 7-ACA at 25 °C using immobilized GL-7-ACA acylase by epoxide silanization would be possible on a large scale.     

Affinity alkylation of the Trp-B4 residue of the beta -subunit of the glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130.

Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the beta-subunit proven by comparative matrix assisted laser desorption/ionization-time of flight mass spectrometry. Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the beta-subunit, Trp-B4, was alkylated by BA-7-ACA. By (1)H-(13)C HSQC spectroscopy of C130 labeled by BA-2-(13)C-7-ACA, it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond. Replacing Trp-B4 with other amino acid residues caused increases in K(m), decreases in k(cat), and instability of enzyme activity. None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA. Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y. Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself. The Trp-B4 residue is located in the middle of a characteristic alphabetabetaalpha sandwich structure. Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis.     

2-Nitro-5-(6-bromohexanoylamino)benzoic acid test paper method for detecting microorganisms capable of producing cephalosporin acylases.

A novel method for detecting microorganisms capable of producing cephalosporin C (CPC) acylase and/or 7-(4-carboxybutanamido)cephalosporanic acid (GL-7-ACA) acylase has been developed. The method is based on the degradation of 2-nitro-5-(6-bromohexanoylamino)benzoic acid (NBHAB), a chromogenic substrate, into yellow 2-nitro-5-aminobenzoic acid by the action of the CPC acylase or the GL-7-ACA acylase. This method is very sensitive and quite specific, and has been successfully applied to screen the acylases from a variety of bacteria. A large number of colonies isolated on a plate surface from more than 67 samples and several known bacteria were tested by the NBHAB paper. Five NBHAB-positive strains and isolates were obtained. They were further examined by the reaction of their bacterial cells upon CPC and GL-7-ACA, respectively, and by thin-layer chromatography in order to distinguish the CPC acylase from the GL-7-ACA acylase.     

假单胞菌sp.130 GL-7-ACA酰化酶的核苷酸和蛋白质序列分析

对来源于假单胞菌sp.130的戊二酰-7-氨基头孢烷酸（GL－7－ACA）酰化酶结构基因的全序列及所编码蛋白质的α,β亚基的N末端和C末端的氨基酸序列进行了测定。将蛋白质序列与其他同类的GL－7－ACA酰化酶进行了同源性比较,结果显示该酶与来源于假单胞菌GK16和C427的酰化酶的序列有较高同源性,而与其它同类酰化酶的同源性较低。这些酶的α亚基N－末端差别较大,但是β－亚基的N－末端有较高的保守性。     

Cloning and co-expression of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli

To convert cephalosporin C to 7-aminocephalosporin (7-ACA), a D-amino acid oxidase (DAAO) gene from Trigonopsis variabilis and a glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) gene from Pseudomonas were cloned and expressed in recombinant Escherichia coli. For DAAO recombinant strain BL21(DE3)/pET-DAAO, a high DAAO activity of 250 U ml⁻¹ was obtained by a fed-batch culture. A GL-7-ACA acylase gene, in which the signal peptide sequence was deleted, was also successfully expressed in a recombinant E. coli BL21(DE3)/pET-ACY with a high expression level of 3000 U l⁻¹. A novel recombinant strain, BL21(DE3)/pET-DA, harboring both genes of DAAO and GL-7-ACA acylase, was further constructed, and a rather high DAAO activity of 140 U ml⁻¹ and GL-7-ACA acylase activity of 950 U l⁻¹ were simultaneously obtained. This recombinant strain, in which two genes are co-expressed, made it possible to catalyze cephalosporin C into 7-ACA directly.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.     

Enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid. Part II: Single-step procedure using a coimmobilized enzyme system

The enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid (7-ACA) using coimmobilized -aminoacid oxidase (DAAO) and 7-β-(4-carboxybutanamido)cephalosporanic acid acylase (Gl-7-ACA acylase) is reported. The results from the coimmobilization of the two enzymes on different carriers and at different ratios of enzyme activities are described. When an inhibitor of catalase activity, such as NaN₃ or H₂O₂, is present, the conversion rate to 7-ACA is higher, but more by-products are obtained. An optimum ratio of 60:1 between the enzymatic activities of DAAO and Gl-7-ACA acylase in the coimmobilized sample at 0.21 Ug⁻¹ Gl-7-ACA acylase activity was determined. The results of using coimmobilized enzymes and of using a mixture of separately immobilized enzymes in the same process are compared.     

头孢菌素酰化酶

7-氨基头孢烷酸(7-amino cephalosporanic acid, 7-ACA)是医药工业合成大多数头孢菌素的重要原料.头孢菌素酰化酶(cephalosporin acylase, CA)催化头孢菌素C(CPC)和戊二酰-7-氨基头孢烷酸(GL-7ACA)的水解反应, 生成7-ACA.根据CA催化底物的不同, 可将其划分为两类：CPC酰化酶和GL-7ACA酰化酶.由CA的同源性、分子质量大小和基因结构, 可以把头孢菌素酰化酶划分为五种;讨论了酶的基本性质.通过CA与N端亲核水解酶(Ntn水解酶)的比较, 推测CA属于Ntn水解酶, 并由此可以进一步理解它们的生理功能.     

Enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid. Part I: Cultivation of Pseudomonas syringae and partial purification and immobilization of 7-β-(4-carboxybutanamido) cephalosporanic acid acylase

The enzymatic transformation of 7-β-(4-carboxybutanamido)cephalosporanic acid (Gl-7-ACA) to 7-amino-cephalosporanic acid (7-ACA) is reported. The optimum conditions for cultivation of the producer strain Pseudomonas syringae, as well as the procedures for isolation, purification, and immobilization of the enzyme Gl-7-ACA acylase, are described. It is shown that when glutaraldehyde is used for immobilization of this enzyme, the yield of immobilization is low. After six hydrolyses of Gl-7-ACA to 7-ACA, the immobilized enzyme activity loss is less than 10%.     

产GL-7-ACA酰化酶重组菌的构建及高表达研究

戊二酰基-7-氨基头孢烷酸(GL-7-ACA)酰化酶是7-氨基头孢烷酸(7-ACA)两步酶法生产中的关键酶。成功构建组成型表达的产GL-7-ACA酰化酶重组大肠杆菌JM105/pMKC-ACY,并对其高表达条件进行了研究,得到了组成简单、廉价的国产培养基配方及操作简便、易于实现工业化的发酵工艺。在优化条件下,上罐补料高密度发酵的酶活高达6668.9U/L,是优化前的12.4倍,产率最高可达275.5U/(L.h),达到了工业生产的要求。     

Bioconversion of cephalosporin C with D-amino acid oxidase from the yeastRhodosporidium toruloides

Summary A D-amino acid oxidase-producing yeast,Rhodosporidium toruloides CCRC 20306, was used to convert cephalosporin C (Ceph C) into -ketoadipyl cephalosporanic acid. A part of Ceph C could be directly converted into glutaryl-7-aminocephalosporanic acid (GL-7-ACA) by permeated cells of CCRC 20306. There were unknown side products formed during the conversion. The side products could be substantially reduced in amount by heating the cell extract containing D-alanine at 55°C for 5 min for five cycles prior to the conversion. Esterase activity present in the cell extract, which might be one of the causes of the side reaction, was greatly diminished by heat treatment.     

Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues

Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), mainly by environmentally toxic chemical deacylation of cephalosporin C (CPC). Thus, the enzymatic conversion of CPC to 7-ACA by cephalosporin acylase (CA) would be very interesting. However, CAs use glutaryl-7-ACA (GL-7-ACA) as a primary substrate and the enzymes have low turnover rates for CPC. The active-site residues of a CA were mutagenized to various residues to increase the deacylation activity of CPC, based on the active-site conformation of the CA structure. The aim was to generate sterically favored conformation of the active-site to accommodate the D-alpha-aminoadipyl moiety of CPC, the side-chain moiety that corresponds to the glutaryl moiety of GL-7-ACA. A triple mutant of the CA, Q50betaM/Y149alphaK/F177betaG, showed the greatest improvement of deacylation activity to CPC up to 790% of the wild-type. Our current study is an efficient method for improving the deacylation activity to CPC by employing the structure-based repetitive saturation mutagenesis.     

Screening and characterization of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp.

Summary Three screening methods were used to isolate GL-7-ACA acylase-producing strains. Three positive isolates were identified with Pseudomonas nitroreducens CCRC 11041 possessing the highest activity, against GL-7-ACA and GL-7-ADCA. No activity was detected when Ceph C or succinyl-7-ACA was used as substrate; glutaric acid was found to be inhibitory. CCRC 11041 could produce maximal GL-7-ACA acylase activity when cultivated on meat extract medium II. The enzyme had a pH optimum of 5.0 and a temperature optimum of 42°C.     

三角酵母整细胞酶促转化头孢菌素C为戊二酰—7—氨基头孢烷酸

研究了利用含D－氨基酸氧化酶(D-amino acid oxidase,DAO EC1.4.3.3）的透性化三角酶母多倍体FA10（Trigonopsis variabilis FA10）细胞酶促转化头孢菌素C（cephalosporin C,CPC）为戊二酰－7－氨基头孢烷酸（Glutaryl-7-ACA,GL-7ACA）的反应过程和细胞中同时存在的过氧化氢酶（Catalase,CAT）通过水解H2O2而对转化反应产生的干扰作用及其对策。实验证明适量添加外源H2O2（6％）或在反应体系中加入过氧化氢酶抑制剂NaN3(0.13mg/mL )可使GL－7ACA生成率分别为73.0％和70.1％。如果将透性化的FA10细胞在pH10.5-11.0,20℃条件下保温30min,CAT被不可逆性完全钝化,以无过氧化氢酶的FA10细胞进行CPC的酶促转化反应GL－7ACA的生成率可达84％。     

Isolation and characterization of a novel soil strain, Pseudomonas cepacia BY21, with glutaryl-7-aminocephalosporanic acid acylase activity

A search was undertaken to screen microorganisms in soil which produce an enzyme capable of deacylating glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) to 7-aminocephalosporanic acid (7-ACA). To facilitate screening, a model substrate, glutaryl-p-nitroanilide, and a 7-ACA sensitive strain, Enterobacter taylorae BY312, were used as a color indicator and bioassay, respectively. An isolate, Pseudomonas cepacia BY21, was found to produce glutaryl-7-ACA acylase, of which the activity was optimal at pH 8.0 and 45°C.     

Two novel engineered bacteria for secretory expression of glutaryl 7-amino-cephalosporanic acid acylase

Two novel engineered bacteria, BL21(DE3)/pETCA1S and TG1/pSuperCA1S, were obtained which can secretory express the gene encoding glutaryl 7-amino-cephalosporanic acid acylase (GL-7ACA acylase) from Pseudomonas sp. 130 with high activity. The growth conditions of transformants for overproduction of GL-7ACA acylase were optimized: in intact cells of BL21(DE3)/pETCA1S and TG1/pSuperCA1S the activity of GL-7ACA acylase was 415 and 600 units g^–1 dry cells, respectively. The highest specific activity of GL-7-ACA acylase is in the intact cell as compared with that of transformants constructed in our laboratory. In fiftieth generation of mutants transferred on agar plates the specific activity of GL-7ACA acylase remained constant.     

GL-7-ACA酰化酶发酵培养基的均匀优化设计

采用国产原料,应用均匀设计优选试验方法,对GL-7-ACA酰化酶生产用的发酵培养基配方进行了优化,取得了良好的效果,最终摇瓶效价达3919．03U／L。     

Morphological,physiological and enzymatic characteristics of cephalosporin acylase-producing Arthrobacter strain 45-8A

A bacterial strain producing cephalosporin acylases was isolated from soil. The morphological and physiological properties of this strain suggest that it belongs to the genus Arthrobacter, and the isolate was therefore designated Arthrobacter strain 45-8A. Substrate specificity of the enzyme was examined. The enzyme can convert both cephalosporin C and 7-(4-carboxylbutan-amino)cephalosporanic acid to 7-aminocephalosporanic acid. An interesting feature of the acylases is their temperature-dependent regulation. Activity of acylases was detected in strain 45-8A grown at temperature below 30 °C, but was not observed at higher temperature. Arthrobacter strain 45-8A did not exhibit -lactamase activity, even though its resistance to cephalosporin C was very strong (>2000 g/ml). This is quite beneficial for its application in the manufacture of 7-aminocephalosporanic acd.Abbreviations used NBHAB 2-Nitro-5-(6-bromohexanoylamino)-benzoic acid - NIPAB 2-Nitro-5-phenylacetaminobenzoic acid - CPC cephalosporin C - GL-7ACA 7-(4-carboxybutanamino)cephalosporanic acid - 6-APA aminopenicillanic acid - 7-ACA 7-aminocephalosporanic acid - PDAB p-Dimethylaminobenzaldehyde