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1.
Glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 catalyzes the cleavage of the amide bond in the GL-7-ACA side chain to produce glutaric acid and 7-aminocephalosporanic acid (7-ACA). The active enzyme is an (alphabeta)(2) heterotetramer of two non-identical subunits that are cleaved autoproteolytically from an enzymatically inactive precursor polypeptide. In this study, we prepared and characterized a chemically modified enzyme, and also examined an effect of the modification on enzyme catalysis and autocatalytic processing of the enzyme precursor. We found that treatment of the enzyme with cyanate ion led to a significant loss of the enzyme activity. Structural and functional analyses of the modified enzyme showed that carbamylation of the free alpha-amino group of the N-terminal Ser-199 of the beta subunit resulted in the loss of the enzyme activity. The pH dependence of the kinetic parameters indicates that a single ionizing group is involved in enzyme catalysis with pK(a) = 6.0, which could be attributed to the alpha-amino group of the N-terminal Ser-199. The carbamylation also inhibited the secondary processing of the enzyme precursor, suggesting a possible role of the alpha-amino group for the reaction. Mutagenesis of the invariant N-terminal residue Ser-199 confirmed the key function of its side chain hydroxyl group in both enzyme catalysis and autoproteolytic activation. Partial activity and correct processing of a mutant S199T were in agreement with the general mechanism of N-terminal nucleophile hydrolases. Our results indicate that GL-7-ACA acylase utilizes as a nucleophile Ser-199 in both enzyme activity and autocatalytic processing and most importantly its own alpha-amino group of the Ser-199 as a general base catalyst for the activation of the hydroxyl group both in enzyme catalysis and in the secondary cleavage of the enzyme precursor. All of the data also imply that GL-7-ACA acylase is a member of a novel class of N-terminal nucleophile hydrolases that have a single catalytic center for enzyme catalysis. 相似文献
2.
Yamada C Kijima K Ishihara S Miwa C Wada K Okada T Fukuyama K Kumagai H Suzuki H 《Applied and environmental microbiology》2008,74(11):3400-3409
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. 相似文献
3.
Jung Soo Lim Seung Won Park Seung Wook Kim 《World journal of microbiology & biotechnology》2006,22(1):39-44
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. 相似文献
4.
Oh B Kim M Yoon J Chung K Shin Y Lee D Kim Y 《Biochemical and biophysical research communications》2003,310(1):19-27
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. 相似文献
5.
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. 相似文献
6.
7.
Zhang W Huang X Zhao G Jiang W 《Biochemical and biophysical research communications》2004,313(3):555-558
7Beta-bromoacetyl amino cephalosporanic acid (BA-7-ACA), an analog of glutaryl-7-amino cephalosporanic acid (GL-7-ACA), can inhibit and specifically alkylate GL-7-ACA acylase (C130) from Pseudomonas sp.130, forming a carbon-carbon bond between BA-7-ACA and the C-2 on indole ring of Trp-beta4 residue of C130. Here we reported that BA-7-ACA labeled C130 (BA-C130) could self-catalyze the hydrolysis of BA-7-ACA during crystallization process. The hydrolysis was confirmed to be a reaction analogous to the one of GL-7-ACA by comparative matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry analysis. BA-C130 was inactive at room temperature, but in the process of crystallization at 18 degrees C it catalyzed the hydrolysis of BA-7-ACA, and thus made the latter become a substrate. Meanwhile, in crystals, 7-ACA was released but the acetic acid still bound with Trp-beta4, and as a result, the enzyme remained to be inactive. These results demonstrated that Trp-beta4 in the alphabetabetaalpha motif was critical and sensitive for the activity of C130 and also suggested that there was a conformational change induced by deacylation during the process of crystallization. 相似文献
8.
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. 相似文献
9.
Isoai A Kimura H Reichert A Schörgendorfer K Nikaido K Tohda H Giga-Hama Y Mutoh N Kumagai H 《Biotechnology and bioengineering》2002,80(1):22-32
The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe. A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to alpha-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Biocatalysts were prepared using ASP327-10 and T. variabilis, and evaluated to demonstrate the feasibility of recombinant S. pombe for industrial application. The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) fixation and permeabilization by alkaline treatment. Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T. variabilis decreased to 20-30%. Heat treatment was required before cell fixation by GDA to inactivate the catalase in S. pombe. This improved the efficiency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S. pombe. To overcome this weakness, a catalase-deficient host strain was obtained by ethylmethansulfate mutagenesis. Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S. pombe even in the absence of G418. The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required. The obtained integrant and its biocatalyst were significantly improved in GL-7ACA conversion ability and mechanical strength. This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme. 相似文献
10.
Two-step immobilized enzyme conversion of cephalosporin C to 7-aminocephalosporanic acid 总被引:10,自引:0,他引:10
Conlon HD Baqai J Baker K Shen YQ Wong BL Noiles R Rausch CW 《Biotechnology and bioengineering》1995,46(6):510-513
The first large-scale production of 7-aminocephalosporanic acid (7ACA) from cephalosporin C (CPC) using a wholly enzymatic synthesis method is reported here. We produced 7ACA from CPC in as high a molar yield as 85% using the immobilized enzymes D-amino acid oxidase (D-AOD) and glutaryl-7-ACA acylase (GL-acylase). In the first reactor, CPC is converted to keto-adipyl-7-aminocephalosporanic acid (keto-7ACA) using an immobilized D-AOD isolated from a yeast, Trigonopsis variabilis. The keto-7ACA is then spontaneously converted to glutaryl-7-aminocephalosporanic acid (GL-7ACA) via a chemical reaction with hydrogen peroxide. The hydrogen peroxide is also a product of the D-AOD reaction. Near quantitative conversion of the keto-7ACA to GL-7ACA was observed. The second reactor converts GL-7ACA to 7ACA using an immobilized GL-acylase, which was isolated from a reconbinant Escherichia coli. The final 7ACA crystalline product is a high quality product. The reactions are conducted under very mild aqueous conditions: pH 8.0 and 20 degrees to 25 degrees C. The production of desacetyl side products is minimal. This process is currently being implemented on an industrial scale to produce 7ACA. (c) 1995 John Wiley & Sons, Inc. 相似文献
11.
Zheng H Zhu T Chen J Zhao Y Jiang W Zhao G Yang S Yang Y 《Journal of biotechnology》2007,129(3):400-405
The main drawback in the industrial production of 7-aminocephalosporanic acid is the accumulation of intermediate (AKA-7-ACA) and destruction of substrate (cephalosporin C) catalyzed by catalase and beta-lactamase. To overcome the adverse effect of these enzymes on the conversion process, Escherichia coli D11 with mutation of katG, katE and ampC genes was constructed by P1 phage transduction, which enabled it not to produce catalase and beta-lactamase, respectively. At the same time, recA mutation in D11 increased the stability of foreign plasmid. With D11 used as host, both d-amino acid oxidase and GL-7-ACA acylase were cloned and expressed by the recombinant plasmids of pMSS or pMSTO, and the production of two enzymes could be increased by addition of 1.0% glucose. Cells of recombinant strain D11/pMSTO could directly convert cephalosporin C into 7-aminocephalosporanic acid at 25 degrees C, with the yield of more than 74%. The data suggested that the constructed D11/pMSTO could be an alternative catalyst for production of 7-aminocephalosporanic acid in one pot. 相似文献
12.
Oh B Kim K Park J Yoon J Han D Kim Y 《Biochemical and biophysical research communications》2004,319(2):486-492
The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively. Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high. However, despite this structural conservation, they catalyze very different substrates. PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA). These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues. In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA. This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA. 相似文献
13.
Isolation and characterization of a novel soil strain, Pseudomonas cepacia BY21, with glutaryl-7-aminocephalosporanic acid acylase activity 总被引:1,自引:0,他引:1
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. 相似文献
14.
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 总被引:2,自引:0,他引:2
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%。 相似文献
15.
A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (GA), one of the two enzymes involved in the biotransformation of cephalosporin C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K(m) value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be optimal at pH between 7 and 9.5 and to increase with temperature and in buffered solutions. To avoid product degradation, optimal reaction conditions were obtained working at 25 degrees C using a 50-mM phosphate buffer, pH 8.0. Immobilized GA showed good stability at pH value below 9 and at temperature up to 30 degrees C. The inactivation of immobilized GA in the presence of different amounts of H(2)O(2), a side product that might be present in the plant-scale industrial solutions of glutaryl-7-ACA, was also investigated, but the deactivation rates were negligible at H(2)O(2) concentration that might be reached under operative conditions. Finally, biocatalyst performance in the complete two-step enzymatic conversion process from CefC to 7-ACA was determined on a laboratory scale. Following the complete conversion of a 75 mM solution of CefC into glutaryl-7-ACA catalyzed by an immobilized D-amino acid oxidase (DAAO), immobilized GA was used for the transformation of this intermediate into the final product 7-ACA. This reaction was repeated for 42 cycles. An estimation of the residual activity of the biocatalyst showed that 50% inactivation of immobilized GA was reached after approximately 300 cycles, corresponding to an enzyme consumption of 0.4 kU per kg of isolated 7-ACA. 相似文献
16.
Two-Step Autocatalytic Processing of the Glutaryl 7-Aminocephalosporanic Acid Acylase from Pseudomonas sp. Strain GK16
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The glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 is an (αβ)2 heterotetramer of two nonidentical subunits. These subunits are derived from nascent polypeptides that are cleaved proteolytically between Gly198 and Ser199 after the nascent polypeptides have been translocated into the periplasm. The activation mechanism of the GL-7-ACA acylase has been analyzed by both in vivo and in vitro expression studies, site-directed mutagenesis, in vitro renaturation of inactive enzyme precursors, and enzyme reconstitution. An active enzyme complex was found in the cytoplasm when its translocation into the periplasm was suppressed. In addition, the in vitro-expressed GL-7-ACA acylase was processed into α and β subunits, and the inactive enzyme aggregate of the precursor was also processed and became active during the renaturation step. Mutation of Ser199 to Cys199 and enzyme reconstitution allowed us to identify the secondary processing site that resides in the α subunit and to show that Ser199 of the β subunit is essential for these two sequential processing steps. Mass spectrometry clearly indicated that the secondary processing occurs at Gly189-Asp190. All of the data suggest that the enzyme is activated through a two-step autocatalytic process upon folding: the first step is an intramolecular cleavage of the precursor between Gly198 and Ser199 for generation of the α subunit, containing the spacer peptide, and the β subunit; the second is an intermolecular event, which is catalyzed by the N-terminal Ser (Ser199) of the β subunit and results in a further cleavage and the removal of the spacer peptide (Asp190 to Gly198). 相似文献
17.
Xi Huang Rong Zeng Xiaoming Ding Xiang Mao Yi Ding Zihe Rao Yong Xie Weihong Jiang Guoping Zhao 《The Journal of biological chemistry》2002,277(12):10256-10264
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. 相似文献
18.
Loredano Pollegioni Elena Rosini Gianluca Molla 《Applied microbiology and biotechnology》2013,97(6):2341-2355
Cephalosporins currently constitute the most widely prescribed class of antibiotics and are used to treat diseases caused by both Gram-positive and Gram-negative bacteria. Cephalosporins contain a 7-aminocephalosporanic acid (7-ACA) nucleus which is derived from cephalosporin C (CephC). The 7-ACA nucleus is not sufficiently potent for clinical use; however, a series of highly effective antibiotic agents could be produced by modifying the side chains linked to the 7-ACA nucleus. The industrial production of higher-generation semi-synthetic cephalosporins starts from 7-ACA, which is obtained by deacylation of the naturally occurring antibiotic CephC. CephC can be converted to 7-ACA either chemically or enzymatically using d-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase. Both these methods show limitation, including the production of toxic waste products (chemical process) and the expense (the enzymatic one). In order to circumvent these problems, attempts have been undertaken to design a single-step means of enzymatically converting CephC to 7-ACA in the course of the past 10 years. The most suitable approach is represented by engineering the activity of a known glutaryl-7-aminocephalosporanic acid acylase such that it will bind and deacylate CephC more preferentially over glutaryl-7-aminocephalosporanic acid. Here, we describe the state of the art in the production of an effective and specific CephC acylase. 相似文献
19.
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. 相似文献
20.
GL-7ACA酰化酶表达检测系统的建立 总被引:1,自引:0,他引:1
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。 相似文献