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1.
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
2.
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
3.
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
4.
Qiang Tan Yewang Zhang Qingxun Song Dongzhi Wei 《World journal of microbiology & biotechnology》2010,26(1):145-152
In this study, d-amino acid oxidase (DAAO) and catalase (CAT) in the permeabilized recombinant Pichia pastori cells were well investigated. It appeared that their thermal stability was negatively correlated with the apparent enzymatic
activities. The frozen-melted cells presented the best stability and the lowest apparent activities of DAAO and CAT, whereas
the cetyltrimethylammonium bromide (CTAB) permeabilized cells displayed the weakest stability and the highest apparent activities
of the two enzymes. Simultaneous action of DAAO and CAT in the CTAB-permeabilized cells and glutaryl-7-aminocephalosporanic
acid acylase (GA) immobilized on carrier contributed to the conversion of cephalosporin C (CPC) to 7-aminocephalosporanic
acid (7-ACA) with a yield of 76.2%. During such a reaction cycle, no visible activity loss occurred at the immobilized GA,
whereas the loss rates of DAAO and CAT activities were about 0.029 and 1.13 U min−1, respectively. Nevertheless, this problem could be easily solved by continuous feeding of the new permeabilized cell suspension
at the rate of 6 ml h−1 to the reactor. Following such a fed-batch strategy, these permeabilized cells and the immobilized GA could be efficiently
reused for 6 and 15 reaction cycles, respectively, yielding around 76% 7-ACA at each reaction cycle. 相似文献
5.
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
6.
7.
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. 相似文献
8.
Hideyuki Suzuki Professor Chiaki Yamada Kyoko Kijima Sayaka Ishihara Kei Wada Keiichi Fukuyama Hidehiko Kumagai 《Biotechnology journal》2010,5(8):829-837
Semisynthetic cephalosporins, the best-selling antibiotics worldwide, are derived from 7-aminocephalosporanic acid (7-ACA). Currently, in the pharmaceutical industrie, 7-ACA is mainly produced from cephalosporin C by sequential application of D -amino acid oxidase and cephalosporin acylase. Here we study the potential of industrially amenable enzyme γ-glutamyltranspeptidase from Bacillus subtilis for 7-ACA production, since the wild-type γ-glutamyltranspeptidase of B. subtilis has inherent glutaryl-7-aminocephalosporanic acid acylase activity with a kcat value of 0.0485 s-1. Its activity has been enhanced by site directed and random mutagenesis. The kcat/Km value was increased to 3.41 s-1 mM-1 for a E423Y/E442Q/D445N mutant enzyme and the kcat value was increased to 0.508 s-1 for a D445G mutant enzyme. Consequently, the catalytic efficiency and the turnover rate were improved up to about 1000-fold and 10-fold, compared with the wildtype γ-glutamyltranspeptidase of B. subtilis. 相似文献
9.
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
10.
Velasco J Luis Adrio J Angel Moreno M Díez B Soler G Barredo JL 《Nature biotechnology》2000,18(8):857-861
Medically useful semisynthetic cephalosporins are made from 7-aminodeacetoxycephalosporanic acid (7-ADCA) or 7-aminocephalosporanic acid (7-ACA). Here we describe a new industrially amenable bioprocess for the production of the important intermediate 7-ADCA that can replace the expensive and environmentally unfriendly chemical method classically used. The method is based on the disruption and one-step replacement of the cefEF gene, encoding the bifunctional expandase/hydroxylase activity, of an actual industrial cephalosporin C production strain of Acremonium chrysogenum. Subsequent cloning and expression of the cefE gene from Streptomyces clavuligerus in A. chrysogenum yield recombinant strains producing high titers of deacetoxycephalosporin C (DAOC). Production level of DAOC is nearly equivalent (75-80%) to the total beta-lactams biosynthesized by the parental overproducing strain. DAOC deacylation is carried out by two final enzymatic bioconversions catalyzed by D-amino acid oxidase (DAO) and glutaryl acylase (GLA) yielding 7-ADCA. In contrast to the data reported for recombinant strains of Penicillium chrysogenum expressing ring expansion activity, no detectable contamination with other cephalosporin intermediates occurred. 相似文献
11.
A search was undertaken to screen microorganisms that produce an enzyme capable of deacylating glutaryl-7-aminocephalosporanic
acid to 7-aminocephalosporanic acid in soil samples. The screening was carried out by preparing enrichment cultures containing
glutaryl-7ACA and cephalosporin C as selective carbon sources. A non-β-lactam model compound, glutaryl-p-nitroanilide, was
synthesized as a substrate suitable for the rapid screening of microorganisms isolated from the enrichment cultures. Two isolates
exhibiting acylase activity, designated BY7.4 and BY8.1, were identified as strains ofPseudomonas species.Pseudomonas BY8.1 showed higher acylase activity toward Gl-7ACA thanPseudomonas BY7.4. Environmental conditions for the optimal acylase activity ofPseudomonas BY8.1 were shown to be pH 9 and 30°C. 相似文献
12.
The gene coding for the glutaryl 7-aminocephalosporanic acid (GL 7-ACA) acylase from Pseudomonas diminuta KAC-1 was cloned and expressed in Escherichia coli. The acylase gene was composed of 2160 base pairs and encoded a polypeptide of 720 amino acid residues. The E. coli BL21 carrying pET2, the plasmid construct for high expression of GL 7-ACA acylase gene, produced this enzyme at approx. 30% of the total proteins with 3.2 units activity mg protein–1. Growth at temperature below 31 °C and deletion of signal peptide increased the processing of precursor acylase to active enzyme in the recombinant E. coli cells. 相似文献
13.
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
14.
In this work, a glutaryl-7-aminocephalosporanic acid acylase (GLA) coding gene was cloned from Pseudomonas diminuta NK703 which was screened from oilfield. The concerted effects of the expression system, inducing condition and culture medium on the expression of NK703 GLA in E. coli were firstly investigated. The best combination was the recombinant E. coli strain of pET-28a + GLA/BL21(DE3) with 2.0% (w/v) lactose inducing in YT medium at 25 °C. Then, by optimizing the components of culture medium, a synthetic medium with dextrin and a feeding medium with glycerol as the carbon sources were developed to further enhance the GLA yield and improve the GLA solubility. In the end, the NK703 GLA activity increased about 50-fold, reached 14,470 ± 465 U/L, and the GLA productivity and the proportion of soluble GLA to the total soluble protein attained 206.0 ± 9.033 U L−1 h−1 and 60.13%, respectively. Scaling up the GLA production in 3.7 L fermenter under the optimized conditions identified in shake flask, the GLA activity also reached 12,406 ± 521 U/L, which was the highest report at fermenter level. 相似文献
15.
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
16.
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
17.
18.
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
19.
Vijay Chintaman Sonawane 《Critical reviews in biotechnology》2013,33(2):95-120
ABSTRACTSemisynthetic cephalosporins are important antibacterials in clinical practice. Semisynthetic cephalosporins are manufactured by derivatizing 7-aminocephalosporanic acid (7-ACA) and its desacetylated form. Microbial enzymes such as D-amino acid oxidase, glutaryl-7-ACA acylase and cephalosporin esterase are being used as biocatalysts for the conversion of cephalosporin C (CEPH-C) to 7-ACA and its desacetylated derivatives. Recent developments in the field of enzymatic modifications of cephalosporin with special emphasis on group of enzymes called as cephalosporin acylase is discussed in this review. Aspects related to screening methods, isolation and purification, immobilization, molecular cloning, gene structure and expression and protein engineering of cephalosporin acylases have been covered. Topics pertaining to enzymatic modifications of cephalosporin by D-amino acid oxidase, cephalosporin methoxylase and β -lactamase are also covered. 相似文献