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1.
B. S. Deshpande S. S. Ambedkar V. K. Sudhakaran J. G. Shewale 《World journal of microbiology & biotechnology》1994,10(2):129-138
-Lactam acylases such as penicillin G acylases, penicillin V acylases and glutaryl 7-aminocephalosporanic acid acylases are used in the manufacture of 6-aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid and 7-aminocephalosporanic acid (7-ACA). Genetically-engineered strains producing 1050 U/g, 3200 U/g and 7000 to 10,000 U/I of penicillin G acylase, penicillin V acylase and glutaryl-7-ACA acylase, respectively, have been developed. The penicillin G acylase studied to date and the glutaryl-7-ACA acylase from Pseudomonas sp. share some common features: the active enzyme molecules are composed of two dissimilar subunits that are generated from respective precursor polypeptide; the proteolytic processing is a post-translational modification which is regulated by temperature; and the Ser residue at the N-terminus of the -sub-unit (Ser290; penicillin G acylase numbering) is implicated as the active site residue. Protein engineering, to generate penicillin G acylase molecules and their precursors with altered sequences, and the structure-function correlation of the engineered molecules are discussed.The authors are with Research and Development, Hindustan Antibiotics Ltd, Pimpri, Pune 411 018, India; 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.
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
4.
Yoo-Seok Jeong Hyo-Jin Yoo Sang-Dal Kim Doo-Hyun Nam Yong-Ho Khang 《Biotechnology and Bioprocess Engineering》2005,10(6):510-515
Pseudomonas cepacia BY21 was found to produce glutaryl acylase that is capable of deacylating glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA)
to 7-aminocephalosporanic acid (7-ACA), which is a starting material for semi-synthetic cephalosporin antibiotics. Amino acids
of the reported glutaryl acylases from variousPseudomonas sp. strains show a high similarity (>93% identity). Thus, with the known nucleotide sequences ofPseudomonas glutaryl acylases in GenBank, PCR primers were designed to clone a glutaryl acylase gene fromP. cepacia BY21. The unknown β-subunit gene of glutaryl acylase from chromosomal DNA ofP. cepacia BY21 was cloned successfully by PCR. The β-subunit amino acids ofP. cepacia BY21 acylase (GenBank accession number AY948547) were similar to those ofPseudomonas diminuta KAC-1 acylase except that Asn408 ofP. diminuta KAC-1 acylase was changed to Leu408. 相似文献
5.
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
6.
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
7.
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
8.
Yong-Gang Zheng Yong Li Jian-Feng Chen Wei-Hong Jiang Guo-Ping Zhao En-Duo Wang 《Biotechnology letters》2001,23(21):1781-1787
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. 相似文献
9.
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
10.
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.
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
13.
14.
GL-7-ACA酰化酶发酵培养基的均匀优化设计 总被引:3,自引:0,他引:3
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。 相似文献
15.
Takimoto A Takakura T Tani H Yagi S Mitsushima K 《Applied microbiology and biotechnology》2004,65(3):263-267
Bacillus subtilis SHS0133 cephalosporin-C deacetylase (CAH) overexpressed in Escherichia coli was immobilized on an anion-exchange resin, KA-890, using glutaraldehyde. The activity yield of immobilized enzyme was approximately 55% of the free enzyme. The pH range for stability of the immobilized enzyme (pH 5–10) was broader than that for free enzyme. The Kmapp value of immobilized enzyme for 7-aminocephalosporanic acid (7-ACA) was similar to that of the free enzyme. This immobilized enzyme obeyed Michaelis–Menten kinetics similar to those of the free enzyme. A batch-type reactor with a water jacket was employed for deacetylation of 7-ACA using CAH immobilized on KA-890. Ten kilograms of 7-ACA were completely converted to deacetyl 7-ACA at pH 8.0 within 90 min. The reaction kinetics agreed well with a computer simulation model. Moreover, the immobilized enzyme exhibited only a slight loss of the initial activity even after repeated use (52 times ) over a period of 70 days. This reaction will thus be useful for the production of cephalosporin-type antibiotics. 相似文献
16.
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
17.
Xiangwei Zhu Hui Luo Yanhong Chang Houbo Su Qiang Li Huimin Yu Zhongyao Shen 《World journal of microbiology & biotechnology》2011,27(4):823-829
Cephalosporin C (CPC) acylase is an enzyme which hydrolyzes CPC to 7-aminocephalosporanic acid (7-ACA) directly, and therefore
has great potential in industrial application. In this study, the CPC acylase from a recombinant Escherichia coli was purified to high purity by immobilized metal affinity chromatography, and the CPC acylase was covalently attached to
three kinds of epoxy supports, BB-2, ES-V-1 and LX-1000EP. The immobilized CPC acylase with LX-1000EP as the support shows
the highest activity (81 U g−1) suggesting its potential in industrial 7-ACA production. The activity of immobilized enzyme was found to be optimal at pH
between 8.5 and 9.5 and to increase with temperature elevation until 55 °C. Immobilized CPC acylase showed good stability
at pH between 8.0 and 9.5 and at temperature up to 40 °C. To avoid product degradation, the production of 7-ACA utilizing
immobilized enzyme was carried out at 25 °C, pH 8.5 in a designed reactor. Under optimal reaction conditions, a very high
7-ACA yield of 96.7% was obtained within 60 min. In the results of repeated batch production of 7-ACA, 50% activity of the
initial cycle was maintained after being recycled 24 times and the average conversion rate of CPC reached 98%. 相似文献
18.
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
19.
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%. 相似文献
20.
Deacetoxycephalosporin C (DAOC) is produced by Acremonium chrysogenum as an intermediate compound in the cephalosporin C biosynthetic pathway, and is present in small quantities in cephalosporin
C fermentation broth. This compound forms an undesirable impurity, 7-aminodeacetoxycephalosporanic acid (7-ADCA), when the
cephalosporin C is converted chemically or enzymatically to 7-aminocephalosporanic acid (7-ACA). In the cephalosporin C biosynthetic
pathway of A. chrysogenum, the bifunctional expandase/hydroxylase enzyme catalyzes the conversion of penicillin N to DAOC and subsequently deacetylcephalosporin
C (DAC). By genetically engineering strains for increased copy number of the expandase/hydroxylase gene, we were able to reduce
the level of DAOC present in the fermentation broth to 50% of the control. CHEF gel electrophoresis and Southern analysis
of DNA from two of the transformants revealed that one copy of the transforming plasmid had integrated into chromosome VIII
(ie a heterologous site from the host expandase/hydroxylase gene situated on chromosome II). Northern analysis indicated that
the amount of transcribed expandase/hydroxylase mRNA in one of the transformants is increased approximately two-fold over
that in the untransformed host.
Received 5 January 1998/ Accepted in revised form 29 May 1998 相似文献