Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In many GL-7ACA acylases, the first Ser residue at the N-terminal of beta-subunit is the catalytic center. In order to investigate relationship between the N-terminal structure and catalytic activities, peptide replacement and site-directed mutagenesis were performed at the N-terminal of beta-subunit of GL-7ACA acylase C130. When the N-terminal 8 amino acid residues of C130 were replaced by the corresponding sequence of penicillin acylases PAC and PGA, respectively, the first mutant B8PAC lost the activity of the acylase, and the second mutant B8PGA had lower activity with the K(m) value increasing from 0.44x10(-3)mol.L(-1) to 0.55x10(-3) mol.L(-1), and the k(cat) decreasing from 4.92 s(-1) to 1.64 s(-1). Although the substitution of Trp (beta4) by Tyr did not change the K(m) value, the k (cat) decreased to 2.29 s(-1). When the Trp was substitued by Leu, both the K ( m ) and k ( cat ) values decreased. Compared with the wild type, mutations of Ser (beta3) to Met, Ala and Cys caused decrease of K(m) values by 52.27%, 43.18% and 38.64%, respectively. Mutation of Asn (beta2) to Gln caused the K ( m ) value being increased by 5-fold, and k ( cat ) decreased by 10-fold. These results suggested that the N-terminal amino acid residues of beta-subunit in GL-7ACA acylase C130 are important for enzyme function.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2001
PMID:Mutagenesis of N-terminal Amino Acid Residues in beta-subunit of Glutaryl-7-amino-cephalosporanic Acid Acylase C130. 1203 60

To study the possibility of oral gene therapy using live attenuated Salmonella, eukaryotic expression vectors EGFPN1, pLCDSN were introduced into a live attenuated AraA(-) auxotrophic mutant of Salmonella typhimurium (SL3261) and were administered orally to BALB/c and C57BL/6 mice. After six weeks, these mice were challenged with 4T(1) and Lewis cancer cells. Until the tumors reached to about 10 mm in diameter, 5-fluorocytosine was given through intraperitoneal injection. Flow cytometry, confocal microscopy and PCR methods were used to detect the integration and expression of the genes. The inhibition of the tumor and the survival time of the mice were also investigated. Results showed that cytosine deaminase gene integration could be detected in almost all kinds of mice tissue. And the GFP expression was much stronger in spleen and tumor than in other tissues. Cytosine deaminase/5-fluorocytosine system had significant antitumoractivities in vivo. The anti-tumor activities of cytosine deaminase/5-fluorocytosine at 500 mg/kg on 4T(1) and Lewis carcinoma in BALB/c and C57BL/6 mice were more potent than the efficiency of 5-fluorouracil 10 mg/kg(P 0.05). Therefor, this experiment demonstrates the potential value of live attenuated Salmonella as carrier for oral gene therapy.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2001
PMID:Treatment of Tumor in Mice by Oral Administration of Cytosine Deaminase Gene Carried in Live Attenuated Salmonella. 1205 Aug 17

To improve the stability of penicillin G acylase(PGA) from Bacillus megaterium, a three-dimensional model of B.megaterium PGA was constructed based on crystal structure of penicillin G acylase from E.coli using PMODELING program. The mutation of Lys at beta427 and 430 to Ala was predicted to enhance the stability of PGA in acidic or organic solvent environment. The results showed that 2 mutant PGA had similar specific activity and Km as the parent PGA. Their optimum pH dropped 0.5 pH units. The stability of Lys430Ala was enhanced obviously at pH 5.2. The half lives of Lys427Ala and Lys430Ala were improved by 60 % and 166 %, respectively, in comparison with the parent PGA.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2000
PMID:Enhancing Penicillin G Acylase Stability by Site-directed Mutagenesis. 1205 68

Glutaryl 7-amino cephalosporanic acid acylase (GL-7ACA acylase) from Pseudomonas sp.130 catalyzes hydrolysis of glutaryl 7-amino cephalosporanic acid to produce 7-amino cephalosporanic acid (7-ACA). 7-ACA is the starting material for the industrial production of most cephalosparonic derivatives. Six plasmids for expression of GL-7ACA acylase were constructed and these recombin ant plasmids presented different expression characteristics in Escherichia coli. The acylase gene from plasmid pKKCA1 was inserted into plasmid pMFT7-5 and the resulting plasmid pMFT7CA1 has higher expression in E.coli. The specific activity of the crude extract of the transformant JM109(DE3)/pMFT7CA1 was near 5 u/g, so the over produced enzyme was easily purified by a single-step anion exchange column chromatography. The enzyme could be purified by immobilized ion affinity chromatography after fused by 6xHis in the N-terminal of its alpha-subunit. Because plasmid pSMLCA1 brings tc(R) and p15A origin, it is special useful plasmid in fermentation. Two secretory expression plasmids, pSUCA1S and pETCA1pelB, could secrete the acylase to periplasmic space of bacteria. The whole cells containing the secretory expression plasmid may be used for production of 7-ACA directly.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002 Jul
PMID:Expression of gene encoding GL-7ACA acylase in Escherichia coli. 1209 81

An artificial strong hydrophobic signal peptide (ASP), containing ten leucines in tandem in the hydrophobic core, was utilized to replace the wild type signal peptide (WTSP) of penicillin G acylase (PAC), by the fusion to its 4 pro site. PAC expression plasmids, including pKKpacdeltaSP, pKKpacWTSP, pKKpacASP, pETpacWTSP and pETpacASP, were constructed. The length and the amino- and carboxyl-terminus amino acid composition of ASP and WTSP were kept identical. The activity assay and Western-blotting analysis were used to study the effect of ASP and WTSP on the secretion of PAC in tac, T7 and dissolved-oxygen regulation expression systems, respectively. Lack of signal peptide in pKKpacdeltaSP resulted in the accumulation of 91 kD PAC precursor (without signal peptide, but with the space peptide between alpha-subunit and beta-subunit) in the cytosol, indicating that the secretion of PAC depends on the signal peptide. In BL21(pKKpacASP) cells, the PAC activity and proprecursor (with signal peptide and space peptide) processing capacity were increased by about 54% and 38.5%, respectively, in comparison with BL21(pKKpacWTSP) cells. Compared with BL21(DE3) (pETpacWTSP), however, the PAC activity and proprecursor processing capacity in BL21(DE3) (pETpacASP) were enhanced by about 69% and 43.5%, respectively. The PAC activity expressed from pETpacASP was about 67% more than that from pETpacWTSP in the dissolved-oxygen-regulated expression system GJ100. Resulting from the strong hydrophobicity of ASP, therefore, the PAC activity and proprecursor processing capacity were increased by about 63% and 41% on average, respectively, in comparison with WTSP. In conclusion, the increase in hydrophobicity of the signal peptide hydrophobic core enhanced the secretion of penicillin G acylase.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2000
PMID:Increase in Hydrophobicity of Signal Peptide Enhances Secretion of Penicillin G Acylase. 1209 95

Site-directed mutagenesis and chemical modification were performed at Ser290 of the penicillin G acylase from E. coli ATCC11105. The Ser290 was substituted by Cys or Secys. Wild type and mutant proteins were purified, and the activities and kinetic constants of penicillin acylases for hydrolysis and synthesis were determined, respectively. Although their K(m) values were not changed, the k(cat) values of the thiol-PGA and seleno-PGA were decreased from 135s(-1) to 0.63s(-1) and 0.38s(-1) against NIPAB, and from 34.38s(-1) to 0.23s(-1) and 0.06s(-1) against penicillin G. Contrary to Choi's report(Choi K S (et al. J Bacteriology), 1992, 10 6270-6276), we found that hydrolysis activity was certainly kept in the mutant of penicillin acylase. In addition, the specific activities of synthesis were decreased by 5-fold and 20-fold, respectively.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 1999
PMID:Site-directed Mutagenesis of the Active Center of Penicillin Acylase from E. coli ATCC 11105. 1211 70

The penicillin G acylase gene (pga) amplified by PCR from Bacillus megaterium was subcloned into an expressing vector pPZW103 (P43 as promoter). The recombinant plasmid was transferred into Bacillus subtilis DB104. Penicillin G acylase production in the B. subtilis transformant was 3-6 u/ml, higher than that of published recombinant strains. Penicillin G acylase production was induced by phenylacetic acid in B. megaterium, whereas the enzyme was produced constitutively in the B. subtilis transformant carrying B. megaterium pga. The recombinant strain showed high stability in antibiotic-free medium for 10 days. Enzyme in crude broth was purified by Al(2)O(3) chromatography and phenyl-Sepharose CL-4B hydrophobic chromatography and the total yield is 79%. The purified enzyme with specific activity of 52 u/mg can be directly immobilized for use.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 1999
PMID:High Expression of Penicillin G Acylase Gene from Bacillus megaterium in Bacillus subtilis. 1211 80

The gene of beta-subunit of GL-7-ACA acylase [7beta-(4-carboxybutanamido) cephalosporanic acid acylase] was cloned into pTrc99B, an IPTG inducible pasmid, to form the recombinant called pTrc-CA1B. Another recombinant plasmid pTrcCA1S was obtained by cloning the gene encoding alpha-subunit of GL-7-ACA acylase, the signal peptide and the expression elements from Pseudomonas sp. into pTrcCA1B. Then, recombinant plasmid pKKCA1S was constructed by cloning the gene encoding the signal peptide, expression elements and GL-7-ACA acylase into the vector pKK235. pTrcCA1S and pKKCA1S were allowed to transform TG1. These two plamids were able to transfer the expression product into the periplasmic space of the host bacteria. As a result, in the whole cell of TG1/pTrcCAIS, the specific activity of GL-7-ACA acylase was 23.9 u/g cell, 8 fold higher than that of TG1/pMR24. And in the whole cell of TG1/pKKCA1S, the specific activity of acylase was 18.3 u/g cell, 6 fold higher than that of TG1/pMR24.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 1998
PMID:Secretory Expression of GL-7-ACA Acylase in E. coli. 1216 43

The pH-dependence in the catalytic reaction of recombinant penicillin G acylase and its mutants from B.megaterium has been studied by using kinetic methods. pK(1) and pK(2)of the residues of the wild type penicillin G a cylase, involved in the catalyzed reaction, were 5.50-5.87 and 10.73, respectively, from the curves of logV(m) and log(V(m)/K(m)) versus pH. Results showed tha t the pK(1) and pK(2) values of these residues of the mutants were similar to that of the wild type. pK(1) of 5.64-5.86 for mutant A and 5.69-6.96 for mutant B were obtained, while pK(2) was 10.61 and 10.48 for mutant A and B, respectively. At the same time, pK values at different temperatures were investigated. The ionization enthalpies(deltaH) were 44.38-59.03 kJ/mol and 147.37 kJ/mol, respectively, from th e curve of pK versus temperature. It was presumed according to the results mentioned above that the ionizing residues, involved in the reaction, wer e histidine and lysine that are localized around the active site.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2002 Nov
PMID:[The pH-dependent catalytic reaction of penicillin G acylase and its mutants]. 1241 25

The Alcaligenes faecalis PGA gene encoding heterodimeric penicillin G acylase (PGA) was cloned and successfully expressed in Escherichia coli and Bacillus subtilis respectively. In contrast to E.coli hosts where the enzymes were retained in the periplasm, B. subtilis cell secreted the recombinant enzyme into the medium. Contrary to limited expression yield of E. coli (pETAPGA), PGA extracellularly expressed by B. subtilis (pBAPGA) and B. subtilis (pMAPGA) reached the highest yield of 653 u/L. This yield increased 109-fold higher than the native expression of A. faecalis CICC AS1.767. The enzyme was fractionated with (NH(4))(2)SO(4) and purified by DEAE-Sepharose CL-6B with a yield of 81%. The purified enzyme had a specific activity of 1.469 u/mg. Furthermore, some enzyme characteristics, such as the pH and temperature optimum, the stability against organic solvent and the ratio of cepholexin synthesis to hydrolysis were determined. By overexpressing A. faecalis PGA in B. subtilis and purifying secreted enzyme from culture medium one could readily obtain a large amount of an alternative source of PGA.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 2003 May
PMID:Purification and characterization of Alcaligenes faecalis penicillin G acylase expressed in Bacillus subtilis. 1276 1


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