Isolation and characterization of soil strains producing glutaryl-7-aminocephalosporanic acid acylase

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.     

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.     

Enhancement of glutaryl-7-aminocephalosporanic acid acylase activity of γ-glutamyltranspeptidase of Bacillus subtilis

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 k_cat value of 0.0485 s^-1. Its activity has been enhanced by site directed and random mutagenesis. The k_cat/K_m 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.     

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.     

Simultaneous expression of glutaryl-7-aminocephalosporanic acid acylase gene and lysis genes of phage λ in a recombinant E. coli

Glutaryl-7-aminocephalosporanic acid acylase (GLA), recommended for use in the form of immobilized-enzyme, is one of the two key enzymes in the two-step synthesis of 7-aminocephalosporanic acid. For simplifying the process of cell disruption and immobilization, the lysis genes of phage λ (S⁻RRz) with the S amber mutation were designed to introduce into the over-expression system of GLA. A novel recombinant strain, E. coli TB1/pMKC-AS, simultaneously containing the maltose binding protein gene (malE), the lysis genes (S⁻RRz) and the target GLA gene (Acy) in a same operon, was successfully constructed. Under neutral pH conditions, cell growth and GLA activity of TB1/pMKC-AS was not affected by the presence of the lysis genes, however, autolysis phenomenon was observed under weak alkaline conditions. Through pH control and fed-batch culture, the GLA activity of TB1/pMKC-AS reached as high as 6810 U/L with 24.8 g/L dry cell density (OD₆₀₀ = 67.9) in a 5 L fermentor. In contrast to the cells of E. coli TB1/pMKC-Acy without the lysis genes, the mild EDTA/Tris buffer (pH 8.0) can cause the lysis of the cells of TB1/pMKC-AS containing the lysis genes. Correspondingly, a mild pH 9.0/42 °C incubation method was developed for conveniently degrading the recombinant cells of TB1/pMKC-AS, based on the expression of the lysis genes. Further experiments showed that the cell lysate after the mild incubation disruption can be directly immobilized by 10% polyacrylamide to make the immobilized enzymes. In comparison with the immobilized GLA from TB1/pMKC-Acy, the immobilized cell lysate of TB1/pMKC-AS has the similar characteristics of catalysis stability, implying a great potential for industrial application of the lysis genes-assisted cell disruption.     

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。     

Construction and application of fusion proteins of d-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase for direct bioconversion of cephalosporin C to 7-aminocephalosporanic acid

Two fusion proteins of D-amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid acylase (GLA) were designed to simplify the bioconversion process of cephalosporin C to 7-aminocephalosporanic acid (7-ACA), which is conventionally produced in a two-step enzymatic process. Two recombinant plasmids, pET-DLA and pET-ALD, were constructed to express fusion proteins of DAAO-linker-GLA (DLA) and GLA-linker-DAAO (ALD), respectively. When the recombinant plasmids were expressed in E. coli, the fusion protein DLA was not correctly folded and only DAAO activity could be detected. ALD, however, possessed activities of both DAAO and GLA, which directly catalyze the conversion of cephalosporin C into 7-ACA.     

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase from Bacillus laterosporus J1, is a member of the alpha/beta-hydrolase fold superfamily

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase (GCA) isolated from Bacillus laterosporus J1, has been conventionally grouped as the only member of class V GCA, although its amino acid sequence shares less than 10% identity with members of other classes of GCA. Instead, it shows higher sequence similarities with Rhodococcus sp. strain MB1 cocaine esterase (RhCocE) and Acetobacter turbidans alpha-amino acid ester hydrolase (AtAEH), members of the alpha/beta-hydrolase fold superfamily. Homology modeling and secondary structure prediction indicate that the N-terminal region of J1 acylase has an alpha/beta-hydrolase folding pattern. The catalytic triads in RhCocE and AtAEH were identified in J1 acylase as S125, D264 and H309. Mutations to alanine at these positions were found to completely inactivate the enzyme. These results suggest that J1 acylase is a member of the alpha/beta-hydrolase fold superfamily with a serine-histidine-aspartate catalytic triad.     

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%.     

Spongiacidin C,a pyrrole alkaloid from the marine sponge Stylissa massa,functions as a USP7 inhibitor

USP7, a deubiquitylating enzyme hydrolyzing the isopeptide bond at the C-terminus of ubiquitin, is an emerging cancer target. We isolated spongiacidin C from the marine sponge Stylissa massa as the first USP7 inhibitor from a natural source. This compound inhibited USP7 most strongly with an IC₅₀ of 3.8 μM among several USP family members tested.