Improvement of catalytic activity of Aspergillus terreus lipase by site-directed mutagenesis / 生物工程学报

The catalytic activity of Aspergillus terreus lipase (ATL) was improved by rational design. According to the sequence analysis and homologous modeling, several amino acids involved in the lid domain and substrate binding pocket domains of the acidic lipase ATL were mutated by site-directed mutagenesis, and eight mutants were constructed. These mutants and the wild type lipase ATL were expressed in Pichia pastoris GS115 and the enzymatic properties were characterized. The mutants ATLLid and ATLV218W exhibited higher hydrolytic activity than ATL towards p-nitrophenyl laurate. The kcat values of ATLLid and ATLV218W towards p-nitrophenyl laurate were 39.37- and 50.79-fold higher, and the kcat/Km values were 2.85- and 8.48-fold higher than the wild type, respectively. Although thermostability of these mutants decreased slightly, ATLLid and ATLV218W still exhibited the maximum activity at pH 5.0 and high stability in a broad range of pH (4.0-8.0), which were similar to the wild type. Using homologous modeling and molecular docking technology the mechanism for the improvement of catalytic activity was analyzed. These findings not only shed light on the relationship between the lid domain/substrate binding pocket domain and catalytic activity but also provided comprehensive scheme for further engineering to gain more efficient lipases.

Characterization of glutamate dehydrogenase SHJG 7666 from Streptomyces hygroscopicus 5008 / 中国药科大学学报

Glutamate dehydrogenase (GDH)a key enzyme in the nitrogen metabolism pathway catalyzes the con-version between α-ketoglutarate and glutamate reversibly using NAD(P)H as a cofactor. Based on genomic stud-ies,it was concluded that SHJG_7666 was a potential GDH in Streptomyces hygroscopicus 5008(S5008),and its expression level in vivo was positively correlated with the biosynthesis of an important aminocyclol compound vali-damycin. Phylogenetic tree analysis showed that the S5008 SHJG_7666 GDH belonged to the Glu/Leu/Phe/Val dehydrogenase family,with conserved glutamate-α-ketoglutarate binding domain and the classical GXGXXG dinu-cleotide binding motif. Further homologous modeling and structural comparison revealed that SHJG_7666 con-tained conserved Lys60,Lys78and Asp120catalytic functional sites and ligand binding residues Ser36,Gly38,Gln119 and Asp166,Asn300,Ala330. Moreover,recombinant expression of SHJG_7666 in E. coli and in vitro enzyme activity demonstrated that glutamate dehydrogenase can convert ammonium salt to glutamate with pH and temperature being optimal at 7. 5 and 37 °C respectively. Enzyme activity under optimum reaction condition has Kmvalue of (25. 3 ±9. 1)μmol/L and kcatof (3 ±0. 8)×10 -5s-1for the substrate α-ketoglutarate. Results of this study further improved the catalytic activity of SHJG_7666,thus laying the foundation for the ultimate increase of vali-damycin production.

The Clone, Expression and Site-directed Mutagenesis of 3-Ketosteroid-Delta (1)-Dehydrogenase from Arthrobacter Simplex / 现代生物医学进展

Objective:In this study,a prokaryotic expression of the 3-ketosteroid-Delta (1)-dehydrogenase (KSDD) which came from Arthrobacter simplex was built.Moreover,in order to investigate the catalytic mechanism of KSDD and improve its stability,the structure of KSDD was predicted by computer and the critical sites were confirmed by site-directed mutations.Methods:The recombinant plasmid was constructed by eukaryotic expression vector pET-22b and the recombinant strain was constructed and expressed in Escherichia coli BL21 (DE3).High-performance liquid chromatography was used to determine the transformation rate of 4-AD to ADD.The KSDD structure and key sites were predicted by SWISS-MODEL.Site-directed mutations for the amino acid residues of key sites were constructed and activities of the mutations were detected.Results:The recombinant strain E.coli pET-22-ksdd was successfully constructed.It was induced to express the dehydrogenase by IPTG and the conversion rate of 4-AD to ADD was 27％ at 21 ℃.The structure of 3-ketosteroid-Delta (1)-dehydrogenase and the four key sites was analyzed by SWISS-MODEL.Four mutants,Y120R,Y320L,Y488F and G492Y were constructed.Mutants Y120R and Y488F were inactivated,so they were proved to be the key active sites of KSDD.The conversion rate of mutant Y320L was consistent with that of wild type,but the stability at 37 ℃ was improved.The conversion rate of mutant G492Y was 1.2 times of the wild type and the stability has been improved at 37 ℃.Conclusions:At present,there are few studies about the structure and catalytic mechanism of dehydrogenase.The active sites of the enzyme were verified by this study,which laid the foundation for the further study of the properties of the enzyme KSDD.

Homologous modeling and function analysis on thioredoxin glutathione reductase from Schistosoma j aponicum / 中国人兽共患病学报

To explore the structure and function of thioredoxin glutathione reductase (TGR) from Schistosoma j aponi-cum ,the homologous model of TGR in Schistosoma j aponicum was constructed by Swiss-Pdbviewer based on sequence and structure alignment .The potential substrates binding sites of TGR were analyzed and these sites of various TGRs were also as-sessed .Our results showed that the homologous model of Schistosoma japonicum TGR based on Schistosoma mansoni TGR structure was proved to be reasonable by PROCHECK program .Analysis of binding sites showed that NADPH and GDS bind-ing sites were conservative sites and GSH binding site was a specific site for parasite .Our data suggested that inhibitors which work in NADPH and GDS binding sites of other various TGRs may also interact with TGR form Schistosoma j aponicum .GSH binding region might be one of the potential targets for design of specific inhibitors of parasite TGRs .In addition ,C-terminal of TGR plays an important role in electron transfer and may participate in the binding of the substrate .Thus compound inhibiting swing of C-terminal could effectively restrain Schistosoma j aponicum TGR activity .