Acidovorax装置72W硝化酶催化效率的提高及其在3-氰吡啶制烟酸生物转化中的应用

Q2 Chemical Engineering Journal of Molecular Catalysis B-enzymatic Pub Date : 2016-11-01 DOI:10.1016/j.molcatb.2017.03.010

He Li , Weiliang Dong , Yue Zhang , Kuan Liu , Wenming Zhang , Min Zhang , Jiangfeng Ma , Min Jiang

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引用次数: 8

摘要

在本研究中，研究了利用Acidovorax设施72W的NitA催化烟酸(NA)生产的效率，并通过定点诱变进一步提高了其效率。结果表明，突变的NitA-C2 (F168V-S192F)对3-氰吡啶的特异性活性增加了5倍，达到35 U mg−1蛋白。进一步对两种腈酶的生化性能进行表征，结果表明，最适pH值为6.0 ~ 8.0℃，温度为60℃，而NitA-C2的pH值和热稳定性均有所降低。最后采用全细胞催化法生产NA，在0.1 mol L−1 3-氰吡啶条件下，两株菌株的NA转化率均达到100%。大肠杆菌BL21 (DE3-pET-nitA- c2)的转化率达到1.0 mmol min−1 g−1湿细胞重，是大肠杆菌BL21 (DE3-pET-nitA)的3倍。这些结果表明，突变的NitA-C2在生物NA生产中具有潜在的应用前景。

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Enhanced catalytic efficiency of nitrilase from Acidovorax facilis 72W and application in bioconversion of 3-cyanopyridine to nicotinic acid

In this study, the catalytic efficiency using NitA from Acidovorax facilis 72W for nicotinic acid (NA) production was investigated and further improved by site-directed mutagenesis. Results showed that the specific activity of mutated NitA-C2 (F168V-S192F) towards 3-cyanopyridine increased 5-fold to 35 U mg⁻¹ protein. Further characterization of the biochemical properties of both nitrilases showed the optimal pH and temperature were 6.0–8.0 and 60 °C, respectively, whereas the pH and thermal stability of NitA-C2 were decreased. Finally, whole cell catalysis was adopted for NA production and a 100% conversion yield was achieved under 0.1 mol L⁻¹ 3-cyanopyridine for both strains. Besides, the conversion rate by E. coli BL21 (DE3-pET-nitA-C2) reached to 1.0 mmol min⁻¹ g⁻¹ wet cell weight, which was 3-fold higher than that by E. coli BL21 (DE3-pET-nitA). These results indicated that the mutated NitA-C2 was a promising candidate which holds potential application in biological NA production.

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来源期刊

Journal of Molecular Catalysis B-enzymatic 生物-生化与分子生物学

CiteScore

2.58

自引率

0.00%

发文量

审稿时长

3.4 months

期刊介绍： Journal of Molecular Catalysis B: Enzymatic is an international forum for researchers and product developers in the applications of whole-cell and cell-free enzymes as catalysts in organic synthesis. Emphasis is on mechanistic and synthetic aspects of the biocatalytic transformation. Papers should report novel and significant advances in one or more of the following topics; Applied and fundamental studies of enzymes used for biocatalysis; Industrial applications of enzymatic processes, e.g. in fine chemical synthesis; Chemo-, regio- and enantioselective transformations; Screening for biocatalysts; Integration of biocatalytic and chemical steps in organic syntheses; Novel biocatalysts, e.g. enzymes from extremophiles and catalytic antibodies; Enzyme immobilization and stabilization, particularly in non-conventional media; Bioprocess engineering aspects, e.g. membrane bioreactors; Improvement of catalytic performance of enzymes, e.g. by protein engineering or chemical modification; Structural studies, including computer simulation, relating to substrate specificity and reaction selectivity; Biomimetic studies related to enzymatic transformations.