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Tan, Chunlin; Zhang, Xian; Zhu, Zhijing; Xu, Meijuan; Yang, Taowei; Osire, Tolbert; Yang, Shangtian; Rao, Zhiming
Microbial cell factories, 01/2019, Letnik: 18, Številka: 1Journal Article
Styrene monooxygenase (SMO) catalyzes the first step of aromatic alkene degradation yielding the corresponding epoxides. Because of its broad spectrum of substrates, the enzyme harbors a great potential for an application in medicine and chemical industries. In this study, we achieved higher enzymatic activity and better stability towards styrene by enlarging the ligand entrance tunnel and improving the hydrophobicity through error-prone PCR and site-saturation mutagenesis. It was found that Asp305 (D305) hindered the entrance of the FAD cofactor according to the model analysis. Therefore, substitution with amino acids possessing shorter side chains, like glycine, opened the entrance tunnel and resulted in up to 2.7 times higher activity compared to the wild-type enzyme. The half-lives of thermal inactivation for the variant D305G at 60 °C was 28.9 h compared to only 3.2 h of the wild type SMO. Moreover, overexpression of SMO in Pseudomonas putida KT2440 with NADH regeneration was carried out in order to improve biotransformation efficiency for epoxide production. A hexadecane/buffer (v/v) biphasic system was applied in order to minimize the inactivation effect of high substrate concentrations on the SMO enzyme. Finally, SMO activities of 190 U/g CDW were measured and a total amount of 20.5 mM (S)-styrene oxide were obtained after 8 h. This study offers an alternative strategy for improved SMO expression and provides an efficient biocatalytic system for epoxide production via engineering the entrance tunnel of the enzyme's active site.
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JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
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