Improving the activity of creatinase from <i>Alcaligenes</i> sp. KS-85 through semi-rational design.

Jiahao Bian; Junyao Hao; Guang-yu Yang

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Improving the activity of creatinase from Alcaligenes sp. KS-85 through semi-rational design.

Author: Jiahao BIAN ¹ ; Junyao HAO ¹ ; Guang-Yu YANG ¹
Author Information

1. State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Publication Type:Journal Article
Keywords: creatinase; creatinine; saturation mutagenesis; semi-rational design
MeSH: Creatinine; Mutagenesis, Site-Directed; Ureohydrolases/genetics*; Catalysis
From: Chinese Journal of Biotechnology 2022;38(12):4601-4614
CountryChina
Language:Chinese
Abstract: Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.