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Appl Biochem Biotechnol. 2016 Feb;178(4):739-52. doi: 10.1007/s12010-015-1906-6. Epub 2015 Nov 7.

Cloning, Expression, Mutagenesis Library Construction of Glycerol Dehydratase, and Binding Mode Simulation of Its Reactivase with Ligands.

Jiang W1,2, Li W3, Hong Y4, Wang S1,2, Fang B5,6,7.

Author information

1
Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.
2
The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China.
3
Hangzhou DAC Biotech Co., Ltd, Hangzhou, 310000, China.
4
Jingdezhen Ceramic Institute of Materials Science and Engineering, Jingde Zhen, 333000, China.
5
Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China. fbs@xmu.edu.cn.
6
The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China. fbs@xmu.edu.cn.
7
The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian, 361005, China. fbs@xmu.edu.cn.

Abstract

The production of 1, 3-propanediol (1, 3-PD) and 3-hydroxypropionaldehyde (3-HPA) by enzyme reaction has been a hot field, and glycerol dehydratase (GDHt) is the key and rate-limiting enzyme involved in their biosynthesis. The gldABC gene encoding GDHt was cloned from Klebsiella pneumoniae, and the activity of the corresponding proteins expressed extracellularly and intracellularly was 6.8 and 3.2 U/mg, respectively, about six and three times higher than that of the wild strain. The change of amino acids for the β subunit can adjust the length of the Co-N bond and affect the homolysis rate of the Co-C bond to change GDHt activity. The expression plasmid, pET-32a-gldAC (containing no gldB which encodes the β subunit of GDHt), was constructed to build the mutagenesis library to improve the GDHt activity. The binding models of glycerol dehydratase reactivation factor (GDHtR) with ATP, CTP, or GTP were simulated by semi-flexible docking, respectively, and there was almost no difference between them. This research provided the basis for studying the quantitative structure-activity relationships between GDHtR and its ligands, as well as searching inexpensive ligands to replace ATP. These results and methods are of great use in economical and highly efficient production of 3-HPA and 1, 3-PD by the enzyme method.

KEYWORDS:

Cellular and extracellular expression; GHDt; GHDtR; Molecular DOCK; Molecular chaperone

PMID:
26547853
DOI:
10.1007/s12010-015-1906-6
[Indexed for MEDLINE]

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