γ-Glutamyl transpeptidase is an enzyme that can catalyze glutamine to generate a γ-glutamyl-enzyme complex.Then the complex can be nucleophilically substituted by an acceptor substrate such as an amino acid or a short peptide, resulting in a transpeptidation reaction that forms a γ-glutamyl peptide.This enzyme has significant application value in the fields of food and medicine.In this research, Bacillus subtilis SCK6 was used as the expression host, based on 173 signal peptides of Bacillus subtilis, a signal peptide screening library was constructed, and the optimal signal peptide (SPcotC) was identified.Promoter screening was then carried out to obtain the optimal promoter (PaprE).Finally, with the optimal signal peptide and prompter, a recombinant strain BS-gts3 was constructed which could express and secrete γ-glutamyl transpeptidase.The maximum extracellular enzyme activity reached 5.04 U/mL in shake flask fermentation.The optimal medium was further determined through one-way and response surface tests.The optimal composition of the medium was 31.73 g/L of lactose, 50.26 g/L of FM802, 1.24 g/L of MgSO4, 1.25 g/L of KH2PO4, and 0.75 g/L of K2HPO4.The culture was then scaled up in a 5 L fermenter, and the maximum extracellular enzyme activity reached 28.26 U/mL after 38 h fermentation, which was the highest reported level.This study provides a foundation for the development and application of γ-glutamyl transpeptidase.
ZHANG Zuzheng
,
MAO Zejing
,
YU Huashun
,
ZHANG Yan
,
YANG Xiao
,
GONG Dachun
,
ZHENG Xianliang
. Efficient expression of γ-glutamyl transpeptidase in Bacillus subtilis and optimization of enzyme-producing culture medium[J]. Food and Fermentation Industries, 2024
, 50(24)
: 75
-83
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DOI: 10.13995/j.cnki.11-1802/ts.038642
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