中国水产科学2025，Vol.32Issue(8)：1079-1092,14.DOI:10.12264/JFSC2025-0122
南极磷虾亮氨酸氨肽酶基因的克隆、异源表达及其酶学性质研究
Gene cloning,heterologous expression and enzymatic properties of leucine aminopeptidase from Euphausia superba
摘要
Abstract
The objective of this study was to screen and clone a leucine aminopeptidase gene(EsLAP)from the transcriptome of Euphausia superba,construct the recombinant expression vector EsLAP-PET-28A,and achieve soluble expression in Escherichia coli through co-expression of a molecular chaperone.The induction conditions were optimized by using the three-factor and three-level response surface method,and the enzymatic properties of the recombinant enzyme were systematically characterized.The total length of the EsLAP gene was 1569 bp,and it encoded 522 amino acids,with a theoretical molecular weight of 55302.67 Da and an isoelectric point of 6.16.Sequence and structural analysis indicated that EsLAP possesses typical peptidase catalytic domains and substrate-specific binding sites of M17 family peptidases.The optimal expression conditions of EsLAP were determined through response surface optimization as follows:IPTG concentration of 0.7 mmol/L,inoculation dose of 3%,and induction time of 19 h.Enzymatic property analysis showed that the optimal reaction temperature of EsLAP was 60℃,the optimal pH was 8.5,the crude enzyme activity was 265.6 U/mL,and the specific enzyme activity was 33.15 U/mg.Metal ions Co2+and Mn2+can significantly enhance enzyme activity,among which Co2+can increase enzyme activity to 296%of the original level.EDTA can significantly inhibit its enzymatic activity,indicating that EsLAP is a metalloproteinase.Strong reducing agents such as β-mercaptoethanol,NaBH4,and DTT significantly inhibit enzyme activity and can reduce the remaining enzyme activity to less than 10%.Substrate specificity analysis showed that EsLAP had the highest catalytic efficiency for Leu-pNA,further verifying that it was leucine aminopeptidase.This is the first report on the heterologous expression and enzymatic characteristics of leucine aminopeptidase derived from Euphausia superba.The related results provide a potential application basis for its use in the food and pharmaceutical industries to degrade N-terminal leucine-containing functional peptides.关键词
南极磷虾/氨肽酶/异源表达/酶学性质Key words
Euphausia superba/aminopeptidase/heterologous expression/enzymatic properties分类
水产学引用本文复制引用
谭佳豪,邹华英,盛军,王芳,陈梦瑶,宋佳,徐甲坤..南极磷虾亮氨酸氨肽酶基因的克隆、异源表达及其酶学性质研究[J].中国水产科学,2025,32(8):1079-1092,14.基金项目
国家重点研发计划项目(2022YFC2807500). （2022YFC2807500）
