食品工业科技2025，Vol.46Issue(6)：206-216,11.DOI:10.13386/j.issn1002-0306.2024050254
Lysinibacillus sphaericus胶原酶的异源表达、鉴定及其在抗氧化活性肽制备中的应用
Heterologous Expression and Characterization of Collagenase from Lysinibacillus sphaericus and Its Application in the Preparation of Antioxidant Peptides
摘要
Abstract
Objective:Collagenase from Lysinibacillus sphaericus(LsCol1)was heterologously expressed and purified,and its enzymatic property and substrate specificity were determined.It was then used to hydrolyze tilapia skin collagen to prepare antioxidant peptides,with the aim of providing a theoretical basis for the development of a new collagenase resource and its application in the preparation of bioactive peptides.Methods:The amino acid sequence of LsCol1 was analyzed using BLAST and Clustal Omega,and the three-dimensional structure was predicted using AlphaFold 3.The gene sequence of LsCol1 was obtained using chemical synthesis technology,and it was then heterologously expressed in Escherichia coli.LsCol1 was purified using nickel affinity chromatography,and its enzymatic properties and substrate specificity were studied.Finally,the antioxidant peptide was prepared by hydrolyzing tilapia skin collagen with LsCol1.Results:The full length of LsCol1 gene was 3219 bp and encoded 1072 amino acids.The N-terminal of LsCol1 had a catalytic domain,and the C-terminal accessory domain consists of a polycystic kidney disease-like domain and two collagen-binding domains.The molecular weight of purified recombinant collagenase LsCol1 was 120 kDa.The optimal reaction temperature and pH of LsCol1 were 37 ℃ and 7.5,respectively,and it was relatively stable in the range below 30 ℃ and pH7.0～10.0.Ca2+promoted LsCol1 activity.The substrate specificity experiment showed that the optimal substrate for LsCol1 was tilapia skin collagen.The hydrolysate prepared from tilapia skin collagen displayed good antioxidant activity,with 1,1-diphenyl-2-picrylhydrazyl radical,2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)radical,and hydroxyl radical,scavenging rates of 65.9％,97.6％,and 32.1％,respectively,at a concentration of 4 mg/mL.Conclusion:The collagenase LsColl from L.sphaericus had high catalytic capacity and potential application in the preparation of bioactive peptides.关键词
胶原蛋白/胶原酶/酶学特性/胶原蛋白水解物/抗氧化活性Key words
collagen/collagenase/enzymatic properties/collagen hydrolysate/antioxidant activity分类
生物科学引用本文复制引用
吴海星,黄安妮,高霞,申铉日,夏光华,张雪莹..Lysinibacillus sphaericus胶原酶的异源表达、鉴定及其在抗氧化活性肽制备中的应用[J].食品工业科技,2025,46(6):206-216,11.基金项目
海南省重点研发计划项目(ZDYF2020172) （ZDYF2020172）
海南省自然科学基金(322RC587) （322RC587）
海南大学科研启动基金项目(KYQD(ZR)20046). （KYQD(ZR）
