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

作　　者：吴海星 黄安妮 高霞 申铉日[1] 夏光华[1,2] 张雪莹 WU Haixing;HUANG Anni;GAO Xia;SHEN Xuanri;XIA Guanghua;ZHANG Xueying(School of Food Science and Engineering,Hainan University,Hainan Engineering Research Center of Aquatic Resources Efficient Utilization in South China Sea,Key Laboratory of Seafood Processing of Haikou,National R&D Branch Center for Prawn Processing Technology(Hainan),Haikou 570228,China;Collaborative Innovation Center of Provincial and Ministerial Co-construction for Marine Food Deep Processing,Dalian Polytechnic University,Dalian 116034,China)

机构地区：[1]海南大学食品科学与工程学院,南海水产资源高效利用工程研究中心,海洋食品精深加工海口市重点实验室,国家对虾加工技术研发分中心(海南),海南海口570228 [2]大连理工大学,海洋食品精深加工关键技术省部共建协同创新中心,辽宁大连116034

出　　处：《食品工业科技》2025年第6期206-216,共11页Science and Technology of Food Industry

基　　金：海南省重点研发计划项目(ZDYF2020172);海南省自然科学基金(322RC587);海南大学科研启动基金项目(KYQD(ZR)20046)。

摘　　要：目的:对来源于Lysinibacillus sphaericus的胶原酶LsCol1进行异源表达、纯化、酶学性质及底物特异性研究,并用于水解罗非鱼皮胶原蛋白制备抗氧化活性肽,以期为新型胶原酶资源的开发及其在生物活性肽制备中的应用提供理论依据。方法:使用序列分析工具BLAST、Clustal Omega等分析LsCol1的氨基酸序列,利用AlphaFold 3预测其三维结构,借助化学合成技术获得LsCol1的基因序列全长并在Escherichia coli中异源表达,使用镍亲和层析色谱分离纯化LsCol1,研究其酶学特性及底物特异性,最后利用LsCol1水解罗非鱼皮胶原蛋白制备抗氧化活性肽。结果:LsCol1基因序列全长为3219 bp,编码1072个氨基酸,N端含有催化域,C端附属结构域由一个多囊肾病样结构域和两个胶原结合结构域组成。纯化后的重组胶原酶LsCol1分子量为120 kDa,最适反应温度和pH分别为37℃和7.5,在低于30℃和pH7.0~10.0范围内稳定性较好,Ca^(2+)对LsCol1的酶活力呈促进作用。底物特异性实验结果表明,LsCol1的最适作用底物为罗非鱼皮胶原蛋白。以罗非鱼皮胶原蛋白为底物制备的水解物具有良好的抗氧化活性,当水解物浓度为4 mg/mL时,其1,1-二苯基-2-三硝基苯肼自由基、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐自由基和羟基自由基清除率分别为65.9%、97.6%和32.1%。结论:L.sphaericus来源的重组胶原酶LsCol1具有良好的催化活性,在抗氧化活性肽制备方面具有潜在的应用价值。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.Ca^(2+)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 LsCol1 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 

分 类 号：Q814[生物学—生物工程]