摘要
为获得高反应温度的壳聚糖酶,本文挖掘了来源于δ变形细菌的壳聚糖酶基因(GenBank:MBN2801850.1)并将其在大肠埃希氏菌(Escherichia coli)中成功异源表达得到新的壳聚糖酶Csn(Db)-1-WSY(Csn1)。Csn1是属于GH8家族的壳聚糖酶,蛋白分子量大小约为52 kDa,比酶活为135.81 U/mg,是典型的内切型壳聚糖酶,其在60℃、pH=10.0的Gly-NaOH缓冲液中表现出最高活性。Csn1的米氏常数Km为1.30 mmol/L,显示其与底物较好的亲和性。在5 mmol/L Cu^(2+)溶液中Csn1的活性增强至初始的131.36%;而Fe^(2+)、Co^(2+)、Mn^(2+)等金属离子均对Csn1表现出活性抑制作用。Csn1水解壳聚糖的主要产物为(GlcN)2至(GlcN)4。研究结果表明,Csn1可为工业制备壳寡糖提供有效的生产工具酶。
In order to exploit high-temperature chitosanase functioning at high temperatures,in this study,we isolated the chitosanase gene(GenBank:MBN2801850.1)from aδ-Proteobacteria and successfully expressed it in Escherichia coli,yielding a chitosanase Csn(Db)-1-WSY(Csn1).Csn1 belongs to GH8 family with a molecular weight of 52 kDa and a specific enzyme activity of 135.81 U/mg.Csn1 was confirmed to be a typical endo chitosanase which exhibited the highest activity at 60℃and pH=10(Gly-NaOH buffer).The Michaelis constant Km of Csn1 was 1.30 mmol/L,indicating that it is good in affinity with substrate.The activity of Csn1 increased to 131.36%of its original activity in Cu^(2+)solution at 5 mmol/L.Moreover,metal ions,such as Fe^(2+),Co^(2+)and Mn^(2+),exhibited inhibitory effects on Csn1 activity.The main hydrolysis products of chitosan catalyzed by Csn1 were GlcN oligomers varying between(GlcN)2 and(GlcN)4.Csn1 may serve as an provides an effective production tool enzyme for the industrial preparation of chitosan oligosaccharides.
作者
王诗媛
苏海鹏
毛相朝
胡阳
Wang Shiyuan;Su Haipeng;Mao Xiangzhao;Hu Yang(State Key Laboratory of Marine Food Processing and Safety Control,College of Food Science and Engineering,Ocean University of China,Qingdao 266404,China;Laboratory for Marine Drugs and Bioproducts,Qingdao Marine Science and Technology Center,Qingdao 266237,China;Qingdao Key Laboratory of Food Biotechnology,Qingdao 266404,China;Key Laboratory of Biological Processing of Aquatic Products,China National Light Industry,Qingdao 266404,China)
出处
《中国海洋大学学报（自然科学版）》
CAS
CSCD
北大核心
2024年第12期42-51,共10页
Periodical of Ocean University of China
基金
国家自然科学基金项目(32201966)
中央高校基本科研业务费专项(202213027)资助。
关键词
内切型壳聚糖酶
壳寡糖
酶学性质
壳聚糖
endo chitosanase
chitosan oligosaccharide
enzymatic property
chitosan
