枯草芽孢杆菌壳聚糖酶基因在毕赤酵母中的表达及酶学性质  

Expression of the chitosanase gene from Bacillus subtilis CH2 and characterization of the recombinant enzyme in Pichia pastoris

作　　者：杨光[1,2] 盛军[1] 陈亚东 沙珍霞 YANG Guang;SHENG Jun;CHEN Yadong;SHA Zhenxia(Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;College of Life Science, Qingdao University, Qingdao 266071, China)

机构地区：[1]中国水产科学研究院黄海水产研究所,农业部海洋渔业可持续发展重点实验室,山东青岛266071 [2]上海海洋大学水产与生命学院,上海201306 [3]青岛大学生命科学学院,山东青岛266071

出　　处：《中国水产科学》2017年第6期1288-1297,共10页Journal of Fishery Sciences of China

基　　金：中国水产科学研究院基本科研业务费资助项目(2014A03XK01,2014C04XK01);青岛大学引进人才科研启动费资助项目

摘　　要：构建了枯草芽孢杆菌CH_2菌株(Bacillus subtilis CH_2)壳聚糖酶基因的真核表达载体p PIC9K-CH2-Cns,在毕赤酵母GS115(Pichia pastoris)中进行重组表达,获得了有生物学活性的重组壳聚糖酶,对重组壳聚糖酶的酶学特性及酶解产物进行了分析。结果显示,重组壳聚糖酶的分子量为29 k D,粗酶的比酶活达到133.60 U/mg,纯化后重组蛋白的比酶活为338.08 U/mg;该酶的最适作用温度为50℃,最适pH为4.5,酶动力学常数Vmax=24.39(μmol/mg)·min^(-1),Km=5.48 mg/m L;Fe2+、K+等对其酶活力有一定的激活作用,其中Mn^(2+)能使酶活力提升2.4倍,Ag^+、Mg^(2+)、Hg^(2+)、EDTA、EGTA和SDS等存在时则对其酶活力有强烈抑制作用。利用重组壳聚糖酶酶解壳聚糖,酶解产物主要为聚合度2~10的壳寡糖,且分布均匀。以上结果表明,枯草芽孢杆菌CH_2菌株壳聚糖酶基因在毕赤酵母GS115中成功表达,获得了一种内切型的高酶活力重组壳聚糖酶,该重组酶具有反应条件温和、酶解产物均一等特点,可被应用于海洋甲壳质加工应用中。Chitosanase is important in carbon and nitrogen recycles that occur extensively in nature,and is useful in the preparation of biofunctional chitooligosaccharides.Chitosanase occurs in a variety of microorganisms,including bacteria and fungi.Transformation by genetic engineering has increased the enzyme activity and enzyme content of recombinant chitosanase.To obtain abundant chitosanases possessing high chitosanolytic activity for large-scale production of chitosan-oligosaccharide,the chitosanase of Bacillus subtilis CH2was best induced by fructose and not induced with chitosan,unlike other chitosanases.This study investigated recombinant expression of chitosanase from B.subtilis CH2,which was cloned and expressed in Pichia pastoris,and characterized the application of the recombinant enzyme for chitosan hydrolysis.Chitosanase may have important industrial applications in the utilization of the enormous chitosan substrates.Mass spectrometry(MS)and thin-layer chromatography(TLC)were used to analyze the enzymatic products.The molecular weight of the recombinant chitosanase was estimated to be29kD using SDS-PAGE.The specific activity of crude enzymes was133.60U/mg.The specific activity of the purified chitosanase was up to338.08U/mg.The optimal pH and temperature of the purified chitosanase was50℃and4.5,respectively.The Km and Vmax values with soluble chitosan as a substrate were5.48mg/mL and24.39(μmol/mg)·min-1,respectively.Fe2+,K+,Na+,Li+,Ca2+,and especially Mn2+enhanced the enzyme activity of the recombinant chitosanase.Whereas,Ag+,Mg2+,Hg2+,EDTA,EGTA,and SDS inhibited the enzyme activity,and the other metal ion tested had no effect on enzyme activity.This characteristic of the recombinant chitosanase was better than the chitosanase of B.subtilis CH2.Furthermore,the enzymatic production of chitooligosaccharides from chitosans of various deacetylation degrees ranged mainly from chitobiose to chitopentamer,and the enzymic products contained2-10degrees of polymerization of chito-oligosaccharide after the recombi

关 键 词：壳聚糖酶 枯草芽孢杆菌CH2菌株 壳寡糖 毕赤酵母 酶学性质 

分 类 号：S96[农业科学—水产养殖]