摘要
壳寡糖具有多种生物活性,是目前仅知的唯一碱性寡糖,在食品、农业和生物医药领域应用广泛。壳聚糖酶可以特异性切割壳聚糖中的β-1,4糖苷键,形成不同聚合度的壳寡糖,因此,获得具有良好稳定性的壳聚糖酶是大规模酶法制备壳寡糖的关键。为了鉴定影响糖苷水解酶(Glycoside hydrolase,GH)46家族壳聚糖酶酸碱耐受性的相关氨基酸位点,选取来自芽孢杆菌(Bacillus sp.)DAU101(最适pH为7.5)的壳聚糖酶为模板,以来自芽孢杆菌的壳聚糖酶Csn-BAC为研究对象,综合同源建模和序列比对的方法,选取了4个候选位点并构建了4个突变体(V1:P68A;V2:A137G;V3:A203M;V4:H234E)。结果显示,与Csn-BAC相比,4个突变体的热稳定性均出现了不同程度的下降,而酸碱耐受性有了明显的提升。结果表明,选取的氨基酸位点对酸碱耐受性均产生了显著的影响,同时表明该策略在改造壳聚糖酶稳定性方面是一种有效的方法。
Chitooligosaccharides,which have a variety of biological activities,are the only known basic oligosaccharide widely used in food,agriculture and biomedicine.Chitosanases can cleave theβ-1,4 glycosidic bonds in chitosan specifically to form chitooligosaccharides with different degrees of polymerization.Therefore,obtaining chitosanases with good stability is the key for the large-scale enzymatic preparation of chitooligosaccharides.In order to identify the amino acid sites affecting the pH stability of GH46 family chitosanases,the chitosanase from Bacillus sp.DAU101(optimal pH 7.5)was selected as template and the chitosanase Csn-BAC from Bacillus sp.MD-5 as the research object.By combining homology modeling and sequence alignments,four candidate sites were selected,and the corresponding mutants were obtained(V1:P68A;V2:A137G;V3:A203M;V4:H234E).Compared with Csn-BAC,the thermal stabilities of four mutants showed varying degrees of reduction,while the pH stability was significantly improved.These results indicate that the selected amino acid sites have an obvious effect on pH tolerance,and this strategy is an effective way to modify the stability of chitosanase.
作者
程伊梦
孙慧慧
刘淇
赵玲
曹荣
CHENG Yimeng;SUN Huihui;LIU Qi;ZHAO Ling;CAO Rong(Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China;College of Food Science and Engineering,Ocean University of China,Qingdao 266003,China)
出处
《南方水产科学》
CAS
CSCD
北大核心
2022年第2期48-56,共9页
South China Fisheries Science
基金
国家重点研发计划项目(2019YFD0901902)。
