【作者】 鲍晶晶； 朱丽英； 黄和； 江凌；

【Author】 Bao Jingjing;Zhu Liying;Huang He;Jiang Ling;College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University;College of Chemical and Molecular Engineering,Nanjing Tech University;College of Pharmaceutical Sciences,Nanjing Tech University;College of Food Science and Light Industry,Nanjing Tech University;

【机构】 南京工业大学生物与制药工程学院； 南京工业大学化学与分子工程学院； 南京工业大学药学院； 南京工业大学食品与轻工学院；

【摘要】 在食品加工生产过程中,会产生诸如虾蟹壳等副产物。在虾蟹壳中,70%的组成为甲壳素,可用于生产氨基葡萄糖(GlcN)。作为一种功能性糖,氨基葡萄糖广泛用于食品、化妆品、药品等行业。目前工业上氨基葡萄糖的生产主要是酸解法及微生物发酵法。前者产生的环境污染严重,而微生物发酵法主要以葡萄糖或者果糖为原料,且产物多为去乙酰度及聚合度不一的壳寡糖混合物。本研究利用酶催化法以甲壳素为原料生产氨基葡萄糖。首先分离纯化了来源于古嗜热菌Thermococcus kodakaraensis KOD1中甲壳素代谢途径中的三个关键酶,三酶可直接催化甲壳素生成单一产物氨基葡萄糖。进一步利用酶固定技术将三酶固定于细胞膜上。以E.coli自身带有的膜蛋白--Curli为锚定蛋白,利用SpyTag-SpyCatcher及SnoopTag-SnoopCatcher双分子肽对之间的特异性结合作用依次将三酶固定于细胞表面,最终获得多酶固定系统Multi-enzyme Assembly Cascade system(MAC system)。多酶固定系统高效催化甲壳素生成氨基葡萄糖,在1L催化体系中转化率达79.02%±3.61%,且具备很好的重复利用性。该法不仅提供了一种生产氨基葡萄糖的方法,也提供了一种多酶固定的新思路。更多还原

【Abstract】 Biomasses such as crab and shrimp shells are produced as the by-products of seafood industry globally.Nearly 70% of shellfishes consist of chitin,which is regarded as an essential raw material in producing glucosamine(GlcN).GlcN is widely used as an additive in fields such as food,cosmetics and pharmacy.Currently,acid hydrolysis and microbial fermentation processes are mainly used in producing GlcN.However,the great environmental threat the acid hydrolysis posed has hampered its development.For fermentation processes,the substrates are normally glucose or fructose,and the problem lies in the purity of products because the products are normally mixtures of chitosans that are of various degrees of polymerizations and deacetylations.In this context,enzyme hydrolysis is an alternative in catalyzing the conversion of chitin into GlcN.In this assay,we first isolated three key enzymes involved in the degradation of chitin from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1.The cluster consists of three enzymes,which can realize the conversion of chitin to GlcN directly.To realize consecutive processes in one pot,we proposed a strategy of immobilizing three enzymes in the biofilm.In this work,curli proteins,which were inherent in E.coli were used as anchors on biofilms for further immobilization of target enzymes.Two pairs of short peptides(SpyTag-SpyCatcher and SnoopTag-SnoopCatcher) provided covalent and specific binding force in immobilizing three target enzymes in sequence.As a consequence,Multi-enzyme Assembly Cascade system(MAC system)was achieved with three enzymes immobilized in the biofilm in sequence.The MAC system showed a great catalytic activity,converting 79.02%± 3.61% of chitin into GlcN with little by-products in a IL mixture.The system also performed well in reusability tests.In this work,we provided a method in producing glucosamine,as well as a novel way of immobilizing multi-enzymes.更多还原