Jamek Shariza B, Nyffenegger Christian, Muschiol Jan, Holck Jesper, Meyer Anne S, Mikkelsen Jørn D
Faculty of Chemical and Natural Resources Engineering, University Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang, Malaysia.
Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark.
Appl Microbiol Biotechnol. 2017 Jun;101(11):4533-4546. doi: 10.1007/s00253-017-8198-4. Epub 2017 Mar 9.
Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β-D-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N'-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-D-glucosaminide (1 → 4)-β-linkages and are thus "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose.
A类几丁质酶（EC 3.2.1.14），属于糖苷水解酶家族18，从还原端攻击几丁质（（1→4）-2-乙酰氨基-2-脱氧-β-D-葡聚糖）和壳寡糖，通过水解N-乙酰-β-D-葡糖胺（1→4）-β-键催化释放壳二糖（N，N'-二乙酰壳二糖），因此是“外切壳二糖水解酶”。在本研究中，粘质沙雷氏菌的A类几丁质酶（SmaChiA）被用作模板，以鉴定分别源自海洋生物巴利阿里铁单胞菌和易变微球藻的两种新型外切壳二糖水解酶A类酶，即FbalChi18A和MvarChi18A。FbalChi18A和MvarChi18A均在大肠杆菌中重组表达，并被证实对壳寡糖具有外切壳二糖水解酶活性，但在温度和pH活性响应曲线方面存在差异。对每种新酶的催化β/α桶结构域的氨基酸序列比较显示出个体差异，但每种与SmaChiA的序列一致性约为69%，且活性位点残基高度保守。将模型底物叠加在酶催化结构域的三维结构模型上，证实了FbalChi18A和MvarChi18A具有A类外切壳二糖水解酶活性，即从还原端攻击底物。这两种新酶的一个主要特征是存在C末端5/12型碳水化合物结合模块（SmaChiA没有C末端碳水化合物结合模块）。这些新酶可能是通过壳二糖将几丁质用作N-乙酰葡糖胺供体底物的有用工具。
