Wong Dominic D W S, Chan Victor J, McCormack Amanda A, Batt Sarah B
Western Regional Research Center, USDA-ARS, 800 Buchanan Street, Albany, CA 94710, USA.
Protein Pept Lett. 2010 Jun;17(6):803-8. doi: 10.2174/092986610791190381.
A novel exo-glucanase gene (xeg5B) was isolated from a rumenal microbial metagenome, cloned, and expressed in E. coli. The 1548 bp gene coded for a protein of 516 amino acids, which assumed an (a/b)(8) fold typical of glycoside hydrolase (GH) family 5. The protein molecule consisted of a loop segment blocking one end of the active site, which potentially provided the enzyme with exo-acting property. The recombinant enzyme showed exclusive specificity towards only xyloglucan and oligoxyloglucan substrates with no detectable activity on unsubstituted linear glucans, CMC, laminarin, and lichenan. The major end products of exhaustive hydrolysis were XX (tetrasaccharide) and XG (trisaccharide). The hydrolysis of tamarind xyloglucan followed the Michaelis-Menten kinetics, yielding K(m) and V(max) of 2.12+/-0.13 mg/ml and 0.17+/-0.01 mg/ml/min (37 degrees C, pH 6.0), respectively.
从瘤胃微生物宏基因组中分离出一个新的外切葡聚糖酶基因（xeg5B），进行克隆并在大肠杆菌中表达。该1548 bp的基因编码一个由516个氨基酸组成的蛋白质，其具有糖苷水解酶（GH）家族5典型的（α/β）8折叠结构。蛋白质分子由一个环段组成，该环段封闭了活性位点的一端，这可能赋予该酶外切活性。重组酶对木葡聚糖和低聚木葡聚糖底物具有专一性，对未取代的线性葡聚糖、羧甲基纤维素、海带多糖和地衣多糖没有可检测到的活性。彻底水解的主要终产物是XX（四糖）和XG（三糖）。罗望子木葡聚糖的水解遵循米氏动力学，在37℃、pH 6.0条件下，米氏常数（K(m)）和最大反应速度（V(max)）分别为2.12±0.13 mg/ml和0.17±0.01 mg/ml/min。
