Jun Hyun S, Ha Jong K, Malburg Laercio M, Verrinder Gibbins Ann M, Forsberg Cecil W
Department of Microbiology, University of Guelph, Guelph, ON N1G 2W1, Canada.
Can J Microbiol. 2003 Mar;49(3):171-80. doi: 10.1139/w03-024.
Xylanase genes xyn10D, xyn10E, and xyn10B, located sequentially on the Fibrobacter succinogenes S85 chromosome, were separately cloned and their properties characterized. Analysis of the sequences documented that xylanases Xyn10D, Xyn10E, and Xyn10B each consist of an N-terminal catalytic domain (glycosyl hydrolase family 10) and a C-terminal carbohydrate-binding module (CBM, family 6) connected by proline-rich linker sequences. The amino acid sequences exhibited similarities of between 53 and 60%. The xyn10D, xyn10E, and truncated xyn10deltaACBM were expressed in Escherichia coli and purified to homogeneity. The purified Xyn10D, Xyn10E, and Xyn10BdeltaCBM exhibited the same temperature optimum (40 degrees C) and pH optimum (6.5) and the highest specific activity against arabinoxylan, oat spelt xylan, and birchwood xylan, respectively. Xyn10D exhibited an affinity for cellulose and xylan with 47 and 33% binding, respectively, while the truncated Xyn10DdeltaCBM did not bind to the substrates. The main hydrolysis products of the three xylanases acting on oat spelt xylan and arabinoxylan were xylose and xylobiose. RT-PCR analysis showed that the three genes were co-transcribed as a single transcript. Western immunoblot analysis revealed that the three xylanases were expressed at a very low level by F. succinogenes grown on either glucose or cellulose as the source of carbohydrate.
依次位于产琥珀酸丝状杆菌S85染色体上的木聚糖酶基因xyn10D、xyn10E和xyn10B被分别克隆，并对其特性进行了表征。序列分析表明，木聚糖酶Xyn10D、Xyn10E和Xyn10B均由一个N端催化结构域（糖基水解酶家族10）和一个C端碳水化合物结合模块（CBM，家族6）组成，二者通过富含脯氨酸的连接序列相连。氨基酸序列的相似度在53%至60%之间。xyn10D、xyn10E和截短的xyn10deltaACBM在大肠杆菌中表达并纯化至均一。纯化后的Xyn10D、Xyn10E和Xyn10BdeltaCBM表现出相同的最适温度（40℃）和最适pH（6.5），并且分别对阿拉伯木聚糖、燕麦spel木聚糖和桦木木聚糖具有最高的比活性。Xyn10D对纤维素和木聚糖具有亲和力，结合率分别为47%和33%，而截短的Xyn10DdeltaCBM不与底物结合。三种木聚糖酶作用于燕麦spel木聚糖和阿拉伯木聚糖的主要水解产物是木糖和木二糖。RT-PCR分析表明，这三个基因作为一个单一转录本共同转录。Western免疫印迹分析显示，以葡萄糖或纤维素作为碳水化合物来源生长的产琥珀酸丝状杆菌对这三种木聚糖酶的表达水平非常低。
