MacKenzie C R, Yang R C, Patel G B, Bilous D, Narang S A
Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Arch Microbiol. 1989;152(4):377-81. doi: 10.1007/BF00425176.
Three genes coding for xylanase synthesis in Clostridium thermocellum were cloned and expressed in Escherichia coli. Genomic DNA from Clostridium thermocellum was digested to completion with HindIII, BamHI, and SalI. The fragments were ligated into the corresponding sites of pUC19 and transformed into Escherichia coli. Two of the genes encoded for xylanases which depolymerized xylans but were unable to extensively convert these substrates to reducing sugar. The third gene encoded for an enzyme that extensively hydrolyzed xylan. The insert containing the latter gene was subjected to extensive mapping and was found to encode for a xylanase with a molecular weight of approximately 25,000. The protein product of the cloned gene was obtained in a relatively pure form by heat treatment, ion exchange and gel permeation steps. The enzyme was quite stable to high temperatures with a half-life of 24 h at 70 degrees C.
对嗜热栖热放线菌中编码木聚糖酶合成的三个基因进行了克隆，并在大肠杆菌中表达。用HindIII、BamHI和SalI将嗜热栖热放线菌的基因组DNA完全消化。将片段连接到pUC19的相应位点并转化到大肠杆菌中。其中两个基因编码的木聚糖酶可使木聚糖解聚，但无法将这些底物大量转化为还原糖。第三个基因编码一种能大量水解木聚糖的酶。对含有后一个基因的插入片段进行了广泛的图谱分析，发现它编码一种分子量约为25,000的木聚糖酶。通过热处理、离子交换和凝胶渗透步骤，以相对纯的形式获得了克隆基因的蛋白质产物。该酶对高温相当稳定，在70℃下的半衰期为24小时。
