以一株降解骨胶原蛋白的菌种蜡样芽孢杆菌(Bacillus cereus)MBL13-U作为出发菌种,通过对菌全基因组测序,针对胶原蛋白酶功能基因进行了定位和结构预测。结果表明,从全基因组序列中得到胶原蛋白酶的功能基因序列长度为 2 916 bp;该功能基因中α螺旋占总氨基酸的37.1%,β转角占12.8%,无规则卷曲占26.0%,且该酶的三维结构预测为镊子状结构。将胶原蛋白酶基因转入大肠杆菌宿主菌株BL21,构建出工程菌pET30a-ColM13/BL21。对工程菌所产的重组蛋白酶ColM13酶解Ⅰ型骨胶原蛋白的结构进行分析表明,酶解作用使骨胶原蛋白产生多肽和大量游离氨基酸,其微观结构发生显著变化,表明ColM13在工程菌中已成功表达。
The genome of Bacillus cereus MBL13-U that degrades bone collagen was sequenced, and a putative gene, ColM13, which encodes a collagenase was closed and its activity was assayed. It consisted of 2 916 bp. Its deduced amino acids sequence contained α-helix, β-rotation, and irregular curl structures, which accounted for 37.1%, 12.8%, and 26.0% of total amino acids, respectively. The mimic three-dimensional structure of the enzyme was to be tweezers. It was cloned and transformed into Escherichia coli BL21 to obtain E. coli BL21/pET30a-ColM13. The recombinant ColM13 degraded type I bone collagen and formed polypeptides as well as a large amount of free amino acids. Besides, the microstructure of collagen changed significantly after treating with the recombinant enzyme. Based on these observations, it was concluded that ColM13 has been successfully expressed in E. coli.
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