Enzyme and Microbial Technology Laboratory, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
J Basic Microbiol. 2010 Apr;50(2):143-9. doi: 10.1002/jobm.200900133.
A gene encoding an organic solvent-stable protease was amplified from Pseudomonas aeruginosa strain K by polymerase chain reaction using consensus primers based on multiple sequence alignment of alkaline and metalloprotease genes from Pseudomonas species. The gene, which consisted of 1440 bp nucleotides and deduced 479 amino acid residues, was successfully expressed in pGEX-4T-1 expression system in the presence of 1.0 mM IPTG, after an incubation of 6 h at 37 degrees C. Under these conditions, the recombinant strain K protease was, subsequently, released into the periplasm of E. coli BL21 (DE3) with an optimum proteolytic activity detected at 1.0112 U/ml. To date, this is the first reported expression of alkaline protease (aprA) with such remarkable property in Escherichia coli.
采用基于多序列比对的通用引物，通过聚合酶链反应（PCR）从铜绿假单胞菌（Pseudomonas aeruginosa）K 株中扩增出一个编码有机溶剂稳定蛋白酶的基因。该基因由 1440 个核苷酸组成，推断编码 479 个氨基酸残基，在存在 1.0 mM IPTG 的情况下，于 37°C 孵育 6 小时后，成功在 pGEX-4T-1 表达系统中表达。在此条件下，重组菌 K 蛋白酶随后被释放到大肠杆菌（Escherichia coli）BL21（DE3）的周质中，在 1.0112 U/ml 时检测到最佳的蛋白水解活性。迄今为止，这是首次在大肠杆菌中报告具有如此显著特性的碱性蛋白酶（aprA）的表达。
