Shakiba Mehrnoush Hadaddzadeh, Ali Mohd Shukuri Mohamad, Rahman Raja Noor Zaliha Raja Abd, Salleh Abu Bakar, Leow Thean Chor
Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Extremophiles. 2016 Jan;20(1):44-55. doi: 10.1007/s00792-015-0796-4.
The gene encoding for a novel cold-adapted enzyme from family II of bacterial classification (GDSL family) was cloned from the genomic DNA of Photobacterium sp. strain J15 in an Escherichia coli system, yielding a recombinant 36 kDa J15 GDSL esterase which was purified in two steps with a final yield and purification of 38.6 and 15.3 respectively. Characterization of the biochemical properties showed the J15 GDSL esterase had maximum activity at 20 °C and pH 8.0, was stable at 10 °C for 3 h and retained 50 % of its activity after a 6 h incubation at 10 °C. The enzyme was activated by Tween-20, -60 and Triton-X100 and inhibited by 1 mM Sodium dodecyl sulphate (SDS), while β-mercaptoethanol and Dithiothreitol (DTT) enhanced activity by 4.3 and 5.4 fold respectively. These results showed the J15 GDSL esterase was a novel cold-adapted enzyme from family II of lipolytic enzymes. A structural model constructed using autotransporter EstA from Pseudomonas aeruginosa as a template revealed the presence of a typical catalytic triad consisting of a serine, aspartate, and histidine which was verified with site directed mutagenesis on active serine.
从嗜冷发光杆菌J15菌株的基因组DNA中克隆出一种编码细菌分类II族（GDSL家族）新型冷适应酶的基因，该基因在大肠杆菌系统中表达，产生一种重组36 kDa的J15 GDSL酯酶，经两步纯化，最终产量和纯化倍数分别为38.6和15.3。生化特性表征显示，J15 GDSL酯酶在20℃和pH 8.0时具有最大活性，在10℃下3小时内稳定，在10℃下孵育6小时后仍保留50%的活性。该酶被吐温-20、吐温-60和曲拉通-X100激活，被1 mM十二烷基硫酸钠（SDS）抑制，而β-巯基乙醇和二硫苏糖醇（DTT）分别使活性提高4.3倍和5.4倍。这些结果表明，J15 GDSL酯酶是一种来自脂解酶II族的新型冷适应酶。以铜绿假单胞菌的自转运酯酶EstA为模板构建的结构模型显示，存在一个由丝氨酸、天冬氨酸和组氨酸组成的典型催化三联体，通过对活性丝氨酸进行定点诱变得到了验证。
