Department of Molecular Biology and Genetics, Gebze Institute of Technology (GIT), 41400 Kocaeli, Türkiye.
J Biotechnol. 2013 Mar 10;164(1):123-9. doi: 10.1016/j.jbiotec.2012.12.016. Epub 2013 Jan 11.
To improve enzymatic activity of Bacillus pumilus lipases, DNA shuffling was applied to two lipase genes from local B. pumilus isolates. Using a high-throughput activity assay, the mutant with highest activity was selected. This chimeric mutant (L3-3), carrying two crossover positions and three point mutations, has a specific activity 6.4 and 8.2 times higher than the two parent enzymes. The mutant also is more tolerant to various detergents and organic solvents, and has a 9 times longer half-life at 50 °C. Homology modeling of mutant L3-3, based on the highly homologous B. subtilis lipase A, shows that the increased thermostability is likely due to structural rigidification and reduced surface hydrophobicity. Increased specific activity may result from the location of mutations close to the active site. Together, our results show that it is possible to evolve, by DNA shuffling, B. pumilus lipase variants with improved applicability as biocatalysts, even if the two parent enzymes are highly similar.
为了提高短小芽孢杆菌脂肪酶的酶活性，应用 DNA 重排技术对来自当地短小芽孢杆菌分离株的两个脂肪酶基因进行了改造。使用高通量活性测定法，选择了活性最高的突变体。该嵌合突变体（L3-3）具有两个交叉点和三个点突变，比两个亲本酶的比活性分别提高了 6.4 倍和 8.2 倍。该突变体还能耐受各种洗涤剂和有机溶剂，在 50°C 下半衰期延长了 9 倍。基于高度同源的枯草芽孢杆菌脂肪酶 A 对突变体 L3-3 进行同源建模表明，其热稳定性的提高可能是由于结构僵化和表面疏水性降低所致。比活性的提高可能是由于突变位置靠近活性位点。总之，我们的研究结果表明，即使两个亲本酶高度相似，也可以通过 DNA 重排技术来进化出具有更好应用价值的短小芽孢杆菌脂肪酶变体，作为生物催化剂。
