Masomian Malihe, Rahman Raja Noor Zaliha Raja Abd, Salleh Abu Bakar, Basri Mahiran
Enzyme and Microbial Technology Research Centre, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
PLoS One. 2016 Mar 2;11(3):e0149851. doi: 10.1371/journal.pone.0149851. eCollection 2016.
Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca(2+)-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65 °C and retained ≥ 97% activity after incubation at 50 °C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.
由于其在高温下固有的稳定性以及耐受苛刻有机溶剂的能力，耐热且耐有机溶剂的酶在工业中的广泛合成反应中具有巨大潜力。在本研究中，通过将嗜热嗜热栖热放线菌菌株HZ的基因组DNA文库构建到大肠杆菌质粒载体中，分离出了一个编码真正脂肪酶的新基因。序列分析表明，HZ脂肪酶与假类芽孢杆菌推定脂肪酶具有62%的同一性。与HZ脂肪酶基因密切相关的已表征脂肪酶来自属于亚家族I.5的耐热芽孢杆菌和嗜热栖热放线菌脂肪酶，同一性≤57%。HZ脂肪酶的氨基酸序列分析确定了该酶中一个包含活性丝氨酸的保守五肽GHSMG和一个Ca(2+)结合基序GCYGSD。蛋白质结构建模表明，HZ脂肪酶由α/β水解酶折叠和一个盖子结构域组成。蛋白质序列比对、保守区域分析、聚类距离矩阵和氨基酸组成说明了HZ脂肪酶与其他耐热脂肪酶之间的差异。系统发育分析表明，这种脂肪酶代表了细菌真正脂肪酶家族I的一个新亚家族，归类为家族I.9。HZ脂肪酶在Plac启动子下使用IPTG进行表达并进行了表征。重组酶在65℃时表现出最佳活性，在50℃孵育1小时后保留≥97%的活性。HZ脂肪酶在各种极性和非极性有机溶剂中均稳定。
