INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
PLoS One. 2012;7(3):e32041. doi: 10.1371/journal.pone.0032041. Epub 2012 Mar 2.
The genome sequence of Streptomyces coelicolor A3(2) contains more than 50 genes coding for putative lipolytic enzymes. Many studies have shown the capacity of this actinomycete to store important reserves of intracellular triacylglycerols in nutrient depletion situations. In the present study, we used genome mining of S. coelicolor to identify genes coding for putative, non-secreted esterases/lipases. Two genes were cloned and successfully overexpressed in E. coli as His-tagged fusion proteins. One of the recombinant enzymes, EstC, showed interesting cold-active esterase activity with a strong potential for the production of valuable esters. The purified enzyme displayed optimal activity at 35°C and was cold-active with retention of 25% relative activity at 10°C. Its optimal pH was 8.5-9 but the enzyme kept more than 75% of its maximal activity between pH 7.5 and 10. EstC also showed remarkable tolerance over a wide range of pH values, retaining almost full residual activity between pH 6-11. The enzyme was active toward short-chain p-nitrophenyl esters (C2-C12), displaying optimal activity with the valerate (C5) ester (k(cat)/K(m) = 737±77 s(-1) mM(-1)). The enzyme was also very active toward short chain triglycerides such as triacetin (C2:0) and tributyrin (C4:0), in addition to showing good primary alcohol and organic solvent tolerance, suggesting it could function as an interesting candidate for organic synthesis of short-chain esters such as flavors.
链霉菌 A3(2) 的基因组序列包含 50 多个编码潜在脂解酶的基因。许多研究表明，这种放线菌在营养耗尽的情况下能够储存重要的细胞内三酰基甘油储备。在本研究中，我们使用链霉菌的基因组挖掘来鉴定编码潜在非分泌酯酶/脂肪酶的基因。克隆了两个基因，并成功地在大肠杆菌中作为 His 标记融合蛋白过表达。两种重组酶之一 EstC 表现出有趣的低温活性酯酶活性，具有生产有价值酯的巨大潜力。纯化的酶在 35°C 下显示出最佳活性，在 10°C 时保留 25%的相对活性，表现出低温活性。其最适 pH 值为 8.5-9，但该酶在 pH 7.5-10 之间保持超过 75%的最大活性。EstC 还在宽 pH 值范围内表现出显著的耐受性，在 pH 6-11 之间保留几乎全部剩余活性。该酶对短链对硝基苯酯（C2-C12）具有活性，对戊酸酯（C5）（k(cat)/K(m) = 737±77 s(-1) mM(-1)）显示出最佳活性。该酶对短链三酰基甘油如醋酸三乙酯（C2:0）和丁酸三丁酯（C4:0）也非常活跃，此外还表现出良好的伯醇和有机溶剂耐受性，表明它可能是短链酯如香料的有机合成的有趣候选物。
