Meilleur Catherine, Hupé Jean-François, Juteau Pierre, Shareck François
INRS-Institut Armand-Frappier, Laval, QC, Canada.
J Ind Microbiol Biotechnol. 2009 Jun;36(6):853-61. doi: 10.1007/s10295-009-0562-7. Epub 2009 Mar 31.
The construction of a cosmid library from the biomass produced in an enriched Sequencing Fed-Batch Reactor allowed the isolation of a new lipase by functional screening. The open reading frame of 928 bp encoded a polypeptide of 308 amino acids with a molecular mass of 32.6 kDa. The amino acid sequence analysis revealed the presence of the conserved pentapeptide GXSXG essential for lipase activity. Alignment with known sequences of proteins showed no more than 52% identity with different lipases, confirming the discovery of a novel gene sequence. The lipase was cloned and expressed in Streptomyces lividans and further purified by a combination of hydrophobic interaction and size-exclusion chromatography. Spectrophotometric assays with different p-nitrophenyl esters demonstrated a preference for long-length acyl chains, especially p-nitrophenylmyristate (C14). Moreover, the enzyme presented an optimal activity at 60 degrees C and at alkaline pH of 10.5.
从富集测序补料分批反应器中产生的生物质构建黏粒文库，通过功能筛选分离出一种新的脂肪酶。928 bp的开放阅读框编码一个由308个氨基酸组成的多肽，分子量为32.6 kDa。氨基酸序列分析显示存在对脂肪酶活性至关重要的保守五肽GXSXG。与已知蛋白质序列比对表明，与不同脂肪酶的同一性不超过52%，证实发现了一个新的基因序列。该脂肪酶在淡紫链霉菌中克隆并表达，然后通过疏水相互作用和尺寸排阻色谱相结合的方法进一步纯化。用不同的对硝基苯酯进行分光光度测定表明，该酶偏好长链酰基，尤其是肉豆蔻酸对硝基苯酯（C14）。此外，该酶在60℃和碱性pH值10.5时呈现最佳活性。
