Chiş Laura, Hriscu Monica, Bica Adriana, Toşa Monica, Nagy Gergely, Róna Gergely, G Vértessy Beata, Dan Irimie Florin
Department of Biochemistry and Biochemical Engineering, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, Cluj-Napoca, RO-400028, Romania.
J Gen Appl Microbiol. 2013;59(2):119-34. doi: 10.2323/jgam.59.119.
A thermophilic strain producing an extracellular esterase/lipase was isolated from a hot spring in Tăşnad, Romania, and was identified phenotypically and by 16S rDNA sequencing as Anoxybacillus flavithermus (GenBank ID: JQ267733). The gene encoding the putative carboxyl esterase (GenBank ID: JX494348) was cloned by direct PCR amplification from genomic DNA. The protein, consisting of 246 amino acids and having a predicted molecular weight of 28.03 kDa, is encoded by an ORF of 741 bps. Expression was achieved in Escherichia coli and a recombinant protein with esterolytic activity and estimated molecular weight of 25 kDa was recovered and purified from the periplasmic fraction by IMAC. The purified enzyme, most active at 60-65°C and in the near-neutral range (pH 6.5-8), displayed a half-life at 60°C of about 5 h. Est/Lip displayed a relative tolerance to methanol, DMSO, acetonitrile, and low detergent concentrations (SDS, Triton) increased its thermostability. Highest activity was attained with p-nitrophenyl butyrate, but the enzyme was also able to hydrolyze long chain fatty acid esters, as well as triolein. The primary sequence and predicted tridimensional structure of the enzyme are very similar to those of other Anoxybacillus and Geobacillus carboxyl esterases in a distinct, recently described lipase family. Est/Lip was highly enantioselective, with preference for the (S)-enantiomer of substrates.
从罗马尼亚塔什纳德的一处温泉中分离出一株产胞外酯酶/脂肪酶的嗜热菌株，通过表型鉴定和16S rDNA测序将其鉴定为嗜热栖热放线菌（GenBank登录号：JQ267733）。通过从基因组DNA直接PCR扩增克隆了编码假定羧基酯酶的基因（GenBank登录号：JX494348）。该蛋白质由246个氨基酸组成，预测分子量为28.03 kDa，由一个741 bp的开放阅读框编码。在大肠杆菌中实现了表达，并从周质部分通过IMAC回收和纯化了一种具有酯解活性、估计分子量为25 kDa的重组蛋白。纯化后的酶在60 - 65°C和近中性范围（pH 6.5 - 8）最具活性，在60°C下的半衰期约为5小时。Est/Lip对甲醇、二甲基亚砜、乙腈表现出相对耐受性，低浓度去污剂（SDS、Triton）可提高其热稳定性。对硝基苯丁酸的活性最高，但该酶也能够水解长链脂肪酸酯以及三油酸甘油酯。该酶的一级序列和预测的三维结构与其他嗜热栖热放线菌和嗜热栖热芽孢杆菌羧基酯酶在一个独特的、最近描述的脂肪酶家族中非常相似。Est/Lip具有高度对映选择性，优先选择底物的（S）-对映体。
