Lee Hyun Woo, Jung Won Kyeong, Kim Yong Ho, Ryu Bum Han, Kim T Doohun, Kim Jungho, Kim Hoon
Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Republic of Korea.
Department of Agricultural Chemistry, Sunchon National University, Suncheon 57922, Republic of Korea.
J Microbiol Biotechnol. 2016 Feb;26(2):315-25. doi: 10.4014/jmb.1509.09081.
A novel esterase gene, est7K, was isolated from a compost metagenomic library. The gene encoded a protein of 411 amino acids and the molecular mass of the Est7K was estimated to be 44,969 Da with no signal peptide. Est7K showed the highest identity of 57% to EstA3, which is an esterase from a drinking water metagenome, when compared with the enzymes with reported properties. Est7K had three motifs, SMTK, YSV, and WGG, which correspond to the typical motifs of family VIII esterases, SxxK, Yxx, and WGG, respectively. Est7K did not have the GxSxG motif in most lipolytic enzymes. Three additional motifs, LxxxPGxxW, PLGMxDTxF, and GGxG, were found to be conserved in family VIII enzymes. The results of the phylogenetic analysis and the alignment study suggest that family VIII enzymes could be classified into two subfamilies, VIII.1 and VIII.2. The purified Est7K was optimally active at 40°C and pH 10.0. It was activated to exhibit a 2.1-fold higher activity by the presence of 30% methanol. It preferred short-length p-nitrophenyl esters, particularly p-nitrophenyl butyrate, and efficiently hydrolyzed glyceryl tributyrate. It did not hydrolyze β-lactamase substrates, tertiary alcohol esters, glyceryl trioleate, fish oil, and olive oil. Est7K preferred an Senantiomer, such as (S)-methyl-3-hydroxy-2-methylpropionate, as the substrate. The tolerance to methanol and the substrate specificity may provide potential advantage in the use of the enzyme in pharmaceutical and other biotechnological processes.
从一个堆肥宏基因组文库中分离出了一个新的酯酶基因est7K。该基因编码一个由411个氨基酸组成的蛋白质，Est7K的分子量估计为44969 Da，且没有信号肽。与已报道特性的酶相比，Est7K与来自饮用水宏基因组的酯酶EstA3的同源性最高，为57%。Est7K有三个基序，分别为SMTK、YSV和WGG，它们分别对应于VIII族酯酶的典型基序SxxK、Yxx和WGG。Est7K在大多数脂解酶中没有GxSxG基序。还发现三个额外的基序LxxxPGxxW、PLGMxDTxF和GGxG在VIII族酶中保守。系统发育分析和比对研究结果表明，VIII族酶可分为两个亚家族，VIII.1和VIII.2。纯化后的Est7K在40°C和pH 10.0时活性最佳。在30%甲醇存在下，它被激活，活性提高2.1倍。它更喜欢短链对硝基苯酯，尤其是对硝基丁酸苯酯，并能有效水解三丁酸甘油酯。它不水解β-内酰胺酶底物、叔醇酯、三油酸甘油酯、鱼油和橄榄油。Est7K更喜欢S型对映体，如(S)-3-羟基-2-甲基丙酸甲酯，作为底物。对甲醇的耐受性和底物特异性可能为该酶在制药和其他生物技术过程中的应用提供潜在优势。
