Purification and characterization of a new cold active lipase, EnL A from Emericella nidulans NFCCI 3643
Purification and characterization of a new cold active lipase, EnL A from Emericella nidulans NFCCI 3643
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DOI：
10.5897/ajb2015.14674
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发表时间：
2015-06
影响因子：
--
通讯作者：
Suseela Lanka;J. Latha
中科院分区：
文献类型：
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作者：
Suseela Lanka;J. Latha
从印度 A.P. 西戈达瓦里区 Pedavegi 棕榈油厂废水倾倒场的土壤样品中分离出一种产生细胞外冷活性脂肪酶的嗜温真菌，并被鉴定为 Emericella nidulans。通过硫酸铵分级纯化酶，然后使用苯基琼脂糖凝胶进行疏水相互作用层析。该酶的纯度是粗品的 35 倍，比活性为 1494.51 U/mg。 SDS PAGE分析表明该蛋白质是单体，MW为约54kDa，并且酶谱分析表明纯化的蛋白质具有活性。表征研究表明，最适温度为 30°C，最适 pH 为 5。K m 和 V max 值分别为 0.61 mM 和 322.58 mM/min.mg。通过 MALDI TOF-MS 分析和随后的 BLAST P 分析对纯化蛋白进行测序，表明该蛋白是来自构巢蛋白的推定秘书脂肪酶。脂肪酶工程数据库(LED)检索表明该蛋白属于新引入的南极假丝酵母脂肪酶A样超家族和曲霉脂肪酶样同源家族。关键词：冷活性脂肪酶，构巢艾美氏菌，疏水作用色谱，南极假丝酵母脂肪酶 A 样。
A mesophilic fungi producing an extracellular cold-active lipase was isolated from the soil samples of palm oil mill effluent dump sites, Pedavegi, West Godavari Dist, A.P. India and was identified as Emericella nidulans . The enzyme was purified by ammonium sulfate fractionation followed by hydrophobic interaction chromatography using phenyl sepharose. The enzyme was 35 fold pure compared to crude with a specific activity of 1494.51 U/mg. SDS PAGE analysis revealed that the protein is monomeric with a MW of ˜54 kDa and zymogram analysis showed that the purified protein was active. Characterization studies revealed that the temperature optimum was at 30°C and an optimum pH of 5. The K m and V max values were found to be 0.61 mM and 322.58 mM/min.mg, respectively. Sequencing of the purified protein by MALDI TOF-MS analysis followed by BLAST P analysis indicated that the protein is a putative secretary lipase from E. nidulans. Search of lipase engineering data base (LED) revealed that this protein belongs to a newly introduced super family of Candida antarctica lipase A like and to the homologous family of Aspergillus lipase like. Key words : Cold active lipase, Emericella nidulans, hydrophobic interaction chromatography, Candida antarctica lipase A like.