Alteration of lipase chain length specificity in the hydrolysis of esters by random mutagenesis
Alteration of lipase chain length specificity in the hydrolysis of esters by random mutagenesis
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DOI：
10.1002/bit.1077
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发表时间：
2001-06-20
影响因子：
3.8
通讯作者：
Vulfson, EN
中科院分区：
文献类型：
--
作者：
Gaskin, DJH;Romojaro, A;Vulfson, EN
通过随机突变米黑根毛霉（Rhizomucor miehei）脂肪酶（Rhizomucor miehei lipase）负责容纳底物酰基链的蛋白质沟中的Phe 94，研究了改变其链长特异性的可行性。重组脂肪酶在E.杆菌选择单个菌落，使其生长，并测定脂肪酶基因的DNA序列。鉴定了19种可能的突变体中的14种，并将这些突变体中的每一种转化到胞外表达该酶的巴斯德毕赤酵母中。使酵母生长，并在几个测定中用长链和短链底物评估上清液。在此基础上，筛选出一个突变体Phe 94 Gly，并将其纯化至均一以用于进一步分析。发现用甘氨酸取代苯丙氨酸94产生的酶对试卤灵酯的活性低约6倍，但对短链底物如丁酸酯的活性高3-4倍。观察到的酶特异性的改变是合理的使用可用的3D结构的脂肪酶。(C)John Wiley & Sons，Inc.
The feasibility of altering the chain length specificity of industrially important Rhizomucor miehei lipase was investigated by randomly mutating Phe94 in the protein groove which is responsible for accommodating the acyl chain of the substrate. The recombinant lipase was initially expressed in E. coli. Individual colonies were selected, grown, and the DNA sequence of the lipase gene determined. Fourteen of the 19 possible mutants were identified and each of these was transformed into Pichia pastoris which expresses the enzyme extracellularly. The yeast was grown and the supernatants assessed in several assays with long and short chain substrates. Based on this preliminary screen, one mutant Phe94Gly, was selected and purified to homogeneity for further analysis. It was found that the substitution of phenylalanine 94 with glycine led to an enzyme which was about six times less active against resorufin ester but displayed 3-4 times higher activity with short chain substrates such as butyric acid esters. The observed alteration to the enzyme specificity was rationalised using the available 3D structure of the lipase. (C) 2001 John Wiley & Sons, Inc.