Sun Huihui, Wang Hualei, Gao Wenyuan, Chen Lifeng, Wu Kai, Wei Dongzhi
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China.
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China.
Biochem Biophys Res Commun. 2015 Dec 25;468(4):820-5. doi: 10.1016/j.bbrc.2015.11.038. Epub 2015 Nov 11.
Nitrilase PpL19 from Pseudomonas psychrotolerans L19 can hydrolyze racemic mandelonitrile to (S)-mandelic acid with an enantiomeric excess (ee) value of 52.7%. In this study, random mutagenesis combined with site-directed mutagenesis was performed to identify the key residues responsible for nitrilase enantioselectivity. Five enzyme mutants exhibiting distinct selectivity were generated and four "hot spots" (M113, R128, A136, and I168) responsible for enantioselectivity toward mandelonitrile were identified and characterized. Furthermore, through saturation mutagenesis, positions 113 and 128 were confirmed to substantially influence the enantioselectivity of PpL19, and certain replacements of the methionine at position 113, in particular, were found to reverse the enantioselectivity of PpL19 from S- to R-selectivity. Two other single mutants of the enzyme, PpL19-A136Y and -I168Y, also showed reversed selectivity and preferentially produced (R)-mandelic acid (ee values: 66.7% and 74.3%, respectively). By combining the beneficial mutations, two enantiocomplementary nitrilase mutants, PpL19-LH and PpL19-GYY, were created, which exhibited high S- and R-selectivity toward mandelonitrile, respectively: PpL19-LH showed the highest S-selectivity toward mandelonitrile ever reported (91.1% ee), and, notably, the PpL19-GYY mutant was identified to be highly R-selective (90.1% ee) and thus an unexpected enantiocomplementary mutant for mandelonitrile.
来自耐冷假单胞菌L19的腈水解酶PpL19可将外消旋扁桃腈水解为对映体过量（ee）值为52.7%的（S）-扁桃酸。在本研究中，进行了随机诱变与定点诱变相结合的实验，以确定负责腈水解酶对映选择性的关键残基。生成了五个表现出不同选择性的酶突变体，并鉴定和表征了负责对扁桃腈对映选择性的四个“热点”（M113、R128、A136和I168）。此外，通过饱和诱变，证实第113位和第128位对PpL19的对映选择性有重大影响，特别是发现第113位甲硫氨酸的某些替换可使PpL19的对映选择性从S型转变为R型。该酶的另外两个单突变体PpL19-A136Y和-I168Y也表现出相反的选择性，并优先产生（R）-扁桃酸（ee值分别为66.7%和74.3%）。通过组合有益突变，创建了两个对映互补的腈水解酶突变体PpL19-LH和PpL19-GYY，它们分别对扁桃腈表现出高S选择性和R选择性：PpL19-LH对扁桃腈表现出有史以来报道的最高S选择性（91.1% ee），值得注意的是，PpL19-GYY突变体被鉴定为具有高R选择性（90.1% ee），因此是一种意想不到的对扁桃腈的对映互补突变体。
