定向进化提高磺酰脲类除草剂脱毒酯酶SulE的活性

Improvement of enzyme activity of sulfonylurea herbicide-detoxifying esterase SulE by directed evolution

磺酰脲除草剂脱毒酯酶SulE对噻磺隆活性极高,但对其他磺酰脲类除草剂活性很低,这限制了在磺酰脲除草剂残留污染生物修复和抗磺酰脲除草剂作物培育中的应用价值.利用Escherichiacoli DH10B (ilvG+)对甲磺隆的敏感性为筛选标记,从sulE基因随机突变库中筛选获得了一个酶活性显著提高的突变体mSulE.与野生型相比,突变体mSulE对甲磺隆、甲嘧磺隆、苯磺隆和胺苯磺隆的比酶活分别提高了8.29、8.61、1.50和4.04倍.测序结果表明,突变体mSulE氨基酸序列发生了2处替换,分别为Y81T和S178N.三维结构模拟表明,2个突变位点都在酶活性中心附近,突变体mSulE酶活提高的原因可能是Y81突变为T增加了催化口袋的面积,以及S178突变为N增加了氨基酸残基之间的氢键作用力.综上所述,突变体mSulE对大部分磺酰脲除草剂的活性要高于野生型,在磺酰脲除草剂残留污染生物修复和抗除草剂转基因工程中具有很好的应用前景.(图6表1参30)

Esterase SulE detoxifies a variety of sulfonylurea herbicides with a methyl or ethyl ester. SulE shows high activity against thifensulfuron-methyl;however, SulE shows low activity against many chemically stable and persistent sulfonylureas, such as metsulfuron-methyl, sulfometuron-methyl, and ethametsulfuron-methyl, which limit its application. In order to improve the enzyme activity of SulE, the sulE gene was subjected to random mutation through error-prone PCR, followed by an efficient screening protocol based on the sensitivity of the host Escherichia coli DH10 B(ilvG+) to metsulfuron-methyl. A mutant, mSulE, with improved activity was screened from the mutation library. The specific activities of mSulE for metsulfuron-methyl, sulfometuron-methyl, tribenuron-methyl, and ethametsulfuron-methyl increased by 8.29-, 8.61-, 1.50-, and 4.04-fold, respectively, in comparison with the wild type SulE. Sequence analysis revealed that mSulE contains two amino acid substitutions(Y81 T and S178 N). Molecular modeling suggested that the activity improvement of mSulE is due to the substitution of Y81 by T and S178 by N, leading to an increase in catalytic pocket area and in hydrogen bonding between amino acids. This study provides a theoretical basis for the elucidation of the structure and function of SulE. In addition, the study provides an improved degradation/detoxification gene for the elimination of sulfonylurea residue and herbicide-resistant transgenic engineering.