Effect of Site-directed Mutagenesis of Amino Acids in Lid Region on the Enzymatic Properties of T1 Lipase

doi:10.13560/j.cnki.biotech.bull.1985.2020-0220

Abstract

Abstract:

T1 lipase from strain Geobacillus zalihae is often used as a biocatalyst in various fields. In order to comprehensively improve the enzymatic properties of T1 lipase,three mutants L188M,A190L and A190Y with better effect were obtained by rational design of lid domain in the early stage of study. On this basis,two combined mutants L188M/A190L and L188M/A190Y were obtained by combining single point mutants. Then the enzymatic properties of the lipase mutants were studied,and the effects of the single and combined mutants were compared. The results showed that the optimal pH of wild ones wt-T1 and mutants was 9.0,and the optimal temperature of the combined mutants increased by 5℃. For other enzymatic properties,both single and combined mutation led to the enzymatic properties of T1 lipase improved at varied degrees,and the effect of the combined mutants was more significant. The combined mutants significantly increased the temperature and pH stability of lipase,and the tolerance to organic solvent dimethyl sulfoxide and n-hexadecane of L188M/A190L was 1.88 times and 2.73 times that of the wt-T1,and that of L188M/A190Y was 1.96 times and 2.58 times that of the wt-T1. The selectivity to C16 and C18 substrates of L188M/A190L was also significantly enhanced,which was 1.65 and 2.7 times that of the wt-T1,and that to L188M/A190Y was 1.5 times and 2.25 times that to the wt-T1. The results broaden the application prospect of T1 lipase to a certain extent,and provide certain theoretical basis for the modification of other lipases.

Key words: T1 lipase, lid domain, single point mutant, combined mutant, enzymatic properties

ZHU Cai-lin, LÜ Xiang, XIA Xiao-le. Effect of Site-directed Mutagenesis of Amino Acids in Lid Region on the Enzymatic Properties of T1 Lipase[J]. Biotechnology Bulletin, 2020, 36(11): 94-102.

Figures/Tables 12

References 33

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突变体	引物（5'-3'）
A190L	上游：5'- AAAGCAGTGTTAGAACTGGCCGCCGTGGC- AAGCAACGTG -3'
	下游：5'- GCTTGCCACGGCGGCCAGTTCTAACACTGC- TTTCTGCAG -3'
L188M	上游：5'- CTGCAGAAAGCAGTGATGGAAGCAGCCGCC- GTGGCA -3'
	下游：5'- TGCCACGGCGGCTGCTTCCATCACTGCTTTC- TGCAG -3'
A190Y	上游： 5'-CAGAAAGCAGTGTTAGAATACGCCGCCGTGG- CAAGC-3'
	下游： 5'-GCTTGCCACGGCGGCGTATTCTAACACTGCTTT- CTG-3'

突变体	引物（5'-3'）
A190L	上游：5'- AAAGCAGTGTTAGAACTGGCCGCCGTGGC- AAGCAACGTG -3'
	下游：5'- GCTTGCCACGGCGGCCAGTTCTAACACTGC- TTTCTGCAG -3'
L188M	上游：5'- CTGCAGAAAGCAGTGATGGAAGCAGCCGCC- GTGGCA -3'
	下游：5'- TGCCACGGCGGCTGCTTCCATCACTGCTTTC- TGCAG -3'
A190Y	上游： 5'-CAGAAAGCAGTGTTAGAATACGCCGCCGTGG- CAAGC-3'
	下游： 5'-GCTTGCCACGGCGGCGTATTCTAACACTGCTTT- CTG-3'

试剂	体积/μL
ddH₂O	18
2×Max Buffer	25
dNTP Mix（10 mmol/L）	1
pET28a-T1	1
上游引物（10 μmol/L）	2
下游引物（10 μmol/L）	2
Phanta Max Super-Fidelity DNA Ploymerase	1

试剂	体积/μL
ddH₂O	18
2×Max Buffer	25
dNTP Mix（10 mmol/L）	1
pET28a-T1	1
上游引物（10 μmol/L）	2
下游引物（10 μmol/L）	2
Phanta Max Super-Fidelity DNA Ploymerase	1

温度/℃	时间	循环数
95	30 s	1
95	15 s
68	15 s	30
72	6 min
72	5 min	1