盖子区域氨基酸的定点突变对T1脂肪酶酶学性质的影响

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

摘要/Abstract

摘要：

T1脂肪酶来源于 Geobacillus zalihae 菌株,常作为生物催化剂广泛应用于各领域。为了综合改善T1脂肪酶的酶学性质,前期通过对该酶盖子结构进行理性设计得到3个效果较好的突变体L188M、A190L、A190Y,在此基础上,将单点突变体进行组合得到L188M/A190L和L188M/A190Y两个复合突变体。在该实验中对突变体脂肪酶酶学性质展开研究,同时将单点与组合突变效果进行比较。结果显示野生型及突变体最适pH均为9.0,复合突变体的最适温度提高5℃。单点突变及组合突变均使T1脂肪酶的酶学性质得到不同程度的改善,且复合突变体效果更显著。组合突变体使脂肪酶的温度、pH稳定性显著提高,L188M/A190L对于有机溶剂二甲基亚砜及正十六烷的耐受性为野生型的1.88倍和2.73倍,L188M/A190Y为野生型的1.96倍和2.58倍。此外,对C16和C18底物的选择性也明显增强,L188M/A190L为野生型的1.65和2.7倍,L188M/A190Y为野生型的1.5倍和2.25倍。该研究成果在一定程度上拓宽了T1脂肪酶的应用前景,同时为其他脂肪酶的改造提供了一定理论依据。

关键词: T1脂肪酶, 盖子结构, 单点突变, 组合突变, 酶学性质

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

朱彩林, 吕祥, 夏小乐. 盖子区域氨基酸的定点突变对T1脂肪酶酶学性质的影响[J]. 生物技术通报, 2020, 36(11): 94-102.

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.

图/表 12

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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