枯草杆菌漆酶重要功能位点的保守性与可变性分析

doi:10.7503/cjcu20160092

摘要/Abstract

摘要：

在对比分析天然枯草杆菌漆酶(CAR)的一级结构与三维空间结构的基础上, 锁定了CAR的底物结合通道和铜离子结合位点, 设计并构建了CAR的4种突变体. 对比分析了野生型酶与4种突变体酶的性质及催化功能, 结果表明, 该漆酶分子中位于第二保守区的H155位点是漆酶催化所必需的, H155即使被其同义氨基酸(如Arg)替换, 也会因氧化还原电势的降低而降低漆酶的催化活力, 同时还会导致漆酶的热稳定性和酸碱稳定性降低, 以及最适pH值酸移; 位于非保守区的A158位点与I224位点的保守性对漆酶的催化是有利的, 而S210G突变增强了漆酶的稳定性与催化功能, 这可能与S210G突变增强了漆酶的底物通道上一段富含Pro片段的柔性有关. 此外, A158/R155H突变(CAHE)可能导致漆酶不耐冷, 在低温(<50 ℃)时引起酶活明显降低. 所研究的漆酶在2,2'-连氮-双(3-乙基苯并噻唑-6-磺酸)(ABTS)介体存在下, 对靛蓝类染料(如靛蓝胭脂红)和三苯甲烷类染料(如结晶紫)的脱色率均高于对偶氮类染料(如甲基红和刚果红)和蒽醌类染料(如活性亮蓝), 说明该漆酶对染料有一定的选择性.

关键词: 漆酶, 结构, 催化, 枯草芽孢杆菌

Abstract:

A laccase from Bacillus subtilis has more than 96% residue identity to most other bacterial laccases, but has lower catalytic activity. Four mutant laccases were designed and their catalytic properties and functions were investigated. The results demonstrated that residue H155 was particularly essential for the catalytic activity of this laccase, and was one of the key residues in the second conserved region of laccases. Replacement of this residue reduced the oxidation-reduction potential and the catalytic activity of the laccase, and also reduces its thermal or pH stability, and caused a shift of its optimum pH from 5.0 to 4.0. Conservation of residues A158 and I224 in non-conserved region of the laccase was also advantageous for the catalysis of the laccase. A158 was conducive to the electron transport in enzymatic oxidation process, while I224 was appropriate to make the substrate into the mononuclear center of the laccase. S210G mutation enhanced the catalytic efficiency of the laccase perhaps by increasing of the flexibility of the pro-rich loop adjacent to the entrance of the substrate channel of this laccase. The laccase in this study decolorized indigo dyes such as indigo carmine more effectively than triphenylmethane dyes such as crystal violet, azo dyes such as methyl red or Congo red, and anthraquinone dyes such as Remazol Brilliant Blue R(RBBR), indicated that the laccase in this study had certain substrate selectivity. These results would provide some insights into the relationship between the structure and function of bacterial laccases.

Key words: Laccase, Structure, Catalysis, Baculus subtilis

中图分类号:

O625.75
Q554

TrendMD:

焦晶, 杨雪, 金兰娜, 高键, 周洋, 肖亚中, 张应玖. 枯草杆菌漆酶重要功能位点的保守性与可变性分析. 高等学校化学学报, 2016, 37(7): 1320.

JIAO Jing, YANG Xue, JIN Lanna, GAO Jian, ZHOU Yang, XIAO Yazhong, ZHANG Yingjiu. Conservative and Variability of the Important Functional Sites in a Laccase from Bacillus Subtilis^†. Chem. J. Chinese Universities, 2016, 37(7): 1320.

图/表 9

Fig.1 3D structure of laccase CARFig.1(B) is an enlarged view of the white box in Fig.1(A). Balls: copper ions; solid arrows: the entrance of substrates; dashed arrows: electron transfer from the mononuclear centre to the trinuclear centre of CAR; orange peptide fragment: pro-rich peptide fragment.

Table 1 Designed primers

Primer	Sequence(5'→3')	Primer	Sequence(5'→3')
CAHS	CGATCACGCCATGGCGCTC	CAHEGS	TTATCCGGGCGCACCGGAA
CAHA	CCATGGCGTGATCGTGATACCAC	CAHEGA	TTCCGGTGCGCCCGGATAA
CAHES	TCACGCCATGGAGCTC	CAHEGFS	AATCCTTCATTCGTTC
CAHEA	TGAGCTCCATGGCGTGATC	CAHEGFA	GAACGAATGAAGGATT

Table 2 Final concentration and maximum absorbance wavelengths of the tested dyes

Dye	M_w/Da	c/(mg·L^-1)	λ_max/nm
Indigo carmine	466.37	40	610
Crystal violet	407.98	5	590
Gongo red	696.67	10	595
Methyl red	269.30	50	410
Remazol brilliant blue R(RBBR)	626.54	100	591

Fig.2 SDS-PAGE analysis of the purified laccases Lane M: protein markers; lanes 1—3: purified laccase CAHEG,CAH and CAHEGF; lanes 4 and 5: purified laccase CAHE.

Table 3 Specific activities and oxidation-reduction potentials of the wild type and mutant laccases

Enzyme	Specific activity/ (U·mg^-1)	Oxidation-reduction potential/V	Enzyme	Specific activity/ (U·mg^-1)	Oxidation-reduction potential/V
CAR	17.1	0.320	CAHEG	99.0	0.395
CAH	220.4	0.420	CAHEGF	67.7	0.345
CAHE	70.2	0.385

Table 4 Kinetic parameters of the wild type and mutant laccases

Enzyme	K_m/(mmol·L^-1)	k_cat/s^-1	k_cat· $K m - 1$ /(L·s^-1·mmol^-1)
CAR	0.17	26.0	153.0
CAHE	0.13	34.0	263.0
CAHEGF	0.14	34.0	243.0
CAHEG	0.12	35.0	292.0
CAH	0.10	85.0	850.0

Table 4 Kinetic parameters of the wild type and mutant laccases

Enzyme	K_m/(mmol·L^-1)	k_cat/s^-1	k_cat· $K m - 1$ /(L·s^-1·mmol^-1)
CAR	0.17	26.0	153.0
CAHE	0.13	34.0	263.0
CAHEGF	0.14	34.0	243.0
CAHEG	0.12	35.0	292.0
CAH	0.10	85.0	850.0

Fig.3 Thermostabilities(A) and acid-base stabilities(B) of the wild type and mutant laccases

Fig.4 Decolorization of several dyes by the wild type and mutant laccases(A) CAH; (B) CAHEG; (C) CAHEGF; (D) CAR. ● Indigo carmine; □ crystal violet; △ gongo red; ▲ methyl red; ○ RBBR.

Fig.5 3D structures and the active sites of wild type and mutant laccases Ball: copper ions; P: Pro residue; arrow: entrance of substrate.

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