聚合物接枝脂肪酶的合成及其对酶活的影响

doi:10.11949/0438-1157.20190254

摘要/Abstract

摘要：

利用原子转移自由基聚合方法将含C₂～C₄烷基链的单体化合物分别接枝到皱褶假丝酵母脂肪酶（CRL）表面合成了HEMA-g-CRL、GMA-g-CRL、nPMA-g-CRL和BMA-g-CRL四种聚合物接枝CRL。CD和荧光光谱学分析显示，聚合物接枝导致HEMA-g-CRL、nPMA-g-CRL和BMA-g-CRL分子的α-螺旋和β-折叠含量增加。与此同时，聚合物接枝CRL分子荧光发射光谱也发生了蓝移，意味着其具有更加紧密的分子构象。酶活测定结果进一步显示，聚合物接枝增强CRL酶活227%～278%。随着单体化合物烷基链长增加，聚合物接枝CRL的K _m值由0.17 mmol·L^-1降至0.09 mmol·L^-1。与此同时，聚合物接枝CRL的k _cat值则提高至106～182 s^-1。BMA-g-CRL的催化效率达到了野生型CRL的3.28倍。这反映出聚合物接枝助力了CRL“盖子”结构开启和CRL活性中心暴露，促进底物的转化。稳定性测试表明，聚合物接枝提升了CRL的热稳定性并拓宽了其pH操作范围。

关键词: 生物催化, 皱褶假丝酵母脂肪酶, 原子转移自由基聚合, 聚合物, 疏水性, 脂肪酶活, 稳定性

Abstract:

In this work, four polymer-grafted Candida rugosa lipase (CRL) were synthesized via atom transfer radical polymerization by grafting hydroxyethyl methacrylate (HEMA), glycidyl methacrylate (GMA), propyl methacrylate (nPMA) and butyl methacrylate (BMA) onto CRL surface using N-(bromoisobutyryloxy) succinimide as the initiator. The results of CD and fluorescence emission spectra showed that the helical and sheet contents of CRL increased with polymer grafting, and exhibited a positive relation with the length of alkyl group in the monomer. Furthermore, a blue shift was also observed in fluorescence emission spectra of polymer-grafted CRL, meaning that polymer-grafted CRL had a more compact structure than wide-type CRL. Activity measurement of polymer-grafted CRL further showed that polymer grafting led to a significant increase of enzymatic activity of CRL in the order of CRL, HEMA-g-CRL = GMA-g-CRL, nPMA-g-CRL and BMA-g-CRL. With an increase of the length of alkyl group in the monomer, moreover, Michaelis parameter of CRL decreased from 0.17 mmol·L^-1 to 0.09 mmol·L^-1 whereas turnover number increased from 67 to 182 s^-1. Therefore, catalytic efficiency of polymer-grafted CRL enhanced greatly and the catalytic efficiency BMA-g-CRL was 3.28 times as high as that of wide-type CRL. It indicated that polymer grafting improved the movement of lid structure, leading to the exposure of the active site in CRL and enhancing the substrate transformation. Stability tests show that polymer grafting enhances the thermal stability of the CRL and broadens its pH range. The research proposed a novel methodology to regulate the CRL activity by polymer grafting, and exhibited the influence of alkyl groups of the monomers to CRL activity. The result in this research provided a benefit guidance for rational screening of monomer molecules to improve the performance of lipase in industrial biotransformation.

Key words: biocatalysis, Candida rugosa lipase, ATRP, polymers, hydrophobility, lipase activity, protein stability

中图分类号:

TQ 925.6

姜紫耀, 刘宗浩, 白姝, 史清洪. 聚合物接枝脂肪酶的合成及其对酶活的影响[J]. 化工学报, 2019, 70(9): 3473-3482.

Ziyao JIANG, Zonghao LIU, Shu BAI, Qinghong SHI. Synthesis of polymer grafted lipase and its effect on enzyme activity[J]. CIESC Journal, 2019, 70(9): 3473-3482.

图/表 7

图1 基于ATRP的聚合物接枝CRL合成

Fig.1 Synthesis of polymer-grafted CRL via ATRP

图2 引发剂BIBS的质谱结果

Fig.2 Mass spectrometry of BIBS initiator

图3 聚合物接枝前后CRL的凝胶过滤色谱及CD和荧光光谱表征结果

Fig.3 Size-exclusion chromatography, CD and fluorescence spectra of native and polymer-grafted CRL

表1 聚合物接枝CRL中α-螺旋和β-折叠含量

Table 1 α-Helix and β-sheet contents of wide-type and polymer-grafted CRLs

酶	α-螺旋含量 /%	β-折叠含量 /%
CRL	29.9	17.3
HEMA-g-CRL	37.0	18.9
GMA-g-CRL	21.1	19.0
nPMA-g-CRL	38.6	21.0
BMA-g-CRL	32.2	24.7

表2 聚合物接枝CRL相对活性及动力学参数

Table 2 Relative activities and kinetic parameters of polymer-grafted CRLs

酶	酶活/%	$V m a x$ ×10^-4/(mmol·s^-1L^-1)	K _m/(mmol·L^-1)	k _cat /s^-1	(k _cat/K _m)/(L·s^-1·mmol^-1)
CRL	100±15	2.99±0.05	0.16±0.01	67±1	429±1
HEMA-g-CRL	227±18	8.12±2.54	0.17±0.01	182±21	1098±24
GMA-g-CRL	228±25	4.69±0.01	0.09±0.01	106±2	1160±12
nPMA-g-CRL	236±19	6.61±0.22	0.13±0.02	149±5	1154±23
BMA-g-CRL	278±11	5.84±0.62	0.09±0.02	131±14	1406±19

表2 聚合物接枝CRL相对活性及动力学参数

Table 2 Relative activities and kinetic parameters of polymer-grafted CRLs

酶	酶活/%	$V m a x$ ×10^-4/(mmol·s^-1L^-1)	K _m/(mmol·L^-1)	k _cat /s^-1	(k _cat/K _m)/(L·s^-1·mmol^-1)
CRL	100±15	2.99±0.05	0.16±0.01	67±1	429±1
HEMA-g-CRL	227±18	8.12±2.54	0.17±0.01	182±21	1098±24
GMA-g-CRL	228±25	4.69±0.01	0.09±0.01	106±2	1160±12
nPMA-g-CRL	236±19	6.61±0.22	0.13±0.02	149±5	1154±23
BMA-g-CRL	278±11	5.84±0.62	0.09±0.02	131±14	1406±19

图4 聚合物接枝对CRL的热稳定性的影响

Fig.4 Effect of polymer grafting on stability of CRL enzymes

图5 pH对聚合物接枝CRL酶活的影响

Fig.5 Effect of pH on activity of polymer-grafted CRLs

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