Synthesis of polymer grafted lipase and its effect on enzyme activity

doi:10.11949/0438-1157.20190254

Abstract

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

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

CLC Number:

TQ 925.6

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.

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.

Figures/Tables 7

Fig.1 Synthesis of polymer-grafted CRL via ATRP

Fig.2 Mass spectrometry of BIBS initiator

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

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

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

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

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

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

References 43

1	Sheldon R A , Van Pelt S . Enzyme immobilisation in biocatalysis: why, what and how [J]. Chem. Soc. Rev., 2013, 42(15): 6223-6235.
2	Hudson S , Cooney J , Magner E . Proteins in mesoporous silicates [J]. Angew. Chem. Intern. Ed., 2008, 47(45): 8582-8594.
3	王梦凡, 齐崴, 苏荣欣, 等 . 交联酶聚体技术研究进展 [J]. 化学进展, 2010, 22(1): 173-178.
	Wang M F , Qi W , Su R X , et al . Advances in cross-linked enzyme aggregates [J]. Prog. Chem., 2010, 22(1): 173-178.
4	Vasic-Racki D, History of industrial biotransformations—dreams and realities[M] //Liese A , Seelbach K , Wandrey C . Industrial Biotransformations. Weinhiem: Wiley-VCH Verlag GmbH & Co. KGaA, 2006:1-36.
5	Hartmann M , Kostrov X . Immobilization of enzymes on porous silicas—benefits and challenges [J]. Chem. Soc. Rev., 2013, 42(15): 6277-6289.
6	Sorensen M H , Ng J B S , Bergstrom L , et al . Improved enzymatic activity of Thermomyces lanuginosus lipase immobilized in a hydrophobic particulate mesoporous carrier [J]. J.Colloid Interf. Sci., 2010, 343(1): 359-365.
7	戈钧, 卢滇楠, 朱晶莹, 等 . 纳米酶催化剂制备方法研究进展 [J]. 化工学报, 2014, 65(7): 2668-2675.
	Ge J , Lu D N , Zhu J Y , et al . Advances in preparation of nanostructured enzyme catalysts [J]. CIESC Journal, 2014, 65(7): 2668-2675.
8	Verma P K , Giri A , Thanh N T K , et al . Superparamagnetic fluorescent nickel-enzyme nanobioconjugates: synthesis and characterization of a novel multifunctional biological probe [J]. J. Mater. Chem., 2010, 20(18): 3722-3728.
9	Duncan R . The dawning era of polymer therapeutics [J]. Nat. Rev. Drug Discov., 2003, 2(5): 347-360.
10	Velonia K , Rowan A E , Nolte R J M . Lipase polystyrene giant amphiphiles [J]. J.Am.Chem.Soc., 2002, 124(16): 4224-4225.
11	Zhu G Y , Wang P . Polymer-enzyme conjugates can self-assemble at oil/water interfaces and effect interfacial biotransformations [J]. J.Am.Chem.Soc., 2004, 126(36): 11132-11133.
12	Lele B S , Murata H , Matyjaszewski K , et al . Synthesis of uniform protein-polymer conjugates [J]. Biomacromolecules, 2005, 6(6): 3380-3387.
13	Murata H , Cummings C S , Koepsel R R , et al . Polymer-based protein engineering can rationally tune enzyme activity, pH-dependence, and stability [J]. Biomacromolecules, 2013, 14(6): 1919-1926.
14	Yang W K , Zhu L J , Cui Y C , et al . Improvement of site-directed protein-polymer conjugates: high bioactivity and stability using a soft chain-transfer agent [J]. ACS Appl. Mater. Inter., 2016, 8(25): 15967-15974.
15	Shimoboji T , Larenas E , Fowler T , et al . Temperature-induced switching of enzyme activity with smart polymer-enzyme conjugates [J]. Bioconjug. Chem., 2003, 14(3): 517-525.
16	Reetz M T . Lipases as practical biocatalysts [J]. Curr. Opin. Chem. Biol., 2002, 6(2): 145-150.
17	Reis P , Holmberg K , Watzke H , et al . Lipases at interfaces: a review [J]. Adv. Colloid Interf. Sci., 2009, 147/148: 237-250.
18	De Maria P D , Sanchez-Montero J M , Sinisterra J V , et al . Understanding Candida rugosa lipases: an overview [J]. Biotechnol. Adv., 2006, 24(2): 180-196.
19	Reetz M T , Zonta A , Simpelkamp J . Efficient heterogeneous biocatalysts by entrapment of lipase in hydrophobic sol-gel materials [J]. Angew. Chem. Intern. Ed., 1995, 34(3): 301-303.
20	Geng J , Biedermann F , Zzyed J M , et al . Supramolecular glycopolymers in water: a reversible route toward multivalent carbohydrate-lectin conjugates using cucurbit[8]uril [J]. Macromolecules, 2011, 44(11): 4276-4281.
21	Cummings C , Murata H , Koepsel R , et al . Tailoring enzyme activity and stability using polymer-based protein engineering [J]. Biomaterials, 2013, 34(30): 7437-7443.
22	Averick S , Simakova A , Park S , et al . ATRP under biologically relevant conditions: grafting from a protein [J]. ACS Macro. Lett., 2012, 1(1): 6-10.
23	Bohlen P , Stein S , Dairman W , et al . Fluorometric assay of proteins in the nanogram range [J]. Arch. Biochem. Biophys., 1973, 155(1): 213-220.
24	Micsonai A , Wien F , Bulyaki E , et al . BeStSel: a web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra [J]. Nucleic. Acids Res., 2018,46(W1): W315-W322.
25	Gupta N , Rathi P , Gupta R . Simplified para-nitrophenyl palmitate assay for lipases and esterases [J]. Anal. Biochem., 2002, 311(1): 98-99.
26	Wang W , Zhou W , Li J , et al . Comparison of covalent and physical immobilization of lipase in gigaporous polymeric microspheres [J]. Bioproc. Biosyst. Eng., 2015, 38(11): 2107-2115.
27	Yong Y , Bai Y X , Li Y F , et al . Characterization of Candida rugosa lipase immobilized onto magnetic microspheres with hydrophilicity [J]. Proc. Biochem., 2008, 43(11): 1179-1185.
28	Du M , Lu D , Liu Z . Design and synthesis of lipase nanogel with interpenetrating polymer networks for enhanced catalysis: molecular simulation and experimental validation [J]. J.Mol.Catal.B-Enzym., 2013, 88: 60-68.
29	Hu Y , Yang J , Jia R , et al . Chemical modification with functionalized ionic liquids: a novel method to improve the enzymatic properties of Candida rugosa lipase [J]. Bioproc. Biosyst. Eng., 2014, 37(8): 1617-1626.
30	Guo Y , Zhu X , Fang F , et al . Immobilization of enzymes on a phospholipid bionically modified polysulfone gradient-pore membrane for the enhanced performance of enzymatic membrane bioreactors [J]. Molecules, 2018, 23(1): 144.
31	Zhang R , Zhao L , Liu R . Deciphering the toxicity of bisphenol a to Candida rugosa lipase through spectrophotometric methods [J]. J.Photoch.Photobio.B, 2016, 163: 40-46.
32	Svendsen A . Lipase protein engineering [J]. BBA-Protein Struct. Mol. Enzymol., 2000, 1543(2): 223-238.
33	Grochulski P , Li Y , Schrag J D , et al . Insights into interfacial activation from an open structure of Candida rugosa lipase [J]. J.Biol.Chem., 1993, 268(17): 12843-12847.
34	Li X , Zhang C , Li S , et al . Improving catalytic performance of Candida rugosa lipase by chemical modification with polyethylene glycol functional ionic liquids [J]. Ind. Eng. Chem. Res., 2015, 54(33): 8072-8079.
35	Du M L , Lu D N , Liu Z . Design and synthesis of lipase nanogel with interpenetrating polymer networks for enhanced catalysis: molecular simulation and experimental validation [J]. J.Mol.Catal.B-Enzym., 2013, 88: 60-68.
36	Keefe A J , Jiang S . Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity [J]. Nature Chemistry, 2012, 4(1): 60-64.
37	Verger R . Interfacial enzyme kinetics of lipolysis [J]. Annual Review of Biophysics and Bioengineering, 1976, 5: 77-117.
38	Gao B , Xu T , Lin J , et al . Improving the catalytic activity of lipase LipK107 from Proteus sp. by site-directed mutagenesis in the lid domain based on computer simulation [J]. J.Mol.Catal.B-Enzym., 2011, 68(3/4): 286-291.
39	张晓凤, 喻晓蔚, 徐岩 . 定点突变提高土曲霉Aspergillus terreus脂肪酶的催化活性 [J]. 生物工程学报, 2018, 34(7): 1091-1105.
	Zhang X F , Yu X W , Xu Y . Improvement of catalytic activity of Aspergillus terreus lipase by site-directed mutagenesis [J]. Chin.J.Biotechnol.,2018, 34(7): 1091-1105.
40	Zhang C Y , Dong X Y , Guo Z , et al . Remarkably enhanced activity and substrate affinity of lipase covalently bonded on zwitterionic polymer-grafted silica nanoparticles [J]. J. Colloid Interf. Sci., 2018, 519: 145-153.
41	Pegram L M , Record M T . Hofmeister salt effects on surface tension arise from partitioning of anions and cations between bulk water and the air-water interface [J]. J.Phys.Chem.B, 2007, 111(19): 5411-5417.
42	曲伟光, 魏荣卿, 何冰芳, 等 . 亲水梳状环氧聚合物载体柔性固定化脂肪酶 [J]. 催化学报, 2011, 32(12): 1869-1874.
	Qu W G , Wei R Q , He B F , et al . Flexible immobilization of lipase on hydrophobilic and comblike polymer support containing epoxy group [J]. Chin. J.Catal., 2011, 32(12): 1869-1874.
43	Lei L , Liu X , Li Y , et al . Study on synthesis of poly(GMA)-grafted Fe₃O₄/SiO _x magnetic nanoparticles using atom transfer radical polymerization and their application for lipase immobilization [J]. Mater. Chem. Phys., 2011, 125(3): 866-871.

[1]	Yepin CHENG, Daqing HU, Yisha XU, Huayan LIU, Hanfeng LU, Guokai CUI. Application of ionic liquid-based deep eutectic solvents for CO₂ conversion [J]. CIESC Journal, 2023, 74(9): 3640-3653.
[2]	Jie LIU, Lisheng WU, Jinjin LI, Zhenghong LUO, Yinning ZHOU. Preparation and properties of polyether-based vinylogous urethane reversible crosslinked polymers [J]. CIESC Journal, 2023, 74(7): 3051-3057.
[3]	Yaxin CHEN, Hang YUAN, Guanzhang LIU, Lei MAO, Chun YANG, Ruifang ZHANG, Guangya ZHANG. Advances in enzyme self-immobilization mediated by protein nanocages [J]. CIESC Journal, 2023, 74(7): 2773-2782.
[4]	Xiaoling TANG, Jiarui WANG, Xuanye ZHU, Renchao ZHENG. Biosynthesis of chiral epichlorohydrin by halohydrin dehalogenase based on Pickering emulsion system [J]. CIESC Journal, 2023, 74(7): 2926-2934.
[5]	Lei MAO, Guanzhang LIU, Hang YUAN, Guangya ZHANG. Efficient preparation of carbon anhydrase nanoparticles capable of capturing CO₂ and their characteristics [J]. CIESC Journal, 2023, 74(6): 2589-2598.
[6]	Shaoyun CHEN, Dong XU, Long CHEN, Yu ZHANG, Yuanfang ZHANG, Qingliang YOU, Chenglong HU, Jian CHEN. Preparation and adsorption properties of monolayer polyaniline microsphere arrays [J]. CIESC Journal, 2023, 74(5): 2228-2238.
[7]	Xuehong WU, Linlin LUAN, Yanan CHEN, Min ZHAO, Cai LYU, Yong LIU. Preparation and thermal properties of degradable flexible phase change films [J]. CIESC Journal, 2023, 74(4): 1818-1826.
[8]	Dong XU, Du TIAN, Long CHEN, Yu ZHANG, Qingliang YOU, Chenglong HU, Shaoyun CHEN, Jian CHEN. Preparation and electrochemical energy storage of polyaniline/manganese dioxide/polypyrrole composite nanospheres [J]. CIESC Journal, 2023, 74(3): 1379-1389.
[9]	Haiqin LIU, Bowen LI, Zhe LING, Liang LIU, Juan YU, Yimin FAN, Qiang YONG. Facile preparation and properties of chemically modified galactomannan films via mild hydroxy-alkyne click reaction [J]. CIESC Journal, 2023, 74(3): 1370-1378.
[10]	Yue HU, Shoujun MA, Xigao JIAN, Zhihuan WENG. Study on curing phthalonitrile resin with novel poly(phthalazinone ether nitrile) [J]. CIESC Journal, 2023, 74(2): 871-882.
[11]	Zhiyuan JIN, Guorong SHAN, Pengju PAN. Preparation and heat and salt resistance of AM/AMPS/SSS terpolymer [J]. CIESC Journal, 2023, 74(2): 916-923.
[12]	Shaojie ZHENG, Jianbin WANG, Jijiang HU, Bo-Geng LI, Wenbo YUAN, Zong WANG, Zhen YAO. Regulation of structure and mechanical properties of poly(propylene-butene) alloys by monomer composition switching [J]. CIESC Journal, 2023, 74(2): 904-915.
[13]	Yajing ZHAO, Jijiang HU, Suyun JIE, Bo-Geng LI. Modification of unsaturated polyester resin by HTPB: effect of introducing method of the rubber [J]. CIESC Journal, 2023, 74(2): 883-892.
[14]	Min LI, Xueru YAN, Xinlei LIU. Advances in benzimidazole-linked polymer adsorbents and membranes [J]. CIESC Journal, 2023, 74(2): 599-616.
[15]	Xin LIU, Jun GE, Chun LI. Light-driven microbial hybrid systems improve level of biomanufacturing [J]. CIESC Journal, 2023, 74(1): 330-341.