醇脱氢酶在聚乙烯膜表面的固定化研究

doi:10.11949/0438-1157.20200509

摘要/Abstract

摘要：

以聚丙烯酸（PAA）改性的聚乙烯（PE）膜为载体，研究了醇脱氢酶（ADH）的两种固定化路线，并以甲醛为底物考察了固定化酶的催化性能。路线1用聚乙烯亚胺（PEI）进一步改性，使用戊二醛（GA）固定化ADH。最优固定化pH为6.0，温度为5～15℃，酶浓度为1.0 mg/ml，GA浓度为0.01%(质量)；固定化酶的最适反应pH为6.5，温度为15～30℃，反应速率最高为9.6 μmol/(L·min)；重复利用10次后可保持47.3%的活性。路线2以PAA-PE为载体，用1-(3-二甲氨基丙基)-2-乙基碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS）为活化剂，固定化ADH。EDC和NHS最优摩尔比为1∶0.5，固定化时间为24 h；固定化酶的最适反应pH为6.5，温度为20～37℃，反应速率为15.58 μmol/(L·min)；重复利用10次后可保持53.8%的活性。

关键词: 醇脱氢酶, 聚乙烯膜, 聚乙烯亚胺, 酶固定化, 共价结合

Abstract:

Using polyacrylic acid (PAA) modified polyethylene (PE) membrane as a carrier, two immobilization routes of alcohol dehydrogenase (ADH) were studied, and the catalytic performance of immobilized enzyme was investigated using formaldehyde as a substrate. In the first route, PAA-PE membrane was further modified by polyethyleneimine (PEI) and then ADH was covalently linked by glutaraldehyde (GA) to the surface of PEI/PAA-PE. The results show that the optimal immobilization pH was 6.0, immobilization temperature was 5—15℃, ADH and GA concentrations were 1.0mg/ml and 0.01%(mass). For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 15—30℃, and the highest reaction rate was 9.6 μmol/(L·min), the remaining activity was 47.3% after 10 use cycles. In the second route, ADH was immobilized on PAA-PE membrane with 1-(3-dimethylaminopropyl)-2-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as activators. The results show that the optimal molar ratio of EDC and NHS was 1∶0.5, and the immobilization time was 24 h. For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 20—37℃, and the highest reaction rate was 15.58 μmol/(L·min), 53.8% activity was remained after 10 cycles.

Key words: alcohol dehydrogenase, polyethylene film, polyethylenimine, enzyme immobilization, covalently bonding

中图分类号:

O 643.36⁺1

郭梦雅, 季书馨, 谷凤娟, 孟子晖, 刘文芳, 王燕子. 醇脱氢酶在聚乙烯膜表面的固定化研究[J]. 化工学报, 2020, 71(9): 4246-4254.

Mengya GUO, Shuxin JI, Fengjuan GU, Zihui MENG, Wenfang LIU, Yanzi WANG. Immobilization of alcohol dehydrogenase on the surface of polyethylene membrane[J]. CIESC Journal, 2020, 71(9): 4246-4254.

图/表 7

图1 PEI/PAA-PE膜的制备及固定化ADH路线(a)与PAA-PE固定化ADH路线(b)

Fig.1 Preparation of PEI/PAA-PE membrane and immobilization route of ADH on it (a) and immobilization route of ADH on PAA-PE (b)

图2 PE(a)、PAA-PE(b)和PEI/PAA-PE(c)膜的红外谱图

Fig.2 FTIR spectra of PE(a), PAA-PE (b) and PEI/PAA-PE (c) membrane

图3 PE中空纤维膜外表面的SEM照片

Fig.3 SEM micrographs of PE, PAA-PE and PEI/PAA-PE

图4 固定化pH(a)、温度(b)、酶浓度(c)和GA浓度(d)对ADH-PEI/PAA-PE催化性能的影响

Fig.4 Effects of pH (a), temperature (b), enzyme (c) and GA concentration (d) in the process of immobilization on the catalytic performance of ADH-PEI/PAA-PE

图5 EDC/NHS摩尔比(a)、固定化时间(b)和pH(c)对固定化效率和ADH-PAA-PE催化性能的影响

Fig.5 Effects of the molar ratio of EDC and NHS (a), immobilized time (b) and pH (c) on immobilization efficiency and the catalytic performance of ADH-PAA-PE

图6 反应pH(a)、温度(b)对固定化和游离ADH催化性能的影响

Fig.6 Effects of pH (a) and temperature (b) in the reaction on the catalytic performance of immobilized and free ADH

图7 ADH-PAA-PE和ADH-PEI/PAA-PE 的重复利用性

Fig.7 Reuseability of ADH-PAA-PE and ADH-PEI/PAA-PE

参考文献 46

1	许松伟, 姜忠义, 吴洪. 醇脱氢酶结构和作用机理研究进展[J]. 有机化学, 2005, 25(6): 629-633.
	Xu S W, Jiang Z Y, Wu H. Progress in structure and kinetic mechanism of alcohol dehydrogenase[J]. Chinese Journal of Organic Chemistry, 2005, 25(6): 629-633.
2	Ramonas E, Ratautas D, Dagys M, et al. Highly sensitive amperometric biosensor based on alcohol dehydrogenase for determination of glycerol in human urine[J]. Talanta, 2019, 200: 333-339.
3	张晓霞, 毛跟年, 张云丽. 乙醇脱氢酶应用研究现状[J]. 动物医学进展, 2007, (12): 92-95.
	Zhang X X, Mao G N, Zhang Y L. Current research and applications of alcohol dehydrogenase[J]. Progress in Veterinary Medicine, 2007, (12): 92-95.
4	胡建强, 刘凤兰. 血清乙醇脱氢酶活性测定及临床应用[J]. 天津医科大学学报, 2001, (1): 110-111.
	Hu J Q, Liu F L. Determination of serum ethanol dehydrogenase activity and its clinical application[J]. Journal of Tianjin Medical University, 2001, (1): 110-111.
5	余带兵, 李红梅, 高露姣, 等. 磁性纳米颗粒固定化乙醇脱氢酶的研究[J]. 工业微生物, 2019, 49(1): 26-32.
	Yu D B, Li H M, Gao L J, et al. Study on immobilized ethanol dehydrogenase by magnetic nanoparticles[J]. Industrial Microbiology, 2019, 49(1): 26-32.
6	Zhou Z, Hartmann M. Progress in enzyme immobilization in ordered mesoporous materials and related applications[J]. Chemical Society Reviews, 2013, 42(9): 3894-3912.
7	吴兆明, 杨敏, 孙颖, 等. 酶固定化载体材料的研究进展[J]. 食品安全质量检测学报, 2018, 9(5): 1031-1037.
	Wu Z M, Yang M, Sun Y, et al. Research progress of immobilized enzyme carrier materials[J]. Journal of Food Safety and Quality, 2018, 9(5): 1031-1037.
8	Ghannadi S, Abdizadeh H, Miroliaei M, et al. Immobilization of alcohol dehydrogenase on titania nanoparticles to enhance enzyme stability and remove substrate inhibition in the reaction of formaldehyde to methanol[J]. Industrial & Engineering Chemistry Research, 2019, (58): 9844-9854.
9	Jia H, Zhu G, Wang P. Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobility[J]. Biotechnology and Bioengineering, 2003, 84(4): 406-414.
10	Kim J, Grate J W, Wang P. Nanostructures for enzyme stabilization[J]. Chemical Engineering Science, 2006, 61(3): 1017-1026.
11	Marques Netto C G C, Andrade L H, Freitas R S, et al. Association of yeast alcohol dehydrogenase with superparamagnetic nanoparticles: improving the enzyme stability and performance[J]. Journal of Nanoscience and Nanotechnology, 2015, 15(12): 9482-9487.
12	Liu L, Yu J, Chen X. Enhanced stability and reusability of alcohol dehydrogenase covalently immobilized on magnetic graphene oxide nanocomposites[J]. Journal of Nanoscience and Nanotechnology, 2015, 15(2): 1213-1220.
13	Li X S, Zhu G T, Luo Y B, et al. Synthesis and applications of functionalized magnetic materials in sample preparation[J]. Trends in Analytical Chemistry, 2013, 45: 233-247.
14	Alsafadi D, Paradisi F. Covalent immobilization of alcohol dehydrogenase (ADH2) from Haloferax volcanii: how to maximize activity and optimize performance of halophilic enzymes[J]. Molecular Biotechnology, 2014, 56(3): 240-247.
15	Shinde P, Musameh M, Gao Y, et al. Immobilization and stabilization of alcohol dehydrogenase on polyvinyl alcohol fibre[J]. Biotechnology Reports, 2018, 19: e00260.
16	Kuo C H, Chen G J, Twu Y K, et al. Optimum lipase immobilized on diamine-grafted PVDF membrane and its characterization[J]. Industrial & Engineering Chemistry Research, 2012, 51(14): 5141-5147.
17	Cen Y K, Liu Y X, Xue Y P, et al. Immobilization of enzymes in/on membranes and their applications[J]. Advanced Synthesis & Catalysis, 2019, 361(24): 5500-5515.
18	Sen D, Sarkar A, Gosling A, et al. Feasibility study of enzyme immobilization on polymeric membrane: a case study with enzymatically galacto-oligosaccharides production from lactose[J]. Journal of Membrane Science, 2011, 378(1): 471-478.
19	Luo J Q, Meyer A S, Jonsson G, et al. Fouling-induced enzyme immobilization for membrane reactors[J]. Bioresource Technology, 2013, 147: 260-268.
20	Marpani F, Luo J, Mateiu R V, et al. In situ formation of a biocatalytic alginate membrane by enhanced concentration polarization[J]. ACS Applied Materials & Interfaces, 2015, 7(32): 17682-17691.
21	Ismail F H, Marpani F, Othman N H, et al. Simultaneous separation and biocatalytic conversion of formaldehyde to methanol in enzymatic membrane reactor[J]. Chemical Engineering Communications, 2019. DOI: 10.1080/00986445.2019.1705795. DOI
22	Hoffmann C, Silau H, Pinelo M, et al. Surface modification of polysulfone membranes applied for a membrane reactor with immobilized alcohol dehydrogenase[J]. Materials Today Communications, 2018, 14: 160-168.
23	Xu T W, Fu R Q, Yan L F. A new insight into the adsorption of bovine serum albumin onto porous polyethylene membrane by zeta potential measurements, FTIR analyses, and AFM observations[J]. Journal of Colloid and Interface Science, 2003, 262(2): 342-350.
24	Lee S Y, Park S Y, Song H S. Lamellar crystalline structure of hard elastic HDPE films and its influence on microporous membrane formation[J]. Polymer, 2006, 47(10): 3540-3547.
25	Shen L Q, Xu Z K, Xu Y Y. Preparation and characterization of microporous polyethylene hollow fiber membranes[J]. Journal of Applied Polymer Science, 2002, 84(1): 203-210.
26	陈创, 于俊荣, 王新威, 等. 超高相对分子质量聚乙烯中空纤维膜的制备与性能研究[J]. 化工新型材料, 2015, 43(1): 43-45.
	Chen C, Yu J R, Wang X W, et al. Preparation and property of ultrahigh molecular weight polyethylene hollow fiber membranes[J]. New Chemical Materials, 2015, 43(1): 43-45.
27	Al-Obeidani S K S, Al-Hinai H, Goosen M F A, et al. Chemical cleaning of oil contaminated polyethylene hollow fiber microfiltration membranes[J]. Journal of Membrane Science, 2008, 307(2): 299-308.
28	Barsbay M, Güven O. RAFT mediated grafting of poly(acrylic acid) (PAA) from polyethylene/polypropylene (PE/PP) nonwoven fabric via preirradiation[J]. Polymer, 2013, 54(18): 4838-4848.
29	Nasef M M, Güven O. Radiation-grafted copolymers for separation and purification purposes: status, challenges and future directions[J]. Progress in Polymer Science, 2012, 37(12): 1597-1656.
30	Virgen-Ortíz J, Santos J, Berenguer-Murcia A, et al. Polyethylenimine: a very useful ionic polymer in the design of immobilized enzyme biocatalysts[J]. Jounral of Materials Chemistry B, 2017, 5(36): 7461-7490.
31	Staros J V, Wright R W, Swingle D M. Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions[J]. Analytical Biochemistry, 1986, 156(1): 220-222.
32	Marshall T, Williams K M. Coomassie blue protein dye-binding assays measure formation of an insoluble protein-dye complex[J]. Analytical Biochemistry, 1992, 204(1): 107-109.
33	Wang Y Z, Chen Y Z, Wang C H, et al. Polyethylenimine-modified membranes for CO₂ capture and in situ hydrogenation[J]. ACS Applied Materials & Interfaces, 2018, 10(34): 29003-29009.
34	Migneault I, Dartiguenave C, Bertrand M J, et al. Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking[J]. BioTechniques, 2004, 37(5): 790-802.
35	Cuatrecasas P. Immobilized enzymes[J]. Science, 1974, 184(4132): 56.
36	Chui W K, Wan L S C. Prolonged retention of cross-linked trypsin in calcium alginate microspheres[J]. Journal of Microencapsulation, 1997, 14(1): 51-61.
37	Nakajima N, Ikada Y. Mechanism of amide formation by carbodiimide for bioconjugation in aqueous media[J]. Bioconjugate Chemistry, 1995, 6(1): 123-130.
38	Sehgal D, Vijay I K. A method for the high efficiency of water-soluble carbodiimide-mediated amidation[J]. Analytical Biochemistry, 1994, 218(1): 87-91.
39	Gilles M A, Hudson A Q, Borders C L. Stability of water-soluble carbodiimides in aqueous solution[J]. Analytical Biochemistry, 1990, 184(2): 244-248.
40	曹林秋. 载体固定化酶: 原理、应用和设计[M]. 北京: 化学工业出版社, 2008: 137.
	Cao L Q. Carrier Immobilized Enzymes: Principle, Application and Design[M]. Beijing: Chemical Industry Press, 2008: 137.
41	陈清西. 酶学及其研究技术[M]. 厦门: 厦门大学出版社, 2015: 123-125.
	Chen Q X. Enzymology and Its Research Techniques[M]. Xiamen: Xiamen University Press, 2015: 123-125.
42	Wang C, Yan Q, Liu H B, et al. Different EDC/NHS activation mechanisms between PAA and PMAA brushes and the following amidation reactions[J]. Langmuir, 2011, 27(19): 12058-12068.
43	Li J, Wu H, Liang Y, et al. Facile fabrication of organic-inorganic hybrid beads by aminated alginate enabled gelation and biomimetic mineralization[J]. Journal of Biomaterials Science, Polymer Edition, 2013, 24(2): 119-134.
44	Hanefeld U, Gardossi L, Magner E. Understanding enzyme immobilisation[J]. Chemical Society Reviews, 2009, 38(2): 453-468.
45	Soni S, Desai J D, Devi S. Immobilization of yeast alcohol dehydrogenase by entrapment and covalent binding to polymeric supports[J]. Journal of Applied Polymer Science, 2001, 82(5): 1299-1305.
46	Cantone S, Ferrario V, Corici L, et al. Efficient immobilisation of industrial biocatalysts: criteria and constraints for the selection of organic polymeric carriers and immobilisation methods[J]. Chemical Society Reviews, 2013, 42(15): 6262-6276.

[1]	王立晖, 刘焕, 李赫宇, 郑晓冰, 姜艳军, 高静. 核壳结构磁性树枝状纤维形有机硅固定化脂肪酶制备及其应用[J]. 化工学报, 2021, 72(9): 4861-4871.
[2]	梁兴唐, 李凤枝, 钟书明, 张瑞瑞, 焦淑菲, 汪双双, 尹艳镇. 聚乙烯亚胺原位改性多孔灯芯草高效吸附废水中的Cr（Ⅵ）[J]. 化工学报, 2021, 72(6): 3380-3389.
[3]	吴玉玲, 邵明龙, 周武林, 高惠芳, 张显, 徐美娟, 杨套伟, 饶志明. 重组大肠杆菌表达17β-羟基类固醇脱氢酶全细胞催化合成宝丹酮的研究[J]. 化工学报, 2020, 71(7): 3229-3237.
[4]	王彩红, 孙婧, 季书馨, 王燕子, 刘文芳. 聚乙烯亚胺/多巴胺改性氧化硅固定碳酸酐酶[J]. 化工学报, 2019, 70(5): 1887-1893.
[5]	梁兴唐, 钟书明, 刘子杰, 郑韵英, 张瑞瑞, 焦淑菲, 廖日权, 王芸, 尹艳镇. 甲壳素/聚乙烯亚胺复合物对水溶液中Cr(Ⅵ)的吸附[J]. 化工学报, 2018, 69(5): 2255-2262.
[6]	孙雪飞, 高勇强, 赵颂, 张文, 王志, 王晓琳. 胍基聚合物接枝改性制备抗菌抗污染超滤膜[J]. 化工学报, 2018, 69(11): 4869-4878.
[7]	杨长青, 郑涛, 郁晨晨, 周俊, 何冰芳, 刘晓宁. 触角状环氧链结构对固定化脂肪酶(YCJ01)酶活的影响[J]. 化工学报, 2014, 65(5): 1815-1820.
[8]	赵道辉, 彭春望, 廖晨伊, 周健. 面向生物能源的酶固定化的计算机模拟[J]. 化工学报, 2014, 65(5): 1828-1834.
[9]	宋菲菲, 王玉军, 骆广生. 拟薄水铝石负载PEI/AMP吸附CO₂性能[J]. 化工学报, 2013, 64(2): 574-580.
[10]	梅佳军，张常青，刘嵘明，马江锋，陈可泉，姜岷. 利用改性载体固定化大肠杆菌产琥珀酸[J]. 化工进展, 2013, 32(01): 161-165.
[11]	赵文瑛, 王丽香, 李振山, 蔡宁生. 不同胺基CO₂固体吸收剂的热稳定性能[J]. 化工学报, 2012, 63(10): 3304-3309.
[12]	岳雅娟, 刘清芝, 伍联营, 胡仰栋. 有机分子在聚乙烯膜中扩散过程的分子动力学模拟[J]. 化工学报, 2012, 63(1): 109-113.
[13]	陶维红，杨立荣，徐刚，邰玉蕾，王立，吴坚平. 磁性多孔微粒对脂肪酶的固定化 [J]. CIESC Journal, 2011, 30(7): 1584-.
[14]	谌容1，2 ，王秋岩1，殷晓浦1，魏东芝2，谢恬1. 醇脱氢酶不对称还原制备手性醇的研究进展 [J]. CIESC Journal, 2011, 30(7): 1562-.
[15]	王翠，姜艳军，周丽亚，高静. 纳米氧化硅固定辣根过氧化物酶处理苯酚废水 [J]. CIESC Journal, 2011, 62(7): 2026-2032.