手性脯氨酸类离子液体化学修饰猪胰脂肪酶催化性能研究
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摘要
通过化学修饰同时提高脂肪酶包括选择性在内的酶学性能具有重要意义。以N-乙酰-脯氨酸作为手性源制备的四种离子液体对猪胰脂肪酶(PPL)进行化学修饰,通过三硝基苯磺酸法(TNBS)测定了修饰度,采用水解4-硝基苯酚棕榈酸酯的反应测定酶活。正如预期的一样,所有修饰酶的酶学性能均得到显著改善。其中修饰酶的热稳定性、有机溶剂耐受性、不同温度和pH下的耐受性甚至对映体选择性均得到同时提升。结果也表明,离子液体的组成及构型对修饰酶的酶学性能有较大程度的影响,经过[BMIM][N-AC-L-pro]修饰后的PPL各项催化性能明显优于[BMIM][N-AC-D-pro],而经过[N-AC-L-pro][Cl]和[N-AC-D-pro][Cl]修饰的PPL催化性能差异较小。经过[N-AC-L-pro][Cl]修饰后PPL展现出最高的酶活,达到原酶的2.5倍。经过[BMIM][N-AC-L-pro]修饰后的PPL展现出最佳的酶学性能,在酶活提高了1.0倍的同时,热稳定性提高了2.6倍(50℃,2.5 h),对映体选择性提高了1.5倍,在强极性质子溶剂甲醇中的稳定性也提升了1.3倍,且在不同温度、pH下也展现出了较好的稳定性与耐受性。通过荧光光谱和圆二色谱表征证明,手性离子液体成功嫁接到PPL的表面,引起脂肪酶构象的改变,从而促使酶的催化性能发生改善。通过该研究,手性离子液体被证实为是一种可用于酶分子改造并提升选择性在内各项催化性能的新型且有效的修饰剂。
It is of great value to improve the enzymatic properties including selectivity by chemical modification at the same time. Four kinds of ionic liquids, prepared from N-acetyl-proline as chiral source, were used to modify porcine pancreatic lipase(PPL). Modification degree was determined by 2,4,6-trinitrobenzene sulfonic acid(TNBS)method, and enzyme activity was determined by the hydrolysis of 4-nitrophenyl palmitate(pNPP). As expected, the catalytic properties of PPL were changed obviously after modification. The thermal stability, organic solvent tolerance, adaptability to temperature and pH changes, even enantio selectivity of all the modified enzymes was improved simultaneously. Research results also showed that the composition and configuration of ionic liquids(Ils)had an important influence on the enzymatic properties of modified enzymes. The PPL modified by [BMIM] [N-ACL-pro] showed obvious better catalytic performance than that modified by [BMIM] [N-AC-D-pro], while the catalytic properties of PPL modified by [N-AC-L-pro] [Cl] and [N-AC-D-pro] [Cl] only showed slight difference.PPL modified by [N-AC-L-pro] [Cl] showed the highest activity, increasing by 2.5 folds as much as the original enzyme. Overall, PPL modified by [BMIM] [N-AC-L-pro] demonstrated the best enzymatic performance: the hydrolysis activity was increased by 1.0 time, the optimum temperature was raised to 55℃, the thermostability was improved by 2.6 times(50℃,2.5 h), the enantio selectivity was increased by 1.5 times, the stability in the strong polar protonic solvent of methanol was enhanced by 1.3 times, and also showed better tolerance of temperature and pH. The fluorescence spectroscopy and circular dichroism spectroscopy(CD) characterizations confirmed that chiral proline ionic liquids were grafted onto the surface of PPL successfully, resulting in the conformation change of PPLs, which led to the improvements of the catalytic performance of modified PPLs. Through this study, chiral ionic liquids have proven to be a new and effective modifier for various catalytic properties of enzyme molecular modification and selectivity enhancement.
It is of great value to improve the enzymatic properties including selectivity by chemical modification at the same time. Four kinds of ionic liquids, prepared from N-acetyl-proline as chiral source, were used to modify porcine pancreatic lipase(PPL). Modification degree was determined by 2,4,6-trinitrobenzene sulfonic acid(TNBS)method, and enzyme activity was determined by the hydrolysis of 4-nitrophenyl palmitate(pNPP). As expected, the catalytic properties of PPL were changed obviously after modification. The thermal stability, organic solvent tolerance, adaptability to temperature and pH changes, even enantio selectivity of all the modified enzymes was improved simultaneously. Research results also showed that the composition and configuration of ionic liquids(Ils)had an important influence on the enzymatic properties of modified enzymes. The PPL modified by [BMIM] [N-ACL-pro] showed obvious better catalytic performance than that modified by [BMIM] [N-AC-D-pro], while the catalytic properties of PPL modified by [N-AC-L-pro] [Cl] and [N-AC-D-pro] [Cl] only showed slight difference.PPL modified by [N-AC-L-pro] [Cl] showed the highest activity, increasing by 2.5 folds as much as the original enzyme. Overall, PPL modified by [BMIM] [N-AC-L-pro] demonstrated the best enzymatic performance: the hydrolysis activity was increased by 1.0 time, the optimum temperature was raised to 55℃, the thermostability was improved by 2.6 times(50℃,2.5 h), the enantio selectivity was increased by 1.5 times, the stability in the strong polar protonic solvent of methanol was enhanced by 1.3 times, and also showed better tolerance of temperature and pH. The fluorescence spectroscopy and circular dichroism spectroscopy(CD) characterizations confirmed that chiral proline ionic liquids were grafted onto the surface of PPL successfully, resulting in the conformation change of PPLs, which led to the improvements of the catalytic performance of modified PPLs. Through this study, chiral ionic liquids have proven to be a new and effective modifier for various catalytic properties of enzyme molecular modification and selectivity enhancement.
引文
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[4]李明,方银军,李在均,等.新型对称烷基咪唑离子液体介质中酶催化合成l-乙酸薄荷酯[J].化学学报, 2009, 67(11):1252-1258.Li M, Fang Y J, Li Z J. et al. Synthesis of l-menthyl acetate catalyzed by lipase in new symmetrical 1, 3-dialkylimidazolium ionic liquids[J]. Acta Chim. Sinica, 2009, 67(11):1252-1258.
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[9] Wahl J, Holzgrabe U. Capillary electrophoresis separation of phenethylamine enantiomers using amino acid based ionic liquids[J]. J. Pharm. Biomed. Anal., 2018, 148:245-250.
[10] Liu W, Zhu X S. Simultaneous determination of Magnolol and Honokiol by amino acid ionic liquid synchronous fluorescence spectrometry[J]. Spectrochim Acta, Part A, 2018, 196:141-147.
[11] Gathergood N, Garcia M T, Scammells P J. Biodegradable ionic liquids(Part I):Concept, preliminary targets and evaluation[J].Green Chem., 2004, 6(3):166-175.
[12] Docherty K M, Kulpa J C F. Toxicity and antimicrobial activity of imidazolium and pyridinium ionic liquids[J]. Green Chem., 2005,7(4):185-189.
[13] Fukumoto K, Ohno H. Design and synthesis of hydrophobic and chiral anions from amino acids as precursor for functional ionic liquids[J]. Chemical Communications, 2006,(29):3081-3083.
[14] Chado G R, Holland E N, Tice A K, et al. Modification of lipase with poly(4-acryloylmorpholine)enhances solubility and transesterification activity in anhydrous ionic liquids[J].Biomacromolecules, 2018, 19(4):1324-1332.
[15] Ueji S, Ueda A, Tanaka H, et al. Chemical modification of lipases with various hydrophobic groups improves their enantioselectivity in hydrolytic reactions[J]. Biotechnol. Lett., 2003, 25(1):83-87.
[16] Zhao H, Song Z Y. Nuclear magnetic relaxation of water in ionicliquid solutions:determining the kosmotropicity of ionic liquids and its relationship with the enzyme enantioselectivity[J]. Journal of Chemical Technology&Biotechnology, 2007, 82(3):304-312.
[17] Evran S, Telefoncu A. Modification of porcine pancreatic lipase with Z-proline[J]. Prep. Biochem. Biotechnol., 2005, 35(3):191-201.
[18] Nwagu T N, Okolo B, Aoyagi H, et al. Chemical modification with phthalic anhydride and chitosan:viable options for the stabilization of raw starch digesting amylase from Aspergillus carbonarius[J]. Int. J. Biol. Macromol., 2017, 99:641-647.
[19] Li C, Huang Z L, Dong L, et al. Improvement of enzymological properties of pepsin by chemical modification with chitooligosaccharides[J]. Int. J. Biol. Macromol., 2018, 118:216-227.
[20] Xu C, Yin X H, Zhang C, et al. Improving catalytic performance of burkholderiacepacia lipase by chemical modification with functional ionic liquids[J]. Chem. Res. Chin. Univ., 2018, 34(2):279-284.
[21] Jia R, Hu Y, Liu L, et al. Enhancing catalytic performance of porcine pancreatic lipase by covalent modification using functional ionic liquids[J]. ACS Catal., 2013, 3(9):1976-1983.
[22] Li X J, 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.
[23]熊亚红,苏健鸿,刘小平.邻苯二甲酸酐修饰脂肪酶的性能研究[J].华南农业大学学报, 2011, 32(2):122.Xiong Y X, Su J H, Liu X P. Study on the property of pocine pancreas lipase modified with phthalic anhydride[J]. Journal of South China Agricultural University, 2011, 32(2):122.
[24]张川,张鲁嘉,张洋,等.基于分子模拟的离子液体修饰Porcine Pancreas脂肪酶催化性能和稳定性的相关研究[J].化学学报, 2016, 74:74-80,Zhang C, Zhang L J, Zhang Y, et al. Study on the stability and enzymatic property improvement of porcine pancreas lipase modified by ionic liquids using molecular simulation[J]. Acta Chim. Sinica, 2016, 74:74-80.
[25] Zhao H. Are ionic liquids kosmotropic or chaotropic? An evaluation of available thermodynamic parameters for quantifying the ion kosmotropicity of ionic liquids[J]. Journal of Chemical Technology&Biotechnology, 2006, 81(6):877-891.
[26] Silman I H, Weissenberg M A, Katchalski E. Some waterinsoluble papain derivatives[J]. Biopolymers, 1966, 4(4):441-448.
[27] Sangeetha K, Abraham T E. Chemical modification of papain for use in alkaline medium[J]. Journal of Molecular Catalysis B:Enzymatic, 2006, 38:171-177.
[28]张静,田亚平.丁二酸酐修饰对枯草芽孢杆菌氨肽酶结构及酶学特性的影响[J].食品与生物技术学报, 2013, 32(6):622-627.Zhang J, Tian Y P. Chemical modification of the Bacillus subtilis aminopeptidase by succinic anhydride and its enzyme properties[J]. Journal of Food Science and Biotechnology, 2013, 32(6):622-627.
[29]贺俊斌,林日辉,龙寒,等.乙二胺四乙酸二酐对草酸脱羧酶的修饰改性[J].化工学报, 2016, 67(10):4389-4398.He J B, Lin R H, Long H, et al. Chemical modification of oxalate decarboxylase with ethylenediaminetetraacetic dianhydride[J].CIESC Journal, 2016, 67(10):4389-4398.
[30] 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.
[31] 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]. Bioprocess Biosyst. Eng., 2014, 37(8):1617-1626.
[32] Ladokhin A S, Jayasinghe S, White S H. How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?[J]. Anal. Biochem., 2000, 285(2):235-245.
[33] Lozano P, De Diego T, Iborra J L. Dynamic structure/function relationships in the alpha-chymotrypsin deactivation process by heat and pH[J]. Eur. J. Biochem., 1997, 248(1):80-85.
[34] Shokri M M, Ahmadian S, Akbari N, et al. Hydrophobic substitution of surface residues affects lipase stability in organic solvents[J]. Mol. Biotechnol., 2014, 56:360-368.
[35] Rahman R N Z A, Tejo B A, Basri M, et al. Reductive alkylation of lipase-experimental and molecular modeling approaches[J].Appl. Biochem. Biotechnol., 2004, 118:11-20.
[2] Vekariya R L. A review of ionic liquids:applications towards catalytic organic transformations[J]. J. Mol. Liq., 2017, 227:44-60.
[3] Itoh T. Ionic liquids as tool to improve enzymatic organic synthesis[J]. Chem. Rev., 2017, 117(15):10567-10607.
[4]李明,方银军,李在均,等.新型对称烷基咪唑离子液体介质中酶催化合成l-乙酸薄荷酯[J].化学学报, 2009, 67(11):1252-1258.Li M, Fang Y J, Li Z J. et al. Synthesis of l-menthyl acetate catalyzed by lipase in new symmetrical 1, 3-dialkylimidazolium ionic liquids[J]. Acta Chim. Sinica, 2009, 67(11):1252-1258.
[5] Tao G H, He L, Sun N, et al. New generation ionic liquids:cations derived from amino acids[J]. Chem. Commun.(Cambridge, U K),2005, 28(28):3562-3564.
[6]吴阳,张甜甜,李静蕊.半胱氨酸阴离子与咪唑阳离子间相互作用的理论研究[J].化学学报, 2009, 67(16):1851-1858.Wu Y, Zhang T T, Li J R. A theoretical investigation of interaction between 1-ethyl-3-methylimidazolium cation and cysteine anion[J]. Acta Chim. Sinica, 2009, 67(16):1851-1858.
[7] Mu X Y, Qi L, Zhang H Z, et al. Ionic liquids with amino acids as cations:novel chiral ligands in chiral ligand-exchange capillary electrophoresis[J]. Talanta, 2012, 97:349-354.
[8] Pereira M P, de Souza Martins R, de Oliveira M A L, et al. Amino acid ionic liquids as catalysts in a solvent-free Morita–Baylis–Hillman reaction[J]. RSC Advances, 2018, 8(42):23903-23913.
[9] Wahl J, Holzgrabe U. Capillary electrophoresis separation of phenethylamine enantiomers using amino acid based ionic liquids[J]. J. Pharm. Biomed. Anal., 2018, 148:245-250.
[10] Liu W, Zhu X S. Simultaneous determination of Magnolol and Honokiol by amino acid ionic liquid synchronous fluorescence spectrometry[J]. Spectrochim Acta, Part A, 2018, 196:141-147.
[11] Gathergood N, Garcia M T, Scammells P J. Biodegradable ionic liquids(Part I):Concept, preliminary targets and evaluation[J].Green Chem., 2004, 6(3):166-175.
[12] Docherty K M, Kulpa J C F. Toxicity and antimicrobial activity of imidazolium and pyridinium ionic liquids[J]. Green Chem., 2005,7(4):185-189.
[13] Fukumoto K, Ohno H. Design and synthesis of hydrophobic and chiral anions from amino acids as precursor for functional ionic liquids[J]. Chemical Communications, 2006,(29):3081-3083.
[14] Chado G R, Holland E N, Tice A K, et al. Modification of lipase with poly(4-acryloylmorpholine)enhances solubility and transesterification activity in anhydrous ionic liquids[J].Biomacromolecules, 2018, 19(4):1324-1332.
[15] Ueji S, Ueda A, Tanaka H, et al. Chemical modification of lipases with various hydrophobic groups improves their enantioselectivity in hydrolytic reactions[J]. Biotechnol. Lett., 2003, 25(1):83-87.
[16] Zhao H, Song Z Y. Nuclear magnetic relaxation of water in ionicliquid solutions:determining the kosmotropicity of ionic liquids and its relationship with the enzyme enantioselectivity[J]. Journal of Chemical Technology&Biotechnology, 2007, 82(3):304-312.
[17] Evran S, Telefoncu A. Modification of porcine pancreatic lipase with Z-proline[J]. Prep. Biochem. Biotechnol., 2005, 35(3):191-201.
[18] Nwagu T N, Okolo B, Aoyagi H, et al. Chemical modification with phthalic anhydride and chitosan:viable options for the stabilization of raw starch digesting amylase from Aspergillus carbonarius[J]. Int. J. Biol. Macromol., 2017, 99:641-647.
[19] Li C, Huang Z L, Dong L, et al. Improvement of enzymological properties of pepsin by chemical modification with chitooligosaccharides[J]. Int. J. Biol. Macromol., 2018, 118:216-227.
[20] Xu C, Yin X H, Zhang C, et al. Improving catalytic performance of burkholderiacepacia lipase by chemical modification with functional ionic liquids[J]. Chem. Res. Chin. Univ., 2018, 34(2):279-284.
[21] Jia R, Hu Y, Liu L, et al. Enhancing catalytic performance of porcine pancreatic lipase by covalent modification using functional ionic liquids[J]. ACS Catal., 2013, 3(9):1976-1983.
[22] Li X J, 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.
[23]熊亚红,苏健鸿,刘小平.邻苯二甲酸酐修饰脂肪酶的性能研究[J].华南农业大学学报, 2011, 32(2):122.Xiong Y X, Su J H, Liu X P. Study on the property of pocine pancreas lipase modified with phthalic anhydride[J]. Journal of South China Agricultural University, 2011, 32(2):122.
[24]张川,张鲁嘉,张洋,等.基于分子模拟的离子液体修饰Porcine Pancreas脂肪酶催化性能和稳定性的相关研究[J].化学学报, 2016, 74:74-80,Zhang C, Zhang L J, Zhang Y, et al. Study on the stability and enzymatic property improvement of porcine pancreas lipase modified by ionic liquids using molecular simulation[J]. Acta Chim. Sinica, 2016, 74:74-80.
[25] Zhao H. Are ionic liquids kosmotropic or chaotropic? An evaluation of available thermodynamic parameters for quantifying the ion kosmotropicity of ionic liquids[J]. Journal of Chemical Technology&Biotechnology, 2006, 81(6):877-891.
[26] Silman I H, Weissenberg M A, Katchalski E. Some waterinsoluble papain derivatives[J]. Biopolymers, 1966, 4(4):441-448.
[27] Sangeetha K, Abraham T E. Chemical modification of papain for use in alkaline medium[J]. Journal of Molecular Catalysis B:Enzymatic, 2006, 38:171-177.
[28]张静,田亚平.丁二酸酐修饰对枯草芽孢杆菌氨肽酶结构及酶学特性的影响[J].食品与生物技术学报, 2013, 32(6):622-627.Zhang J, Tian Y P. Chemical modification of the Bacillus subtilis aminopeptidase by succinic anhydride and its enzyme properties[J]. Journal of Food Science and Biotechnology, 2013, 32(6):622-627.
[29]贺俊斌,林日辉,龙寒,等.乙二胺四乙酸二酐对草酸脱羧酶的修饰改性[J].化工学报, 2016, 67(10):4389-4398.He J B, Lin R H, Long H, et al. Chemical modification of oxalate decarboxylase with ethylenediaminetetraacetic dianhydride[J].CIESC Journal, 2016, 67(10):4389-4398.
[30] 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.
[31] 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]. Bioprocess Biosyst. Eng., 2014, 37(8):1617-1626.
[32] Ladokhin A S, Jayasinghe S, White S H. How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?[J]. Anal. Biochem., 2000, 285(2):235-245.
[33] Lozano P, De Diego T, Iborra J L. Dynamic structure/function relationships in the alpha-chymotrypsin deactivation process by heat and pH[J]. Eur. J. Biochem., 1997, 248(1):80-85.
[34] Shokri M M, Ahmadian S, Akbari N, et al. Hydrophobic substitution of surface residues affects lipase stability in organic solvents[J]. Mol. Biotechnol., 2014, 56:360-368.
[35] Rahman R N Z A, Tejo B A, Basri M, et al. Reductive alkylation of lipase-experimental and molecular modeling approaches[J].Appl. Biochem. Biotechnol., 2004, 118:11-20.