修饰纤维素酶生物催化对薯蓣皂苷元转化的影响研究
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摘要
进入21世纪以来,随着化石资源与能源短缺、环境危机日益加剧,人类必须对传统的基于化学过程的物质加工模式(化学加工或化学制造)进行革命性转变,转向以生物可再生资源为原料的、环境友好的、高效的生物加工或生物制造模式。生物催化技术成为绿色化学与绿色化工发展的重要手段之一,它的发展必将对医药、能源、农业、食品、生命科学等领域产生巨大的影响。
天然酶具有蛋白质的特性,在催化底物的过程中,受到环境和其它条件的影响,容易变性失活,降低催化效率。在天然产物有效成分的提取转化中应用最广泛的生物催化剂-纤维素酶,由于热作用及酶与底物之间的吸附脱附作用,变得极不稳定,纤维素酶活性的稳定化成为亟需解决的问题之一。
本文选用三聚氯氰活化聚乙二醇(PEG)和单甲氧基聚乙二醇(MPEG),活化PEG和活化MPEG作为修饰剂,修饰天然纤维素酶,制备了不同分子量不同修饰度的修饰纤维素酶。通过实验发现,PEG-纤维素酶、MPEG-纤维素酶和天然纤维素酶均符合米氏酶的特性,并且米氏常数Km的关系为PEG-纤维素酶 改变实验条件,考察各种修饰酶和天然酶的热稳定性。实验结果表明,采用PEG2000修饰的修饰度为49%的修饰酶具有较高的热稳定性,同时修饰酶的稳定性均高于天然酶的稳定性,PEG修饰酶稳定性高于MPEG修饰酶。修饰酶还表现出具有和天然酶相同的适宜催化温度和pH。
将修饰酶应用于盾叶薯蓣中薯蓣皂苷元的提取转化,所得产品的质量和纯度都较高,催化效果优于阶梯生物催化。高效液相色谱分析产品纯度高达97.28%。并且修饰酶催化工艺中酸的用量得到了减小,降低了废物排放和环境污染。
本文还对PEG修饰酶的稳定性机理作了初步探讨,对PEG能够提高纤维素酶的稳定性机理从亲水性憎水性、吸附脱附、分子热动能理论上进行了深入分析。
Along with the petrochemical resources and the energy crisis, environment and the society crisis becomes worse increasingly, it is important to change the traditional chemical manufacturing which based on chemical process to the recycled, less- polluted, efficient biological process. The biocatalysis has become one of the important methods for develpoment of the green chemistry and green chemical engineering, and its development will have significant effect on medicine, agriculture, food, life science and so on.
For the reason that natural enzymes have properties of protein, natural enzymes are easy to deactivate and the efficiency of biocatalysis will be reduced consequently under the effect of reaction conditions. Cellulase, which is used widely in the extraction and transformation of useful component from natural plants, become instable under the action of heat and the adsorption and desorption between cellulase and substrates. Therefore it is very important to find efficient methods to improve the stability of natural cellulase.
Cyanuric chloride was applied to active polyethylene glycol (PEG) and monomethoxy polyethylene glycol (MPEG). Modified cellulases with different kinds and molecular weight were synthesized by activated PEG, activated MPEG and natural cellulase. Experimental data show that PEG-cellulase, MPEG-cellulase and natural cellulase have Michaelis-Menten characteristic, and the order of kinetics constant Km is PEG-cellulase The thermostability of modified cellulase and natural cellulase were studied through changing the parameters of reaction. The results show that the thermostability of modified cellulase which were modified by PEG2000 and had degree of modification 49% is the best. And the optimum temperature and pH of modified cellulases are equal to natural cellulase.
Modified cellulase was employed in the extraction and transformation of diosgenin from Dioscorea zingiberensis C. H. Wright. The yield and melting point of product by modified cellulase catalysis is better than stepwise enzyme catalysis, and the purity of it is more than 97% according to its HPLC chromatogram. Meanwhile, the amount of sulfuric acid decreased in this technology and reduced the emission of contamination.
The stability mechanisms of PEG-modified cellulase were analyzed, and the hydrophilic/hydrophobic groups, adsorption/desorption and molecule kinetic energy hypothesis were proposed to explain the stability of modified cellulase.
天然酶具有蛋白质的特性,在催化底物的过程中,受到环境和其它条件的影响,容易变性失活,降低催化效率。在天然产物有效成分的提取转化中应用最广泛的生物催化剂-纤维素酶,由于热作用及酶与底物之间的吸附脱附作用,变得极不稳定,纤维素酶活性的稳定化成为亟需解决的问题之一。
本文选用三聚氯氰活化聚乙二醇(PEG)和单甲氧基聚乙二醇(MPEG),活化PEG和活化MPEG作为修饰剂,修饰天然纤维素酶,制备了不同分子量不同修饰度的修饰纤维素酶。通过实验发现,PEG-纤维素酶、MPEG-纤维素酶和天然纤维素酶均符合米氏酶的特性,并且米氏常数Km的关系为PEG-纤维素酶
将修饰酶应用于盾叶薯蓣中薯蓣皂苷元的提取转化,所得产品的质量和纯度都较高,催化效果优于阶梯生物催化。高效液相色谱分析产品纯度高达97.28%。并且修饰酶催化工艺中酸的用量得到了减小,降低了废物排放和环境污染。
本文还对PEG修饰酶的稳定性机理作了初步探讨,对PEG能够提高纤维素酶的稳定性机理从亲水性憎水性、吸附脱附、分子热动能理论上进行了深入分析。
Along with the petrochemical resources and the energy crisis, environment and the society crisis becomes worse increasingly, it is important to change the traditional chemical manufacturing which based on chemical process to the recycled, less- polluted, efficient biological process. The biocatalysis has become one of the important methods for develpoment of the green chemistry and green chemical engineering, and its development will have significant effect on medicine, agriculture, food, life science and so on.
For the reason that natural enzymes have properties of protein, natural enzymes are easy to deactivate and the efficiency of biocatalysis will be reduced consequently under the effect of reaction conditions. Cellulase, which is used widely in the extraction and transformation of useful component from natural plants, become instable under the action of heat and the adsorption and desorption between cellulase and substrates. Therefore it is very important to find efficient methods to improve the stability of natural cellulase.
Cyanuric chloride was applied to active polyethylene glycol (PEG) and monomethoxy polyethylene glycol (MPEG). Modified cellulases with different kinds and molecular weight were synthesized by activated PEG, activated MPEG and natural cellulase. Experimental data show that PEG-cellulase, MPEG-cellulase and natural cellulase have Michaelis-Menten characteristic, and the order of kinetics constant Km is PEG-cellulase
Modified cellulase was employed in the extraction and transformation of diosgenin from Dioscorea zingiberensis C. H. Wright. The yield and melting point of product by modified cellulase catalysis is better than stepwise enzyme catalysis, and the purity of it is more than 97% according to its HPLC chromatogram. Meanwhile, the amount of sulfuric acid decreased in this technology and reduced the emission of contamination.
The stability mechanisms of PEG-modified cellulase were analyzed, and the hydrophilic/hydrophobic groups, adsorption/desorption and molecule kinetic energy hypothesis were proposed to explain the stability of modified cellulase.
引文
[1]孙志浩,生物催化工艺学,北京:化学工业出版社,2005,1～10
[2]卢定强,韦萍,周华等,生物催化与生物转化的研究进展,化工进展,2004,23(6):585～589
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[39]Jessica G, Jefferson D R, Matteo C, et al, Peptide–polyethylene glycol conjugates: Synthesis and properties of peptides bearing a C-terminal polyethylene glycol chain, Bioorganic and Medicinal Chemistry, 2006, 14(18): 6197–6201
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[41]叶建新,肖成祖,蛋白质的化学修饰,生物技术通讯,1995,6(4):181～184
[42]Sariaslani F Sima, Rosazza John P N, Biocatalysis in natural products chemistry, Enzyme and Microbial Technology, 1984, 6(6):242-253
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[44]侯嵘峤,孟宪纾,徐育新,以酶解法从中药及药渣中制备 β-葡萄糖的研究,沈阳药学院学报,1994,11(4):289~293
[45]赵立亚,金凤燮,鱼红闪,酶法生产稀有人参皂苷极其产物成分的分析,大连轻工业学院学报,2002,21(2):112~115
[46]陶卫平,热稳定性酶,生物学通报,1998,33(1):20～21
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[2]卢定强,韦萍,周华等,生物催化与生物转化的研究进展,化工进展,2004,23(6):585～589
[3]沈同,王镜岩,生物化学,北京:高等教育出版社,1990,232～293
[4]May S W, Biocatalysis in the 1990s, A Perspective, Enzyme and Microbial Technology, 1992, 14(1):80-84
[5]许根俊,酶的作用原理,北京:科学出版社,1984,1～15
[6]熊振平,酶工程,北京:化学工业出版社,1989,1～15
[7]朱跃钊,卢定强,万红贵等,工业生物技术的研究现状与发展趋势,化工学报,2004,55(12):1950~1956
[8]Olga K, Frances H A, Directed evolution of enzyme catalysts, Trends in Biotechnology, 1997, 15(12):523~530
[9]杜晨宇,李春,曹竹安等,工业生物技术的核心——生物催化,生物工程进展,2002,22(1):9~14
[10]何坤荣,生物催化技术前景广阔,精细与专用化学品,2002,10(22):13～14
[11]Haiyan T, Virginia W C, Milestones in directed enzyme evolution, Current Opinion in Chemical Biology, 2002, 6(6):858~864
[12]居乃琥,酶工程研究和酶工程产业的新进展,食品与发酵工业,2000,26(13):54～62
[13]Bertus B, Extremophiles as a source for novel enzymes, Current Opinion in Microbiology, 2003, 6(3):213~218
[14]马晓建,白净,任珂等,酶工程研究的新进展,化工进展,2003,22(8):813~817
[15]Straathof A J J, Panke S, Schmid A, The production of fine chemicals by biotransforrnations, Current Opinion in Biotechnology, 2002, 13(6):548~556
[16]Andreas S B,Bettina R R,生物催化-基础与应用,孙志浩,许建和译,北京:化学工业出版社,2006,5～6
[17]Schmid A, Dordick J S, Hauer B, Industrial biocatalysis today and tomorrow, Nature, 2001, 409(6817):258~268
[18]Cowan D A, Microbial genomes-the untapped resource, Trends in Biotechnology, 2000, 18(1):14~16
[19]陶文沂,李江华,生物催化剂在制药工业的应用,无锡轻工大学学报,2002,21(5):538~545
[20]周孟津,张榕林,沼气实用技术,北京:化学工业出版社,2004,15～46
[21]吴创之,马隆龙,生物质能现代利用技术,北京:化学工业出版社,2003,35～75
[22]张作良,王凡业,刘均洪,污染物的生物催化降解研究进展,化工生产与技术,2005,12(3):26~30
[23]Whitehead I M, Challenges to biocatalysis from flavor chemistry, Food Technology, 1998, 52(2):40~46
[24]Vieille C, Zeikns J G, Thermozymes: identifying molecular determinants of protein structural and functional stability, TIBTECH, 1996, 14(6): 183~190
[25]Janecek S, Strategies for obtaining stable enzymes, Process Biochemistry, 1993, 28(7):435~445
[26]来鲁华,蛋白质的结构预测与分子设计,北京:北京大学出版社,1993,20～28
[27]Brugger R, Kronenberger A, Bischoff A, et al, Thermostability engineering of fungal phytases using low-Mr additives and chemical crosslinking, Biocatalysis and Biotransformation, 2001, 19(5):505~516
[28]Govardhan C P, Crosslinking of enzymes for improved stability and performance, Current Opinion in Biotechnology, 1999, 10(4):331~335
[29]徐金库,张媛媛,刘均洪,酶的稳定性研究,化学与生物工程,2004,3(1):1～3
[30]潘力,林炜铁,姚汝华,交联酶晶体制备及其稳定性的研究,生物工程学报,1999,15(3):401~403
[31]Kawamura Y, Nakanishi K, Matsuno R, et al, Stability of immobilized α-chymotrypsin, Biotechnology and Bioengineering, 1981, 23(6):1219~1236
[32]Triantafyllou A O, Wehtje E, Adlercreutz P, et al, How do additives affect enzyme activity and stability in nonaqueous media, Biotechnol Bioeng, 1997, 54(1):67~76
[33]Kazan D, Erarslan A, Stabilization of escherichia coli penicillin G acylase by polyethylene glycols against thermal in activation, Applied Biochemistry and Biotechnology,1997, 62(1):1~13
[34]Lee L L, Lee J C, Thermal stability of proteins in the presence of poly(ethylene glycols), Biochemistry, 1987, 26(24):7813~7819
[35]Hernaiz M J, Sanchez-Montero J M, Sinisterra J V, Modification of purified lipases from Candida rugosa with polyethylene glycol: A systematic study, Enzyme and microbial technology, 1999, 24(3):181~190
[36]潘力,林炜铁,姚汝华,交联酶晶体制备及其稳定性的研究,生物工程学报,1999,15(3):401~403
[37]周海梦,王洪睿,蛋白质的化学修饰,北京:清华大学出版社,2000,19~47
[38]罗贵民,酶工程,北京:化学工业出版社,2002,83～114
[39]Jessica G, Jefferson D R, Matteo C, et al, Peptide–polyethylene glycol conjugates: Synthesis and properties of peptides bearing a C-terminal polyethylene glycol chain, Bioorganic and Medicinal Chemistry, 2006, 14(18): 6197–6201
[40]申冸文，化学生物学与生物技术，北京：科学出版社，2005,405～412
[41]叶建新,肖成祖,蛋白质的化学修饰,生物技术通讯,1995,6(4):181～184
[42]Sariaslani F Sima, Rosazza John P N, Biocatalysis in natural products chemistry, Enzyme and Microbial Technology, 1984, 6(6):242-253
[43]姜彬慧,胡筱敏,左小红,酶技术与中药现代化,世界科学技术-中药现代化,2004,6(2):46~49
[44]侯嵘峤,孟宪纾,徐育新,以酶解法从中药及药渣中制备 β-葡萄糖的研究,沈阳药学院学报,1994,11(4):289~293
[45]赵立亚,金凤燮,鱼红闪,酶法生产稀有人参皂苷极其产物成分的分析,大连轻工业学院学报,2002,21(2):112~115
[46]陶卫平,热稳定性酶,生物学通报,1998,33(1):20～21
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