【作者】 李滨县；

【导师】 张裕卿；

【作者基本信息】 天津大学 ， 化学工程， 2007， 硕士

【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<MPEG-纤维素酶<天然酶,说明修饰酶与底物的结合能力要高于天然酶,修饰酶具有更高的催化活性。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。更多还原

【Abstract】 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<MPEG-cellulase<natural cellulase. This order reflects that the combination between enzyme and substrates of modified cellulase is stronger than natural cellulase and the catalysis activity of modified cellulase is higher.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.更多还原