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题名	纳米纤维固定化beta-D-半乳糖苷酶的制备及其应用
作者	郑国建
学位类别	硕士
答辩日期	2010-06-01
授予单位	中国科学院研究生院
授予地点	北京
导师	王平
关键词	beta-D-半乳糖苷酶 静电纺丝 纳米纤维 苯乙烯马来酸酐共聚物 己基半乳糖苷 两相反应体系
其他题名	Immobilization of β-D-galactosidase onto Nanofibers and Its Application
学位专业	生物化工
中文摘要	b-D-半乳糖苷酶可以催化乳糖和脂肪醇之间的转糖苷化反应合成重要的表面活性剂烷基糖苷。由于该反应通常为油水两相体系，水相中的酶分子与疏水底物不能有效接触、界面处酶分子遇有机溶剂易失活等问题限制了其工业化生产。针对该问题，本论文将b-D半乳糖苷酶固定化在苯乙烯马来酸酐共聚物（PSMA）纳米纤维膜上，并考察了纳米纤维酶膜用于催化乳糖水解以及烷基糖苷合成反应中的应用。具体研究包括如下三部分内容：（1）通过静电纺丝法制备了PSMA纳米纤维，优化PSMA电纺液浓度、电纺液流速、电场距离和外加电压等参数，结果表明以上各因素对纳米纤维形态均有显著影响。当电纺液浓度为35%（w/v）、流速为250 mL/h、距离为25 cm时，采用21 kV电压进行电纺即可得到直径在300 nm左右且分布均匀的纳米纤维；（2）以PSMA纳米纤维为载体进行固定化b-D-半乳糖苷酶的研究，经优化确定最佳固定化酶条件为pH 4.0（0.2 M醋酸-醋酸钠缓冲液）、固定化温度为15 oC、酶浓度为1 mg/mL。固定化4 h后固定化酶量可达到16.5±1.3 mg/g 膜，固定化酶催化2-硝基苯酚-b-D-半乳吡喃糖苷（ONPG）水解反应的米氏常数Km值为2.7 mM，略大于游离酶的Km值（2.2 mM）；固定化酶的Vmax为97.2 mmol/(min·mg)，为游离酶的47.8%。固定化酶在37 oC重复使用21次后活性损失仅为15%。在连续搅拌式反应器中将固定化酶用于催化乳糖的水解反应，连续使用18天仍能稳定运行；（3）利用固定化在纳米纤维上的b-D-半乳糖苷酶在己醇/水两相体系中催化己醇和乳糖之间的转糖苷化反应制备己基半乳糖苷。研究表明剧烈搅拌和高温对固定化酶的活性影响较大，在50 oC、强搅拌的条件下反应，固定化酶使用两次后仅剩余0.83%的活力。将反应温度降至37 oC，辅以温和振荡反应条件时，固定化酶的重复使用性能得到较大提升，重复使用6次仍剩余44.2%的活力。使用聚丙烯夹套可以实现负载有b-D-半乳糖苷酶的PSMA纳米纤维酶膜均匀分布于油水两相界面上，从而显著降低了乳糖水解这一副反应的发生。
英文摘要	Alkyl polyglycosides is an important nonionic surfactants group that can be enzymatically synthesized via transgalactosylation reaction between fatty alcohol and lactose by b-D-galactosidase. Normally this reaction was carried out in biphasic medium that the inefficient contact between enzyme dissolved in aqueous phase and hydrophobic substrate, as well as the deactivation of enzyme upon contacting with organic solvent at the interface severely limited the enzymatic biosynthesis of alkyl polyglycosides in large scale. To solve this problem, in this study, b-D-galactosidase was immobilized onto poly(styrene-co-maleic anhydride) nanofibers and its application for both lactose hydrolysis reaction and the synthesis of alkyl polyglycoside were investigated. The thesis includes three parts: (1) Poly(styrene-co-maleic anhydride) nanofibers with a uniform diameter of about 300 nm were prepared by eletrospinning a solution containing 35% (w/v) polymer, 25 cm gap between ejection needle tip and collector, a flow rate of 250 mL/h, and an applied voltage of 21 kV. (2) The electrospun nanofibers were applied subsequently for immobilization of b-D-galactosidase. The immobilized condition was optimized as following: 0.2M, pH 4.0 acetate buffer, 15oC, and enzyme concentration of 1.0mg/ml. Under this condition, an enzyme loading of 16.5 ± 1.3 mg protein/g membrane was obtained. The immobilized enzyme demonstrated a Michealis constant (Km) of 2.7 mM, which is slightly higher than that of the native enzyme (2.2 mM). The Vmax of the immobilized enzyme was found as 97.2 mmol/(min·mg-enzyme), 47.8% that of native enzyme. The immobilized enzyme showed an excellent reusability such that 21 reusing cycles only led to 15% loss of the enzyme activity. Furthermore, the immobilized b-D-galactosidase was suitable for hydrolysis of lactose in a continuous stirring tank reactor that was operated steadily for 18 days. (3) b-D-galactosidase immobilized on PSMA nanofiber was used for the synthesis of hexyl-galactoside in hexanol/aqueous biphasic system. The results indicated that intensive magnetic stirring and higher temperature led to sever loss of activity of the immobilized enzyme such that only 0.83% of the initial activity was retained after two times usage operated at 50oC with strong magnetic stirring. While operated at 37 oC and gentle rotation, the reusability of the nanofibrous enzyme was remarkably improved that 44.2% of the initial activity was retained after 21 reusing cycles. When a polypropylene frame was applied to support the membrane, the nanofibrous enzyme membrane was found spread evenly at the interface of the biphasic system, and the side reaction of lactose hydrolysis was greatly suppressed.
公开日期	2013-09-17
页码	83
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1518]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	郑国建. 纳米纤维固定化beta-D-半乳糖苷酶的制备及其应用[D]. 北京. 中国科学院研究生院. 2010.

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