脂肪酶的化学修饰及其界面催化性能研究
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摘要
脂肪酶作为一种水解酶,不仅催化水解反应而且催化有机相中酯合成和酯交换反应。有机相酶催化自上世纪八十年代开创以来,已获得了巨大的发展,得到广泛应用,但还存在酶在有机相中溶解性、稳定性差、催化效率低等缺点。化学修饰是克服上述缺点,提高酶催化功能的有效方法之一。本章研究的目的是:用具有长链烷基的硬脂酸对Lipolase脂肪酶进行化学修饰得到具有两亲分子结构的修饰脂肪酶,提高修饰酶的表/界面活性及其在有机相或两相界面的催化性能。本文研究的主要内容如下:
1.选择合适的修饰剂并对其进行活化:为了提高酶在有机相中的溶解性,应在酶表面引入疏水性物质,我们选择长链硬脂酸作为修饰剂。通过查阅文献,比较多种活化方法,本实验采用N-羟基琥珀酰亚胺活化法活化硬脂酸,此方法成本低、毒性小且反应温和。通过红外光谱和质谱对硬脂酸琥珀酰亚胺酯进行了结构分析。
2.脂肪酶的修饰:由于蛋白质分子表面游离氨基较多并且具有较高的反应活性,因此氨基常被选为修饰基团。此反应即为硬脂酸琥珀酰亚胺酯与脂肪酶表面氨基的反应。反应条件:pH值7.4、常温磁力搅拌。分离方法:透析。通过改变脂肪酶与硬脂酸琥珀酰亚胺酯的质量比和反应时间得到不同修饰度的修饰酶。
3.酶活测定:对修饰酶的水解活性和有机溶剂中的酯化活性和界面催化活性进行了测定,并与未修饰酶进行了比较。实验结果表明修饰酶的水解活性略有下降,但在有机溶剂中的酯化活性和在油/水界面催化活性明显提高。
4.有机溶剂溶解性、稳定性及热稳定性测定:脂肪酶修饰后在表面存在长链烷基,分子具有两亲性,亲油性增加。实验结果表明修饰酶在正己烷中的溶解度增加,稳定性及热稳定较之未修饰酶均有所提高。
5.界面化学性能表征:酶修饰后结构发生了变化:一端是亲水基,一端是疏水基,类似于表面活性剂,应有一定的表面活性。本论文考察了修饰酶与未修饰酶水溶液的表面张力与界面张力。结果表明:与未修饰酶相比,修饰酶降低溶液表面张力与界面张力的能力提高。
6. LB膜的制备及其结构表征:酶经修饰后表面活性提高,更容易在界面吸附。LB单分子膜由LB成膜装置制备,并在原子力显微镜(AFM)下观察其形貌特征。LB膜制备的同时得到∏-A曲线。实验结果表明:修饰酶的∏-A曲线上移;由AFM图象分析发现在修饰酶的分子表面有暴露的长链烷基。
Lipases catalyze not only the hydrolysis of lipids in water but synthetic reactions in organic solvent, such as esterification and interesterification. The application of lipase for synthetic purpose has been under intensive study in recent years in the food, pharmaceutic cosmetics and so on. However, the stability and dispersibility of native lipase in organic solvent is much poorer than in water. It seems reasonable to attempt to prepare more stable enzymes in organic solvents to overcome its problems. Chemical modification as a rapid and inexpensive method can overcome problems above and improve its dissolution and catalysis property in organic solvent. The introduction of hydrophobic or amphoteric compounds onto the enzyme surface can promote dissolution into organic solvent through favorable interaction between these compounds and the solvent.
In the paper, we introduce fatty acids onto the enzyme surface. The stearic acid was activated by N-hydroxysuccinimide. The lipolase was modified with activated stearic acid-hydroxysuccinimide ester through the reaction between the amino groups and activated fatty acids. The reaction was carried out at room temperature and in the case of the pH 7.4. The modification ratio can be controlled by changing the molar ratio and reaction time in the reaction system.
The enzymatic activity, surface activity and interface behavior were studied. The hydrolysis activity, interfacial hydrolysis activity and esterification activity of the modified enzyme were assayed. The results showed that the interfacial hydrolysis activity at the interface of oil-water phase and esterification activity in hexane of the modified enzyme increased apparently, although the hydrolysis activity decreased little compared with the unmodified enzyme. After modification the surface activity of modified enzyme was improved. The surface tension and the interfacial tension of the modified and unmodified enzymes were also investigated. The surface tension and interfacial tension of the solution of modified enzyme was much lower than that of unmodified enzyme. The reduction in surface tension and interfacial tension is due to the adsorption of enzyme at two-phase interface. As enzyme adsorb to an interface, the reduction of surface/interfacial tension gives an indication of the characteristic adsorption.The Langmuir-Blodgtt monolayer film of modified enzyme was gained by use of LB technique and the surface pressure was higher than that of unmodified one, shown which was much more easer to congregate on interface. The AFM figure of modified enzyme showed a difference from that of unmodified enzyme. We can find the alkyl on the surface of the modified enzyme.
From the above experimental results, it could be concluded that the lipolase modified with fatty acid had good behaviors in organic solvent and at interface of two-phase comparing to unmodified lipolase.
1.选择合适的修饰剂并对其进行活化:为了提高酶在有机相中的溶解性,应在酶表面引入疏水性物质,我们选择长链硬脂酸作为修饰剂。通过查阅文献,比较多种活化方法,本实验采用N-羟基琥珀酰亚胺活化法活化硬脂酸,此方法成本低、毒性小且反应温和。通过红外光谱和质谱对硬脂酸琥珀酰亚胺酯进行了结构分析。
2.脂肪酶的修饰:由于蛋白质分子表面游离氨基较多并且具有较高的反应活性,因此氨基常被选为修饰基团。此反应即为硬脂酸琥珀酰亚胺酯与脂肪酶表面氨基的反应。反应条件:pH值7.4、常温磁力搅拌。分离方法:透析。通过改变脂肪酶与硬脂酸琥珀酰亚胺酯的质量比和反应时间得到不同修饰度的修饰酶。
3.酶活测定:对修饰酶的水解活性和有机溶剂中的酯化活性和界面催化活性进行了测定,并与未修饰酶进行了比较。实验结果表明修饰酶的水解活性略有下降,但在有机溶剂中的酯化活性和在油/水界面催化活性明显提高。
4.有机溶剂溶解性、稳定性及热稳定性测定:脂肪酶修饰后在表面存在长链烷基,分子具有两亲性,亲油性增加。实验结果表明修饰酶在正己烷中的溶解度增加,稳定性及热稳定较之未修饰酶均有所提高。
5.界面化学性能表征:酶修饰后结构发生了变化:一端是亲水基,一端是疏水基,类似于表面活性剂,应有一定的表面活性。本论文考察了修饰酶与未修饰酶水溶液的表面张力与界面张力。结果表明:与未修饰酶相比,修饰酶降低溶液表面张力与界面张力的能力提高。
6. LB膜的制备及其结构表征:酶经修饰后表面活性提高,更容易在界面吸附。LB单分子膜由LB成膜装置制备,并在原子力显微镜(AFM)下观察其形貌特征。LB膜制备的同时得到∏-A曲线。实验结果表明:修饰酶的∏-A曲线上移;由AFM图象分析发现在修饰酶的分子表面有暴露的长链烷基。
Lipases catalyze not only the hydrolysis of lipids in water but synthetic reactions in organic solvent, such as esterification and interesterification. The application of lipase for synthetic purpose has been under intensive study in recent years in the food, pharmaceutic cosmetics and so on. However, the stability and dispersibility of native lipase in organic solvent is much poorer than in water. It seems reasonable to attempt to prepare more stable enzymes in organic solvents to overcome its problems. Chemical modification as a rapid and inexpensive method can overcome problems above and improve its dissolution and catalysis property in organic solvent. The introduction of hydrophobic or amphoteric compounds onto the enzyme surface can promote dissolution into organic solvent through favorable interaction between these compounds and the solvent.
In the paper, we introduce fatty acids onto the enzyme surface. The stearic acid was activated by N-hydroxysuccinimide. The lipolase was modified with activated stearic acid-hydroxysuccinimide ester through the reaction between the amino groups and activated fatty acids. The reaction was carried out at room temperature and in the case of the pH 7.4. The modification ratio can be controlled by changing the molar ratio and reaction time in the reaction system.
The enzymatic activity, surface activity and interface behavior were studied. The hydrolysis activity, interfacial hydrolysis activity and esterification activity of the modified enzyme were assayed. The results showed that the interfacial hydrolysis activity at the interface of oil-water phase and esterification activity in hexane of the modified enzyme increased apparently, although the hydrolysis activity decreased little compared with the unmodified enzyme. After modification the surface activity of modified enzyme was improved. The surface tension and the interfacial tension of the modified and unmodified enzymes were also investigated. The surface tension and interfacial tension of the solution of modified enzyme was much lower than that of unmodified enzyme. The reduction in surface tension and interfacial tension is due to the adsorption of enzyme at two-phase interface. As enzyme adsorb to an interface, the reduction of surface/interfacial tension gives an indication of the characteristic adsorption.The Langmuir-Blodgtt monolayer film of modified enzyme was gained by use of LB technique and the surface pressure was higher than that of unmodified one, shown which was much more easer to congregate on interface. The AFM figure of modified enzyme showed a difference from that of unmodified enzyme. We can find the alkyl on the surface of the modified enzyme.
From the above experimental results, it could be concluded that the lipolase modified with fatty acid had good behaviors in organic solvent and at interface of two-phase comparing to unmodified lipolase.
引文
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[3]万柱礼,刁家升,常文瑞,等.B链氨端去二肽(B1-2)羧端去五肽(B26-30)胰岛素的初步晶体学研究[J].科学通报,1994, 12: 1130-1132.
[4] DeSantis G, Berglund P, Stabile M R, el a1. Site-directd mutagenesis combined with chemical modification as a strategy for altering the specificity of the SI and Sl’s pockets of subtilisin bacillus lentus [J]. Biochemistry, 1998, 37: 5968-5973.
[5] Berglund P, DeSanntis G, Stabile MR, el a1. Chemical modification of cysteine mutants of subtilisin bacillus lentus can creat better catalysts than the Wild-type enzyme [J]. J Am Chem Soc, 1997, 119 (5): 265-266.
[6] Plettner E, DeSantis G, Stabile MR, el a1. Modulation of esterase and amidase activity of subtilisin bacillus lentus by chemical modification of cysteine mutants [J]. J Am Chem Soc, 1999, 121: 4 977-4 981.
[7] St Clair NL. Navia MA. Cross-1inked enzyme crystals as robust biocatalysts [J]. J Am Chem Soc, 1992, 114: 7 314-7316.
[8] Haring D, Schreier P. Cross-linked enzyme crystals [J]. Current Opin Chem Biol, 1999, 3: 35-38.
[9]严锐,黄金营.蛋白质化学修饰的研究进展.化学工业与工程[J].2005, 22(1): 53-55.
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