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基于中空纳米纤维的多酶体系:创新设计与应用
Alternative TitleHollow Nanofiber Supported Multienzyme System:Innovation Designs And Applications
姬晓元
Subtype硕士
Thesis Advisor王平 ; 张松平
2013-05-01
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline生物工程
Keyword中空纳米纤维   同轴共纺   多酶体系   胆汁酸检测   葡萄糖检测试纸
Abstract包括辅酶再生的多酶体系的构建和固定化是生物催化领域的一个具有挑战性的课题。针对目前多酶催化剂均是以颗粒形态的微囊或纳、微球作为载体,具有传质阻力大、酶稳定性差、制备过程复杂等不足之处。本课题首次利用同轴共纺静电纺丝技术,制备具有中空腔室结构的纳米纤维,并实现多酶体系在中空腔室内的原位包埋,构建基于中空纳米纤维的多酶催化体系。这种新型的多酶体系不仅具有传统纳米纤维比表面积大、制备简单以及宏观成膜形态等特性,而且其独特的可控的中空腔室可以使多酶和辅酶之间更好地进行分子协同作用,获得更高的活性;同时纳米尺度的腔室,又可以为所包埋的酶提供有效的稳定性机制。本论文的研究主要包括如下几个部分: (1)采用同轴共纺技术,制备聚氨酯中空纳米纤维。考察了内外相电纺液、针头以及内外相流速对纤维内外相直径及形貌的影响。以含水量为5%(v/v)的甘油溶液作为内相电纺液,以溶于N,N-二甲基乙酰胺(DMAC)的30 wt %聚氨酯溶液作为外相电纺液,内外相电纺液流速分别为0.07 mL/h和0.5 mL/h,电压17 kV,针头到接收板的距离为25 cm,温度25℃,湿度10%条件下,可以制备出结构均一的中空结构纳米纤维。 (2)以α-胰凝乳蛋白酶(α-CT)为模型酶,深入考察了中空纳米纤维对固定化酶的影响,并与以传统实心纳米纤维为载体,以表面吸附、表面交联多层固定化、直接混纺固定化酶相比较,进一步研究了固定化酶微环境对酶分子催化效率以及稳定性的影响。结果表明同轴共纺固定化酶由于固定化条件温和,其催化水解反应的活性回收率达到游离酶的87%;而在有机相中催化转酯反应的活性比游离酶提高了约1000倍。基于中空纳米纤维的固定化α-CT的储存稳定性、热稳定性以及有机溶剂稳定性均得到了大幅度的提高,充分显示了以中空纳米纤维固定化酶的突出优势。 (3)构建了包括3-α-类固醇脱氢酶(3-α-HSD)、黄递酶(DP)和NAD(H)的多酶体系,用于胆汁酸的检测。通过同轴共纺技术,将其原位包埋在中空纳米纤维中,固定化3-α-HSD和黄递酶的活性回收率分别达到了游离酶的76%和82%。基于中空纳米纤维的多酶体系用于胆汁酸浓度的检测, 在0-200umol.L-1范围内都呈现良好的线性,高于游离多酶体系0-150umol.L-1的检测范围。固定化多酶体系的稳定性得到了大幅度的提高,在4℃和25℃下储存的半衰期分别达到160天和80天。 (4)以含有葡萄糖氧化酶和过氧化物酶的含水量20% (v/v)的甘油溶液作为同轴共纺的内相电纺液,以含有20 mg/mL的邻联茴香胺的聚氨酯溶液作为外相电纺液,通过同轴共纺制备出了基于中空纳米纤维的葡萄糖检测的试纸条,滴加20 ?L的葡萄糖溶液就可以迅速显色,采用分光光密度法可以精确检测0-50 mM的葡萄糖浓度。试纸条具有优异的稳定性,4℃下储存3个月没有活性损失,25℃储存3个月后活性保留仍在50%以上。
Other AbstractHighly efficient immobilization of multienzyme systems involving cofactor regeneration represents one of the greatest challenges in bioprocessing. Multienzyme systems that have been reported to date are almost exclusively in discrete spherical capsule formations and nano or micro particles, which have limitations in some significant applications owing to high diffusion resistance, poor enzyme stability, and usually complicated preparation process. To break through those limitations, the present work proposed a novel hollow nanofiber supported multienzyme system through co-axial electrospinning technology. By choosing bio-friendly solution with dissolved multienzyme as core solution and immiscible solvent with dissolved polymer as shell solution of co-axial electrospinning, an in-situ encapsulation of multienzyme inside the nano domain of hollow nanofibers were realized. Compared to the existing multienzyme system in the form of particulates, novel hollow nanofiber supported multienzyme system is expected to offer several advantages: i.e., the woven-membrane formation of electrospun nanofibers enable an easily recycling and operation, facilitating molecular interactions between enzymes and possibly shared cofactor, as well as enhancing stability due to confining multienzyme system inside the nano-scale compartments. The thesis includes the following parts: (1) Polyurethane (PU) hollow nanofibers were produced by co-axial electrospinning. The effects of coaxial spinneret structure, composition of internal and external phase of electrospinning solution and their flow rate on structure of hollow nanofibers were studied. The results showed that uniform hollow PU nanofibers could be successfully fabricated by co-axial electrospinning with glycerol containing 5 % (?) water and N, N-Dimethylacetamide solution of 30 wt% PU as core solution and shell solution under the following condition: core-shell flow rate of 0.07 and 0.5 mL/h, voltage 17 kV, distance between spinneret and collector 25 cm, temperature 25℃, humility 10 %. (2) With α-chymotrypsin (α-CT) as a model enzyme, relationship between performance of nanofibrous α-CT and the immobilization protocols applied as well as the structure of nanofiber were studied. Compared with nanofibrous α-CT prepared via the following three protocols: 1) physically attaching α-CT onto the outer surface of electrospun nanofibers; 2) multilayer immobilization by attaching BSA as first layer followed by crosslinking α-CT as the second layer; 3) preparing organic solvent-soluble enzyme, and then entrapping the enzyme inside nanofiber via electrospinning polymer solution containing enzymes, hollow nanofiber-supported α-CT was the most efficient one in terms of both activity and stability. Its hydrolytic activity was 87% of that of native one, and its activity for synthesis in organic solvent was more than 1000 times higher than that of native enzyme. The hollow nanofiber-supported α-CT also exhibited significantly improved storage and thermal stability, as well as the stability in anhydrous methanol. All these results indicated that hollow nanofiber-supported α-CT had significant advantages over other nanofibrous enzymes. (3) A multienzyme system for bile acid assay, which includes 3-α-hydroxysteroid dehydrogenase (3-α-HSD), diaphorase (DP) and NAD(H) was constructed and encapsulated in-situ inside lumen of hollow nanofiber via co-axial electrospinning. The activity recovery of immobilized 3-α-HSD and diaphorase reached to 76% and 82%, respectively. By using this nanofiber-supported multienzyme system for bile acid assay, a good linearity was obtained by varying bile acid concentrations within 0-200uM, and this linear range was slightly broader than that obtained by using free multienzyme system (0-150uM). Investigation on storage stability of the multienzyme system showed that its half-life at 4℃ and 25℃ reached to 160 days and 80 days, respectively. (4) The hollow nanofibers based glucose test strips were fabricated by co-axial electrospinning, with PU solution containing 20 mg/mL chromogenic agent as external solution and glycerol solution containing glucose detection multienzyme system, glucose oxidase and horseradish peroxidase as inner solution. The test strips changed color quickly upon contacting with 20 uL glucose solution, thus enabling precise spectrophotometric detection of glucose concentration ranging from 0-50 mM. The test strips also showed excellent storage stability, with no loss of activity observed after storage for three months at 4℃, and about 50% of original activity was retained after storage for three months at 25℃.
Pages127
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/8376
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
姬晓元. 基于中空纳米纤维的多酶体系:创新设计与应用[D]. 北京. 中国科学院研究生院,2013.
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