肠球菌中β-D-葡萄糖苷酶的分离纯化及酶学性质研究

发布时间：2018-04-24 14:04

本文选题：β-D-葡萄糖苷酶 + 肠球菌　；参考：《山东轻工业学院》2010年硕士论文

【摘要】： 银杏提取物(EGb761)含有24 %银杏黄酮内酯(Ginkgo-flavone glycosides)及6 %萜类化合物(Terpenoids),它可望成为治疗阿尔兹海默病(Alzheimer's disease, AD)的有效药,但是其临床前期试验,包括代谢等还没有能够按照药物研发的现代标准来进行。银杏提取物的代谢主要是通过肠道微生物菌群分泌的水解酶来进行的,由此,本文选定肠球菌为模式菌株,通过系列研究不但能够初步阐明机体代谢吸收银杏提取物中主要有效成分的酶学及分子生物学的机制,而且为按照现代药物标准研发银杏系列产品做好方法上的准备并提供理论依据,因此具有理论研究和实际应用双重重要意义。目前,国内外尚未见此方面成功的研究报道。首先通过响应面法优化设计得到了肠球菌最佳培养基配方为:葡萄糖1.87896 %、酵母膏0.77474 %、牛肉膏0.3 %、蛋白胨1 %、KNO36.25 mmol/L、NaCl0.5 %,最适培养条件为:装液量60 mL、初始pH7.0、接种量3 %、温度36℃,接种后置于170 r/min恒温摇床中培养20 h后,于OD560处测定吸光度值为0.748,比优化前的0.682提高了9.7 %。其次确定了肠球菌中β-D-葡萄糖苷酶的酶活测定方法:在10mL具塞比色管中加入1.5 mL pH5.0的0.02 mol/L柠檬酸-磷酸氢二钠缓冲液,再用适当刻度的移液枪吸取0.1 mL粗酶提取液加入其中,于45℃恒温水浴中预热一定时间,再加入已预热5～10 min的0.4 mL1 mmol/L底物pNPG溶液,用秒表精确计时,14 min后取出立即加入2 mL1 mol/L的Na2CO3溶液终止反应,在比色管架上冷却至室温,于400nm处测光吸收值。再次通过单因素和正交实验对肠球菌产β-D-葡萄糖苷酶的发酵条件进行了优化,得到基础产酶培养基的最佳配比为:玉米粉3 %,酵母浸粉1.2 %,蛋白胨1 %,葡萄糖2 %,K2HPO4 0.1 %,MgSO47.5 mmol/L;相应的最佳肠球菌产酶发酵条件为:初始pH7.0,发酵温度37℃,发酵时间168 h,摇床转速180 r/min,装液量50 mL(250 mL三角瓶),接种量为3%,此时肠球菌产β-D-葡萄糖苷酶的酶活最高,达到19.1242 U/(mL·min)。肠球菌产β-D-葡萄糖苷酶发酵液经过40 %～80 %硫酸铵分级沉淀、DEAE-纤维素52阴离子交换层析、Sephadex G-100凝胶过滤层析等步骤分离纯化得到了β-D-葡萄糖苷酶纯品,该酶的比活力由3.37 U/mg提高到33 U/mg,纯化倍数为9.79倍,且酶的回收率为5.33 %。提纯酶蛋白经NATURE-PAGE和SDS-PAGE电泳,确定来源于肠球菌的β-D-葡萄糖苷酶相对分子量约为121KD,由两个亚基组成(65KD和54KD)。提纯后的β-D-葡萄糖苷酶经酶学性质研究发现酶耐酸性较强,具有较强的热稳定性。5 mmol/L的Mg2+、Ba2+、Ca2+对β-葡萄糖苷酶有激活作用;而Cu2+、Zn2+、Fe3+、Ag+对酶有显著的抑制作用。此外,pNPG对该酶有很强底物特异性,动力学研究表明肠球菌β-D-葡萄糖苷酶的米氏常数Km为0.05 mmol/L,Vmax为18.0375 U/L。
[Abstract]:Ginkgo biloba extract (EGb761) contains 24% ginkgo flavone (Ginkgo-flavone glycosides) and 6% terpenoids (Terpenoids). It is expected to be an effective drug for the treatment of Alzheimer's disease (AD), but its preclinical trials, including metabolism, have not been carried out according to the modern standard of drug development. The metabolism of the extract is mainly carried out through the hydrolase secreted by the microbial flora of the intestinal tract. Therefore, this paper selects Enterococcus as a model strain. Through a series of studies, it can not only elucidate the mechanism of enzyme and molecular biology of the main effective components in the body metabolism and absorption of Ginkgo biloba extract, but also be developed according to the modern drug standard. The products of Ginkgo biloba series are prepared and provided with theoretical basis. Therefore, it is of both theoretical and practical significance. At present, there are no successful research reports at home and abroad.
First, the optimum medium of Enterococcus was obtained by the response surface method. The optimum formula was as follows: glucose 1.87896%, yeast extract 0.77474%, beef paste 0.3%, peptone 1%, KNO36.25 mmol/L, NaCl0.5%. The optimum conditions for culture were 60 mL, initial pH7.0, 3% inoculation and temperature 36, after inoculation in 170 r/min rocking bed incubating 20 h in constant temperature rocking bed, The absorbance value at OD560 was 0.748, which was 9.7 higher than that before the optimization of 0.682.
Secondly, the enzyme activity determination method of beta -D- glucosidase in Enterococcus is determined: adding 1.5 mL pH5.0 to 0.02 mol/L citric acid hydrogen phosphate buffer solution in the CECD tube and adding 0.1 mL crude enzyme extract from the appropriate scale transfer gun, preheating a certain time in the constant temperature water bath at 45 C, and adding the preheating 5~10 MI in the 45 C constant temperature water bath. The 0.4 mL1 mmol/L substrate pNPG solution of n is timed accurately with a stopwatch. After 14 min, the termination reaction of Na2CO3 solution, which is immediately added to 2 mL1 mol/L, is cooled to room temperature on the colorimetric tube frame, and the absorption value at 400nm is measured at 400nm.
The fermentation conditions of Enterococcus producing beta -D- glucosidase were optimized by single factor and orthogonal experiment. The optimum ratio of the base producing enzyme culture medium was as follows: corn flour 3%, yeast extract 1.2%, peptone 1%, glucose 2%, K2HPO4 0.1%, MgSO47.5 mmol/L; the optimum fermentation conditions for Enterococcus were: initial pH7.0, fermentation The temperature was 37 C, the fermentation time was 168 h, the rotational speed of the rocking bed was 180 r/min, the loading amount was 50 mL (250 mL triangle bottle) and the inoculation amount was 3%. The enzyme activity of Enterococcus producing beta -D- glucosidase was the highest, reaching 19.1242 U/ (mL. Min).
The fermentation broth of Enterococcus producing beta -D- glucosidase was fractionated by 40% to 80% ammonium sulfate, DEAE- cellulose 52 anion exchange chromatography and Sephadex G-100 gel filtration chromatography were separated and purified to obtain pure beta -D- glucosidase. The specific activity of the enzyme was increased from 3.37 U/mg to 33 U/mg, and the purification multiplier was 9.79 times, and the recovery rate of the enzyme was 5. .33% purified enzyme protein was determined by NATURE-PAGE and SDS-PAGE electrophoresis. The relative molecular weight of beta -D- glucosidase derived from Enterococcus was about 121KD, which was composed of two subunits (65KD and 54KD). After purification, the enzymatic properties of the purified beta glucosidase were found to be more acid resistant, with a stronger thermal stability of.5 mmol/L Mg2+, Ba2+. Glucosidase is activated; Cu2+, Zn2+, Fe3+, Ag+ have significant inhibitory effects on the enzyme. Furthermore, pNPG has a strong substrate specificity. Kinetic studies show that the Michaelis constant Km of Enterococcus beta -D- glucosidase is 0.05 mmol/L and Vmax is 18.0375 U/L..

【学位授予单位】：山东轻工业学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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