本文選題：β-D-葡萄糖苷酶 + 腸球菌　； 參考：《山東輕工業(yè)學院》2010年碩士論文

【摘要】： 銀杏提取物(EGb761)含有24 %銀杏黃酮內酯(Ginkgo-flavone glycosides)及6 %萜類化合物(Terpenoids),它可望成為治療阿爾茲海默病(Alzheimer's disease, AD)的有效藥,但是其臨床前期試驗,包括代謝等還沒有能夠按照藥物研發(fā)的現(xiàn)代標準來進行。銀杏提取物的代謝主要是通過腸道微生物菌群分泌的水解酶來進行的,由此,本文選定腸球菌為模式菌株,通過系列研究不但能夠初步闡明機體代謝吸收銀杏提取物中主要有效成分的酶學及分子生物學的機制,而且為按照現(xiàn)代藥物標準研發(fā)銀杏系列產(chǎn)品做好方法上的準備并提供理論依據(jù),因此具有理論研究和實際應用雙重重要意義。目前,國內外尚未見此方面成功的研究報道。 首先通過響應面法優(yōu)化設計得到了腸球菌最佳培養(yǎng)基配方為: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 %,最適培養(yǎng)條件為:裝液量60 mL、初始pH7.0、接種量3 %、溫度36℃,接種后置于170 r/min恒溫搖床中培養(yǎng)20 h后,于OD560處測定吸光度值為0.748,比優(yōu)化前的0.682提高了9.7 %。 其次確定了腸球菌中β-D-葡萄糖苷酶的酶活測定方法:在10mL具塞比色管中加入1.5 mL pH5.0的0.02 mol/L檸檬酸-磷酸氫二鈉緩沖液,再用適當刻度的移液槍吸取0.1 mL粗酶提取液加入其中,于45℃恒溫水浴中預熱一定時間,再加入已預熱5～10 min的0.4 mL1 mmol/L底物pNPG溶液,用秒表精確計時,14 min后取出立即加入2 mL1 mol/L的Na2CO3溶液終止反應,在比色管架上冷卻至室溫,于400nm處測光吸收值。 再次通過單因素和正交實驗對腸球菌產(chǎn)β-D-葡萄糖苷酶的發(fā)酵條件進行了優(yōu)化,得到基礎產(chǎn)酶培養(yǎng)基的最佳配比為: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;相應的最佳腸球菌產(chǎn)酶發(fā)酵條件為:初始pH7.0,發(fā)酵溫度37℃,發(fā)酵時間168 h,搖床轉速180 r/min,裝液量50 mL(250 mL三角瓶),接種量為3%,此時腸球菌產(chǎn)β-D-葡萄糖苷酶的酶活最高,達到19.1242 U/(mL·min)。 腸球菌產(chǎn)β-D-葡萄糖苷酶發(fā)酵液經(jīng)過40 %～80 %硫酸銨分級沉淀、DEAE-纖維素52陰離子交換層析、Sephadex G-100凝膠過濾層析等步驟分離純化得到了β-D-葡萄糖苷酶純品,該酶的比活力由3.37 U/mg提高到33 U/mg,純化倍數(shù)為9.79倍,且酶的回收率為5.33 %。提純酶蛋白經(jīng)NATURE-PAGE和SDS-PAGE電泳,確定來源于腸球菌的β-D-葡萄糖苷酶相對分子量約為121KD,由兩個亞基組成(65KD和54KD)。提純后的β-D-葡萄糖苷酶經(jīng)酶學性質研究發(fā)現(xiàn)酶耐酸性較強,具有較強的熱穩(wěn)定性。5 mmol/L的Mg2+、Ba2+、Ca2+對β-葡萄糖苷酶有激活作用;而Cu2+、Zn2+、Fe3+、Ag+對酶有顯著的抑制作用。此外,pNPG對該酶有很強底物特異性,動力學研究表明腸球菌β-D-葡萄糖苷酶的米氏常數(shù)Km為0.05 mmol/L,Vmax為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..

【學位授予單位】：山東輕工業(yè)學院
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R378

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