摘要
α-L-鼠李糖苷酶是一种水解酶,可以水解人们日常饮食中常见的黄酮苷类化合物,广泛分布于自然界的细菌和真菌中。α-L-鼠李糖苷酶在实际工艺中具有很多潜在的应用价值。本论文选用Enterococcus durans作为发酵菌种,对α-L-鼠李糖苷酶的活性条件测定进行了研究,重点讨论了从发酵液中分离提纯得到α-L-鼠李糖苷酶的主要过程,并研究了纯化后的α-L-鼠李糖苷酶的酶学性质。主要实验结果如下:
(1)确定了α-L-鼠李糖苷酶的活性测定条件:在10 mL的比色管中,加入2 mL pH 5.0的柠檬酸-磷酸氢二钠缓冲溶液后,再加入0.1 mL酶液或粗酶液,置于45℃水浴锅中预热5 min,加入0.2mL已经预热的对硝基苯基-α-L-鼠李糖苷作为底物,反应4 min,反映结束后立即取出并加入2 mL 1 mol/L的Na2CO3终止反应,冷却至室温后,于400 nm波长处测定吸光值。
(2)通过单因素实验以及正交试验对Enterococcus durans产α-L-鼠李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:蔗糖1.25%,大豆蛋白胨1.25%,磷酸二氢钾2 mmol/L,牛肉膏0.3%,蛋白胨1%,氯化钠0.5%,硫酸铁3 mmol/L;相应的产酶发酵条件为:培养基的初始pH为7.0,培养温度为34℃,发酵周期为4 d,摇床转速为180 r/min,250 mL三角瓶装液量为70 mL,接种量为3%。
(3)α-L-鼠李糖苷酶的分离纯化:
①实验确定了硫酸铵分级沉淀浓度范围为50~80%;
②硫酸铵盐析后的沉淀用20 mL 4℃蒸馏水溶解后,装入透析袋,置于4℃蒸馏水中透析约30h后,浓缩至5mL;
③采用DEAE-纤维素52阴离子交换层析纯化α-L-鼠李糖苷酶:采用平衡缓冲液的pH为5.0,KCl梯度线性化洗脱浓度为0.2～0.6 mol/L,在KCl浓度低于0.2 mol/L或高于0.6 mol/L时,基本没有蛋白峰洗出。洗脱流速为0.6 mL/min,15 min收集一管,发现收集的第12管中酶活最高,第一蛋白峰没有目的酶活性,为杂蛋白峰,KCl浓度为0.3～0.5 mol/L时,即可把α-L-鼠李糖苷酶洗脱下来。
④采用Sephadex G-100凝胶过滤层析纯化α-L-鼠李糖苷酶:用pH 5.0、浓度0.02 mol/L的醋酸缓冲液洗脱,流速为0.4 mL/min,15 min收集一管,得到三个蛋白峰,第三个为峰为目标峰。
(4)对盐析后和凝胶层析纯化后的酶蛋白进行SDS-聚丙烯酰胺凝胶电泳,分离胶浓度为10%,浓缩胶浓度为6%。结果表明,盐析并透析后的酶液仍含有很多杂蛋白,经过离子交换层析和凝胶层析后,SDA-PAGE凝胶电泳显示单条带,表明酶蛋白已经得到纯化,并确定酶蛋白的分子量约为90 KDa。
(5)纯化后的α-L-鼠李糖苷酶经酶学性质研究发现耐酸性较强并具有较强的耐热性;K~+、Mg~(2+)对α-L-鼠李糖苷酶具有明显的激活作用,而Ca~(2+)、Fe~(2+)则对酶具有显著的抑制作用。多数有机溶剂对α-L-鼠李糖苷酶均有一定程度的抑制作用,其中丙酮对α-L-鼠李糖苷酶的抑制作用最强。以对硝基苯基-α‐L‐鼠李糖苷为底物的动力学常数为:Vmax﹦16.13 U/L,Km﹦4.58 mmol/L。
α-L-rhamnosidase is a kind of hydrolase , it can hydrolyze compounds of flavonoids nucleoside which are common compounds in daily diet.α-L-rhamnosidase is widely distributed in bacteria and fungi. It has a lot of potential applications in actual processes. Enterococcus durans was selected as fermentation strain. Enzyme activity, purification and enzymatic property ofα-L-rhamnosidase were studied in this paper.
The main experimental results are as follows:
(1) This work identified the determination method ofα-L-rhamnosidase from Enterococcus durans as following: in a 10 mL colorimetric tube accessed 2 mL 0.02 mol/L pH 5.0 citric acid-disodium hydrogen phosphate buffer solution, then add crude enzyme extract in it, keep in water bath at 45℃for 5 min, and then incorporated 0.2 mL the preheated substrate pNPR solution into them, after 14 min get out immediately and add 2 mL 1 mol/L Na2CO3 solution to terminate the reaction, then measure the absorbence at 400 nm when it was cooled to room temperature.
(2) With a single factor and orthogonal experiment, the fermentation conditions ofα-L-rhamnosidase producing of Enterococcus durans were optimized to be the best basis for enzyme production medium nutrient as : saccharose 1.25%, soybean peptone 1.25%, K2HPO3 2 mmol/L, beef extract 0.3%, peptone 1%, NaCl 0.5%, Fe2(SO4)3 3 mmol/L; the correspondent fermentation conditions of Enterococcus duransα-L-rhamnosidase producing were as following: initial pH 7.0 of nutrient medium, fermentation temperature is 34℃, fermentation time is 4 days, rotation speed is 180 r/min, 250 mL flask with 50 mL liquid volume, inoculation size 3%.
(3) Purification ofα-L-rhamnosidase
①It is determined that the concentration for the ammonium sulphate was between 50% and 80%.
②In order to concentrate the precipitation after ammonium sulphate to 5 mL, dissolved it with 4℃distilled water, and then enclosed in dialysis membrane and sink it into 4℃distilled for 30 hours.
③Purify theα-L-rhamnosidase by means of anion-exchange chromatography on DEAE-Sephadex 52: The balance buffer at pH 5.0 was used as eluent and the concentration range of sodium chloride was controlled between 0.2 and 0.6 mol/L. Below 0.2 mol/L or above 0.6 mol/L, there was almost no protein eluted out. The velocity eluate is 0.6 ml/min, and collect each tube every 15 min. The NO.12 tube displayed the highest activity among all tubes, and the first protein peak has no activity. When the sodium chloride concentration was between 0.3 mol/L and 0.5 mol/L, theα-L-rhamnosidase can be eluted completely.
④The unrefinedα-L-rhamnosidase was applied to the column of gel filtration chrmatography on Sephadex G-100. The velocity was 0.4 mL/min, and collect each tube every 15 min. Three protein peaks were eluted out, the third one is target peak.
(4) The homogeneity of the enzyme was verified by using SDS-PAGE after salting out and Gel-filtration chromatography. The concentration of separate glue is 10% and the concentrate glue is 4%. The result showed that after the ammonium suphate fractional precipitation it contained some proteins with different molecular weights. However, after gel-filtration chromatography on Sephadex G-100, theα-L-rhamnosidase was purified completely and it showed homogeneity by SDS-PAGE. The molecular weight was about 90 K Da.
(5) Enzymatic properties study indicate that this enzyme has high acid tolerance and thermal stability. The enzyme activity synergistically increased by the addition of metal cations such as K~+ ,and Mg~(2+), meanwhile, Ca~(2+) and Fe~(2+) can significantly inhibit its activity. Some kinds of organic solvents can inhibit its activity as well, especially acetone. Kinetic studies showed that Michaelis constant Km is 4.58 mmol/L, Vmax is 16.13 U/L for the enzyme, Pnpr was used as substrate.
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