右旋糖酐酶高产菌株选育及其酶学性质研究

发布时间：2018-05-10 16:46

本文选题：右旋糖酐酶 + 圆弧青霉　；参考：《安徽工程大学》2017年硕士论文

【摘要】：右旋糖酐酶(Dextranase;EC 3.2.1.11)可专一性催化裂解右旋糖酐(Dextran)分子中的α-1,6葡萄糖苷键,用于制糖工业降低甘蔗汁粘度、医药工业中低分子量药用右旋糖酐制备、牙医业去除牙龈糖苷沉积等领域,工业应用广泛,市场需求巨大。当前,右旋糖酐酶生产菌株产酶水平偏低,使得规模工业发酵受限,生产效率不高。为提高右旋糖酐酶的产量以适应实际应用的需要,本研究开展圆弧青霉产右旋糖酐酶菌株的诱变选育工作。首先采用常压室温等离子体(ARTP)和紫外辐照(UV-ray)技术,对右旋糖酐酶产生菌圆弧青霉Penicilliumcyclopium(P.cyclopium 4022)开展多轮诱变处理,研究蓝色葡聚糖平板透明圈圈径比与摇瓶酶活的相关性,建立右旋糖酐酶平板透明圈法检出突变体的方法,以获得右旋糖酐酶高产突变株;在此基础上,对突变株P.cyclopium12p27产右旋糖酐酶的培养基营养条件及产酶培养条件开展优化研究,以提升圆弧青霉产右旋糖酐酶的产酶能力;并对P.cyclopium 12p27所产右旋糖酐酶进行分离与纯化,考察其温度、pH稳定性及其酶学性质。主要研究结果如下:1.采用ARTP辐照和紫外诱变技术对P.cyclopium 4022诱变处理,经过平板透明圈法初筛及摇瓶复筛,获得一株遗传性能稳定的右旋糖酐酶高产突变株P.cyclopium 12p27,该菌株较出发菌株酶活力分别提高了49.64%(ARTP)和 128.18%(UV-ray)。构建了 Blue-Dextran 2000 平皿透明圈初筛与摇瓶发酵复筛相结合的突变株筛选方法:Blue-Dextran 2000平皿透明圈圈径比与摇瓶发酵产酶酶力相关性良好。2.通过单因素实验研究碳源、氮源以及表面活性物质对酶活的影响,进而结合数理统计法对圆弧青霉产右旋糖酐酶发酵条件进行优化研究。采用响应面实验设计对产酶条件进行优化,得到圆弧青霉产右旋糖酐酶最佳发酵条件为:D70:蔗糖49.5g/L,牛肉膏4.1g/L,山梨醇19.70mmol/L。MgSO4·7H2O0.5g/L、KCl0.5g/L、FeSO4·4H2O0.01g/L、K2HPO41.0g/L、pH6.5。在该条件下右旋糖酐酶产酶酶活达到10.95U/mL,较优化提升了 173.07%。3.对产酶培养条件中的不同因素进行优化处理,进一步提高经诱变选育得到的高产菌株12P27的产酶酶活。包括对发酵过程中的培养温度、摇床转速、培养基初始pH、摇瓶装液量与接种量等培养条件进行择优筛选。获得最有利于发酵菌株产酶的培养条件为:在30℃下,控制摇床转速为230r/min,摇瓶装液量40mL,初始pH为7,接种量0.5mL,产酶酶活达到14.48U/mL,较优化前的10.95U/mL,提高了 1.32倍。4.利用75%饱和度的硫酸铵沉淀与DEAE Cellulose DE-52离子交换层析,对P.cyclopium12p27右旋糖酐酶进行分离纯化。使右旋糖酐酶的比活力从124.53U/mg提高到1052.71U/mg,纯化了 8.45倍,回收率达到10.98%。经SDS-PAGE电泳鉴定,纯化后的右旋糖酐酶纯度已达到电泳纯,表观分子量约65 kDa。5.P.cyclopium 12p27右旋糖酐酶的酶学性质研究表明:该酶在温度30℃时表现出良好的热稳定性,最适催化温度为40℃;最适pH为6.0,偏碱性条件下右旋糖酐酶活力保留效果优于偏酸条件,在pH4～7范围内,右旋糖酐酶稳定性良好。Fe2+和K+对其酶活性具有促进作用,而Pb2+、Al3+、Ag+、Mn2+、Co2+和Zn2+对酶活性有一定的抑制作用。该酶对于不同分子量右旋糖酐的水解性能有差异,对大分子右旋糖酐的水解能力强于中低分子量右旋糖酐,对D70以上的右旋糖酐水解能力高于D3、D40等小分子右旋糖酐底物。采用米氏方程的转换形式,作Lineweaver-Burk双倒数图,得到米氏常数Km值为4.4827mmol/L,Vmax值为 1.1461μmol·mL~(-1)·min~(-1)。
[Abstract]:Dextranase (EC 3.2.1.11) can be used to catalyze the catalytic cracking of alpha -1,6 glucoside bonds in the Dextran molecule, which is used in sugar industry to reduce the viscosity of sugarcane juice, the preparation of low molecular weight medicinal dextran in the pharmaceutical industry, and the removal of gingival glycosides from the medical industry. The industry is widely used and the market demand is huge. The production strain of dextran producing strain is low, which makes the industrial fermentation limited and the production efficiency is not high. In order to improve the production of dextran enzyme to meet the needs of practical application, this study carried out the mutagenesis and breeding of the strain of dextran produced by Penicillium arcus. First, the ambient temperature plasma (ARTP) and ultraviolet radiation (UV-ray) were used. Technology, a multi wheel mutagenesis was carried out on the dextran producing strain of Penicillium Penicilliumcyclopium (P.cyclopium 4022), and the correlation between the transparent circle diameter ratio of the blue dextran plate and the enzyme activity of the shake flask was studied. The method of detecting the mutant by the transparent ring method of the dextran enzyme plate was established in order to obtain the mutant strain with high dextran enzyme production. The nutrient conditions of mutant P.cyclopium12p27 producing dextran and the cultivation conditions of enzyme production were optimized to improve the enzyme production capacity of dextran produced by Penicillium ARCIS, and to separate and purify the dextran enzyme produced by P.cyclopium 12p27, and investigate its temperature, pH stability and its enzymology properties. As follows: 1. the mutagenesis of P.cyclopium 4022 was treated by ARTP irradiation and ultraviolet mutagenesis. A mutant strain P.cyclopium 12p27 with high genetic performance and stable dextran enzyme was obtained by a transparent plate screening and shake flask rescreening. The strain was 49.64% (ARTP) and 128.18% (UV-ray) higher than the enzyme activity of the starting strain, and Blue was constructed. Selection of mutant strain screening methods combined with -Dextran 2000 Petri dish transparent ring initial screening and shake flask fermentation: a good correlation between Blue-Dextran 2000 plate transparent circle diameter ratio and shake flask fermentation production of enzyme force.2. through single factor experiment to study the effect of carbon source, nitrogen source and surfactant on enzyme activity, and then combined with mathematical statistics method for circular arc green. Optimization of fermentation conditions of mildew producing dextranase. The optimum fermentation conditions were obtained by the response surface experiment design. The optimum fermentation conditions of the enzyme were as follows: D70: sucrose 49.5g/L, beef paste 4.1g/L, sorbitol 19.70mmol/L.MgSO4. 7H2O0.5g/L, KCl0.5g /L, FeSO4. 4H2O0.01g/L, K2HPO41.0g/L, pH6.5. in this condition The enzyme activity of lower dextran enzyme production reached 10.95U/mL, optimized and improved 173.07%.3. to optimize the different factors in the culture conditions of enzyme production, and further improved the enzyme production of high yield strain 12P27, which was induced by mutagenesis, including the culture temperature of the fermentation process, the rotational speed of the rocking bed, the initial pH of the medium, the volume of the shake bottling liquid and the connection. The optimum conditions for the cultivation of the fermentation conditions were as follows: at 30, the rotation speed of the rocking bed was 230r/min, the volume of the shake bottled liquid was 40mL, the initial pH was 7, the inoculation amount was 0.5mL, the enzyme production reached 14.48U/mL, and the optimized 10.95U/mL was improved by 1.32 times the 75% saturation of the ammonium sulfate precipitation and DE. AE Cellulose DE-52 ion exchange chromatography was used to separate and purify P.cyclopium12p27 dextran. The specific activity of dextran was increased from 124.53U/mg to 1052.71U/mg, purified 8.45 times, and the recovery rate was identified by SDS-PAGE electrophoresis. The purity of the purified dextran has reached pure electrophoresis and the apparent molecular weight is about 65 kDa.5.P. The enzymatic properties of cyclopium 12p27 dextran showed that the enzyme exhibited good thermal stability at 30 C, the optimum catalytic temperature was 40, the optimum pH was 6, and the activity retention of dextran was better than the partial acid condition under the alkaline condition. In the range of pH4 ~ 7, the stability of dextran enzyme was good.Fe2+ and K+ on its enzyme activity. Pb2+, Al3+, Ag+, Mn2+, Co2+ and Zn2+ have a certain inhibitory effect on the enzyme activity. The hydrolysis of dextran with different molecular weight is different, the hydrolysis ability of the macromolecular dextran is stronger than that of low molecular weight dextran, and the hydrolysis ability of dextran above D70 is higher than that of D3, D40 and other small molecules. Using the conversion form of the Michaelis equation, the Lineweaver-Burk double reciprocal graph is used to obtain the Km value of 4.4827mmol/L and the Vmax value of 1.1461 Mu mol. ML~ (-1). Min~ (-1).

【学位授予单位】：安徽工程大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q93

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