重组草酸青霉生淀粉糖化酶的酶学特性鉴定

Characterization of a Recombinant Raw Starch-digesting Glucoamylase from Penicillium oxalicum

关键词:  草酸青霉  生淀粉糖化酶  基因克隆与表达  酶学性质

Key words:  Penicillium oxalicum  raw starch digesting glucoamylase  gene cloning and expression  enzymatic properties

摘要:

[目的]了解在毕赤酵母中表达的来源于草酸青霉(Penicillium oxalicum)GXU20的重组生淀粉糖化酶的酶学特性。[方法]用3,5-二硝基水杨酸法测定pH值、温度、金属离子和化学试剂对纯化的重组生淀粉糖化酶的活力的影响,同时测定酶的底物特异性、酶对生淀粉的吸附能力以及酶对不同生淀粉的水解效率等,用高效液相色谱法对该酶水解生木薯淀粉的产物进行分析鉴定,用扫描电子显微镜观察重组酶对不同生淀粉颗粒的水解方式。[结果]重组生淀粉糖化酶rPoGA15A的最适pH值为4.5,最适温度为65℃,在pH值为2.0~10.0时,重组酶具有较好的pH耐受性,温度小于50℃时,酶的稳定性好。除Ag⁺、Cu²⁺和SDS之外,其他大部分金属离子和化学试剂对重组酶的酶活力影响不大。重组生淀粉糖化酶对大米和玉米生淀粉的活性较高,对木薯和马铃薯生淀粉的活性次之,重组生淀粉糖化酶rPoGA15A对不同生淀粉的吸附能力与其对相应不同生淀粉的水解活性大小呈正相关。扫描电子显微镜观察表明重组生淀粉糖化酶对不同生淀粉颗粒的降解作用明显,水解方式各有特点。高效液相色谱分析表明该重组生淀粉糖化酶水解生木薯淀粉的产物仅有葡萄糖。重组生淀粉糖化酶rPoGA15A在40℃下对大米和玉米生淀粉水解72 h后水解率分别达到86.5%和71.9%。[结论]重组生淀粉糖化酶具有广泛的pH耐受性,对生淀粉具有高水解活性,在生淀粉的水解和生料同步糖化发酵生产酒精中有一定的应用潜力。

Abstract:

[Objective] In order to investigate enzymatic properties of a recombinant raw starch-digesting glucoamylase rPoGA15A from Penicillium oxalicum GXU20, which is expressed in Pichia pastoris, the recombinant enzyme rPoGA15A is biochemically characterized in this study.[Methods] The enzymatic properties of rPoGA15A, such as the effects of pH, temperature, metal ions and chemical reagents on enzyme activity, substrate specificity, raw starch adsorption ability, and hydrolysis efficiency of the enzyme towards different raw starches are measured by the 3, 5-dinitrosalicylic acid method.Moreover, the hydrolysates of raw cassava starch by the enzyme rPoGA15A are analyzed by high performance liquid chromatography (HPLC), and different raw starch granules degraded by the enzyme rPoGA15A are observed by scanning electron microscopy (SEM).[Results] The recombinant enzyme rPoGA15A displays optimal pH and temperature at pH 4.5 and 65℃, respectively, and exhibits great stability over a broad pH range from 2.0~10.0, and is stable below 50℃.Most of metal ions and chemical reagents have little effect on the enzymatic activity except Ag⁺, Cu²⁺ and SDS. The enzyme rPoGA15A shows higher enzymatic activity towards raw rice and corn starches than that towards raw cassava and potato starches. The adsorption capacity of the enzyme rPoGA15A to different raw starches is consistent with the corresponding substrate specific activities. In addition, SEM observation demonstrates that the enzyme rPoGA15A has great capacity to degrade different raw starches and has respective characteristics of hydrolysis mode against different raw starch granules. HPLC analysis of hydrolysate of raw cassava starch by the enzyme shows that glucose is the only product. Furthermore, the recombinant enzyme rPoGA15A can hydrolyze 86.5% of raw rice starch and 71.9% of raw corn starch after 72 h hydrolysis at 40℃.[Conclusion] The recombinant raw starch-digesting glucoamylase rPoGA15A exhibits good stability over broad pH range, and shows high ability to digest different raw starches, thus indicating its potential in application in raw starch hydrolysis, and simultaneous saccharification and fermentation of raw starch to ethanol.