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首页> 外文期刊>Bioresources and Bioprocessing >Use of combined UV and chemical mutagenesis treatment of Aspergillus terreus D34 for hyper-production of cellulose-degrading enzymes and enzymatic hydrolysis of mild-alkali pretreated rice straw
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Use of combined UV and chemical mutagenesis treatment of Aspergillus terreus D34 for hyper-production of cellulose-degrading enzymes and enzymatic hydrolysis of mild-alkali pretreated rice straw

机译：结合使用紫外线和化学诱变处理的土壤曲霉D34用于高产纤维素降解酶和酶水解轻度碱预处理的稻草

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Background: Microbial production of cellulose-degrading enzymes could be significantly improved using traditional mutagenesis treatment. Development of high-titre cellulase producing mutants drastically reduces the costs involved in cellulase production and downstream processing in commercial-scale enzyme production. Here, we have evaluated the efficacy of different Aspergillus terreus D34 mutants for hyper-production of improved cellulase enzymes utilizing locally available lignocellulosic biomass residues as growth substrates in solid state fermentation conditions. Further, enzymatic hydrolysis of mild-alkali pre-treated rice straw was performed using the improved cellulases. Results: A 4.9-fold higher β-glucosidase activity was obtained from ethyl methyl sulphonate (EMS) treated mutant strain (EMS2) when grown on mixed rice straw/sugarcane bagasse (RSBG) biomass growth substrate. Similarly with the EMS2 mutant and BG-grown culture extract a 1.1-fold higher xylanase activity was observed. Irrespective of the growth substrates and the mutant strains, the maximum cellulase (FPase, carboxymethyl cellulase, avicelase, β-glucosidase) and xylanase activities (U mL~(-1)) were 2.34, 39.8, 2.46, 19.9 and 655, respectively. Further, external supplementation of 20% bovine serum albumin (BSA), 3% tween 80 and 20% polyethylene glycol (PEG) 6000 to the crude enzyme extract increased the FPase activity nearly 4.0-, 2.8- and 2.2-fold. Addition of 0.05% sodium benzoate marginally increased the stability of cellulase enzyme and retained more than 60% of the initial activity after 96 h incubation at 37℃. While at 4℃, no loss in enzyme activity was observed even after prolonged incubation period (up to 90 days). Further, maximum reducing sugars of 0.842 g g~(-1) at a rate of 0.25 mM g~(-1) h~(-1) at 10% biomass loading of mild-alkali pretreated rice straw was produced using the BG-grown culture extract of EMS2 mutant strain. Conclusion: The extracellular protein production and corresponding cellulase activities of A. terreus D34 were significantly enhanced after combined UV and chemical mutagenesis treatments. In the present study, besides accelerating the rate of cellulase production, we have also demonstrated production of high reducing sugars by enzymatic saccharification of pretreated lignocellulosic biomass using hyper-produced cellulase enzymes. Due to high enzyme activity of the cellulase enzymes produced from the mutant strains, the volume of enzyme loadings in enzymatic hydrolysis could be reduced up to 7-fold. These studies clearly show the potential of the developed hyper-cellulase producing mutants in decreasing the overall process economics of cellulosic ethanol technology.
机译：背景：使用传统的诱变处理可以显着提高纤维素降解酶的微生物产量。产生高滴度纤维素酶的突变体的开发大大降低了纤维素酶生产和商业规模酶生产中下游加工所涉及的成本。在这里，我们评估了不同的曲霉D34突变体在固态发酵条件下利用本地可用的木质纤维素生物质残基作为生长底物，过量生产改良的纤维素酶的功效。此外，使用改良的纤维素酶进行了弱碱预处理的稻草的酶水解。结果：当在混合的稻草/蔗糖蔗渣（RSBG）生物量生长基质上生长时，从甲基磺酸乙酯（EMS）处理的突变菌株（EMS2）获得的β-葡萄糖苷酶活性高4.9倍。类似地，对于EMS2突变体和BG生长的培养物提取物，观察到木聚糖酶活性高1.1倍。无论生长底物和突变菌株如何，最大纤维素酶（FPase，羧甲基纤维素酶，avicelase，β-葡萄糖苷酶）和木聚糖酶活性（U mL〜（-1））分别为2.34、39.8、2.46、19.9和655。此外，在粗酶提取物中外部补充20％的牛血清白蛋白（BSA），3％的吐温80和20％的聚乙二醇（PEG）6000可使FPase活性提高了近4.0倍，2.8倍和2.2倍。在37℃孵育96小时后，添加0.05％的苯甲酸钠略微增加了纤维素酶的稳定性，并保留了超过60％的初始活性。在4℃下，即使延长孵育时间（长达90天），酶活性也没有降低。此外，使用BG种植的稻草，在轻度碱预处理的稻草的10％生物量负载下，以0.25 mM g〜（-1）h〜（-1）的速率，最大还原糖为0.842 gg〜（-1）。 EMS2突变菌株的培养提取物。结论：结合紫外线和化学诱变处理后，土曲霉D34的胞外蛋白产生和相应的纤维素酶活性显着提高。在本研究中，除了加快纤维素酶的生产速度外，我们还证明了通过使用超量生产的纤维素酶对预处理的木质纤维素生物质进行酶促糖化来生产高还原糖。由于由突变菌株产生的纤维素酶的高酶活性，在酶促水解中的酶负载量可以减少多达7倍。这些研究清楚地表明，开发的产生高纤维素酶的突变体在降低纤维素乙醇技术的整体工艺经济性方面具有潜力。

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