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重离子辐照结合α-纤维素适应性进化提高黑曲霉真菌纤维素酶活性研究

Heavy Ion Irradiation Combined with Alpha-cellulose Adaptive Evolution to Improve Cellulase Activity of Aspergillus niger

  • 摘要: 为获得可有效降解植物细胞壁中纤维素的高产纤维素酶菌株,将具有安全可靠、不产生毒素、生长速度快且发酵周期短等优势的黑曲霉作为出发菌株。利用重离子12C6+束流按照0 , 40 , 70 , 100 , 130 , 160, 190 Gy的辐照剂量对黑曲霉原始菌株进行辐照,经平板初筛后得到两株优良菌株;然后添加α-纤维素进行了8周自适应性进化,通过连续5代发酵该菌株生长性能稳定;将得到的每代菌株进行胞内蛋白分子量的对比,得到一株最优菌株CJH-JWSFZh-W122,将其摇瓶发酵测定纤维素酶活性,其产纤维素酶的滤纸酶活(FPA)到了223.5 U/mL,内切葡聚糖酶活(CMC)达到了440.8 U/mL,分别比原始菌株提高了6.07%和8.01%。突变株在连续5代发酵后仍保持了良好的发酵性能。对突变菌株CJH-JWSFZh-W122和原始菌株进行了RAPD分析和SDS-PAGE电泳分析,证实了该菌株存在基因突变。

     

    Abstract: In order to obtain high-yielding cellulase strains that can effectively degrade cellulose in plant cell walls, Aspergillus niger, which has the advantages of safety and reliability, non-toxicity, fast growth and short fermentation period, was used as the starting strain. The original strain, Aspergillus niger, was irradiated by heavy ion beam 12C6+ (irradiation doses: 40, 70, 100, 130, 160 and 190 Gy) , and two excellent strains were obtained after primary screening on the plate; 8 weeks of self-adaptive evolution, the strain has stable growth performance through 5 consecutive generations of fermentation; the obtained strains of each generation were compared for the molecular weight of intracellular proteins, and an optimal mutant strain CJH-JWSFZh-W122 was obtained, which was determined by shaking flask fermentation Cellulase activity, the filter paper activity(FPA) of cellulase production reached 223.5 U/mL, and the carboxymethyl cellulase activity(CMCase) reached 440.8 U/mL, which were 6.07% and 8.01% higher than the original strains, respectively. The excellent fermentation performance was still maintained after 5 consecutive generations; RAPD analysis and SDS-PAGE electrophoresis analysis were applied to compare the mutant strain CJH-JWSFZh-W122 and original strain, which confirmed the gene mutation of this strain.

     

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