野生多孔菌分离、鉴定与漆酶的分离纯化和基因克隆
发布时间：2019-05-09 08:55
【摘要】：多孔菌是指子实层体呈孔状、质地为革质至木质的一类大型担子菌,主要生长在各类木材上。部分多孔菌具有产酶活性、药用活性、降解活性等应用价值。开展多孔菌的采集、分离与功能活性研究,可为多孔菌资源的开发利用,奠定理论和实验基础。本文针对采集的野生多孔菌,开展分离鉴定、培养条件、抗氧化活性、胞外漆酶活性、农药降解作用研究。具体内容如下:1.对两株野生多孔菌子实体进行分离纯化获得纯培养SS菌株和BJ菌株,并对其分类学地位、最适培养条件和液体发酵产物抗氧化活性进行分析。结果表明:结合形态学特征和ITS鉴定,确定SS菌株为多瘤木层孔菌(Phellinus tuberculosus)。菌丝体最适培养条件研究表明,最适碳源为葡萄糖,最适氮源为黄豆粉,最适C/N比为20/1,最适生长因子为维生素C,最适温度为28℃,最适pH为7.0。发酵液抗氧化活性为0.102 mM(FeSO4);BJ菌株为石榴嗜蓝孢孔菌(Fomitiporia punicata)。菌丝体最适培养碳源为葡萄糖和麦芽糖,最适氮源为酵母浸粉,最适C/N比为10/1,最适温度为28℃,最适pH为7.0。发酵液总抗氧化活性为0.517 mmol/L(维生素E),菌体的总超氧化物歧化酶活力为770.37 U/g。2.分离获得一株高产漆酶活性的菌株,编号F1635,经过形态学和分子学鉴定为硬毛栓菌(Trametes trogii),利用DEAE-Sepharose、SP-Sepharose和凝胶过滤层析等手段,对发酵液漆酶进行纯化,最终得到电泳纯级的酶蛋白(TsL)。进一步开展酶学特性、降解作用、基因克隆的研究。结果表明:该漆酶是分子量为64.8 kDa的单亚基蛋白。当以ABTS为作用底物时,Tsl的最适pH为2.6,最适温度为50℃,酶促动力学常数Km和Vmax值分别为18.58μM和1.125μmol/min。TsL漆酶对溴百里酚蓝、甲基橙、铬黑T、伊文思蓝等染料具有较强的降解作用,在12 h内降解率均达到78%以上;对农药乙酰甲胺磷和高效氯氰菊酯无明显降解作用。利用简并引物对漆酶的保守区进行克隆,得到长度分别为1246 bp和1264 bp的两条漆酶部分基因的cDNA序列。
[Abstract]:Porous bacteria are a kind of large basidiomycetes with pore shape and leathery to wood texture, which mainly grow on all kinds of wood. Some porous bacteria have the application value of enzyme production activity, medicinal activity, degradation activity and so on. The study on the collection, isolation and functional activity of porous bacteria can lay a theoretical and experimental foundation for the exploitation and utilization of porous bacteria resources. In this paper, the isolation and identification, culture conditions, antioxidant activity, extracellular laccase activity and pesticide degradation of wild porous bacteria were studied. The details are as follows: 1. Pure cultured SS and BJ strains were obtained by isolation and purification of two wild porous bacteria fruiting bodies, and their taxonomic status, optimum culture conditions and antioxidant activity of liquid fermentation products were analyzed. The results showed that combined with morphological characteristics and ITS identification, SS strain was identified as (Phellinus tuberculosus). The optimum culture conditions of mycelium showed that the optimum carbon source was glucose, the optimum nitrogen source was soybean powder, the optimum C / N ratio was 20 渭 1, the optimum growth factor was vitamin C, the optimum temperature was 28 鈩，
本文编号：2472641
[Abstract]:Porous bacteria are a kind of large basidiomycetes with pore shape and leathery to wood texture, which mainly grow on all kinds of wood. Some porous bacteria have the application value of enzyme production activity, medicinal activity, degradation activity and so on. The study on the collection, isolation and functional activity of porous bacteria can lay a theoretical and experimental foundation for the exploitation and utilization of porous bacteria resources. In this paper, the isolation and identification, culture conditions, antioxidant activity, extracellular laccase activity and pesticide degradation of wild porous bacteria were studied. The details are as follows: 1. Pure cultured SS and BJ strains were obtained by isolation and purification of two wild porous bacteria fruiting bodies, and their taxonomic status, optimum culture conditions and antioxidant activity of liquid fermentation products were analyzed. The results showed that combined with morphological characteristics and ITS identification, SS strain was identified as (Phellinus tuberculosus). The optimum culture conditions of mycelium showed that the optimum carbon source was glucose, the optimum nitrogen source was soybean powder, the optimum C / N ratio was 20 渭 1, the optimum growth factor was vitamin C, the optimum temperature was 28 鈩，
本文编号：2472641
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