产纤维素酶菌株的筛选及酶学性质研究

发布时间：2018-02-27 18:11

  本文关键词： 纤维素酶 放线菌 酶学性质 酶的纯化　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：自然界中的纤维素资源极为丰富,如果能对这些纤维素资源加以利用,不仅保护了环境,又可实现资源回收利用,因此国内外对于纤维素酶的研究一直是个研究热点。本研究从腐植土里筛选出具有降解纤维素酶能力的放线菌一株,通过单因素及正交试验确定其最适的产酶发酵培养基;用生理生化及分子生物学方法鉴定该放线菌;对该纤维素酶进行酶学研究;最后通过分离纯化以获得较高纯度的内切酶。研究为放线菌所产纤维素酶的特性提供理论依据,对高纯度纤维素酶的获取及开发利用具有指导意义。1.试验通过对腐植土中具降解纤维素能力的菌进行筛选,最终确定筛得放线菌一株,并编号NO.11,其在CMC-Na液体培养基中,内切型-β-葡聚糖酶酶活为30.06U·mL-1,外切型-β-葡聚糖酶酶活为10.40U·mL-1,β-葡萄糖苷酶酶活为8.05U·mL-1,FPA酶酶活为 13.88U·mL-1。2.通过对菌株NO.11进行生理生化及分子生物学鉴定,最终确定该菌为暗灰链霉菌。3.通过正交实验,得出菌株NO.11最适的产酶培养基配方为:0.2%的牛肉膏,2%的稻草粉,初始pH7.5,0.3%的KH2PO4,接种量为6%,发酵温度为32℃,发酵时间为5 d。在此条件下菌株NO.11的CMC酶活力为55.28 U·mL-1,其产酶能力明显提高。4.为获得更高纯度的纤维素酶,试验通过硫酸铵分级盐析、透析、葡聚糖凝胶G-100柱层析分离纯化纤维素酶。试验结果显示,最终纯化得到的酶液酶活为53.09U·mL-1,比活力为211.22 U·mg-1,纯化倍数为1.90倍。通过SDS-PAGE可知,该放线菌所产CMC酶的分子量在65kDa左右,经硫酸铵盐析、透析、Sephadex G-100柱层析后,电泳检测其可达到电泳纯。5.在菌株NO.11所产内切酶、外切酶、β-葡萄糖苷酶的酶促反应中,其最适pH值为4.5;滤纸酶、外切酶及β-葡萄糖苷酶的最适反应温度均为50℃;内切酶的最适反应温度为55℃;内切酶在pH值4.0-6.5间其相对酶活在70%以上;在30-60℃的反应温度下,其相对酶活在70%以上;金属离子Mg2+,Ca2+,Mn2+对各酶均有激活作用。Zn2+对外切酶有激活作用,但对内切酶、β-葡萄糖苷酶都产生抑制作用。Fe3+对β-葡萄糖苷酶有抑制作用。
[Abstract]:The cellulose resources in nature are extremely rich. If these cellulose resources can be utilized, not only the environment can be protected, but also the resources can be recycled. Therefore, the research on cellulase has been a hot topic at home and abroad. In this study, a strain of actinomycetes with cellulase degradation ability was selected from the decomposed soil, and the optimum fermentation medium for cellulase production was determined by single factor and orthogonal test. The actinomycetes were identified by physiological, biochemical and molecular biological methods; the cellulase was studied by enzyme; finally, the endonuclease with high purity was obtained by separating and purifying the cellulase from actinomycetes. The study provides a theoretical basis for the characteristics of cellulase produced by actinomycetes. It is of guiding significance to obtain, develop and utilize high purity cellulase. By screening the bacteria with the ability of degrading cellulose in the decomposed soil, a strain of actinomycetes was identified and numbered no. 11 in CMC-Na liquid medium. The activity of 尾 -glucanase was 30.06U 路mL-1, the activity of extraneous 尾 -glucanase was 10.40U 路mL-1, the activity of 尾 -glucosidase was 8.05U 路mL-1FPA was 13.88U 路mL-1.2.The results of physiological, biochemical and molecular biological identification of strain NO.11 were as follows: (1) the enzyme activity of 尾 -glucanase was 30.06 U 路mL ~ (-1), 10.40 U 路ml ~ (-1) and 8.05 U 路m ~ (-1) 路m ~ (-1), respectively. Finally, the strain was determined as Streptomyces dark ash. By orthogonal experiment, the optimum enzyme production medium of strain NO.11 was obtained as follows: 0.2% beef paste 2% straw powder, initial pH 7.5% 0.3% KH 2PO 4, inoculation amount 6%, fermentation temperature 32 鈩，

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