本文關(guān)鍵詞： 纖維素酶 放線菌 酶學(xué)性質(zhì) 酶的純化　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：自然界中的纖維素資源極為豐富,如果能對這些纖維素資源加以利用,不僅保護了環(huán)境,又可實現(xiàn)資源回收利用,因此國內(nèi)外對于纖維素酶的研究一直是個研究熱點。本研究從腐植土里篩選出具有降解纖維素酶能力的放線菌一株,通過單因素及正交試驗確定其最適的產(chǎn)酶發(fā)酵培養(yǎng)基;用生理生化及分子生物學(xué)方法鑒定該放線菌;對該纖維素酶進行酶學(xué)研究;最后通過分離純化以獲得較高純度的內(nèi)切酶。研究為放線菌所產(chǎn)纖維素酶的特性提供理論依據(jù),對高純度纖維素酶的獲取及開發(fā)利用具有指導(dǎo)意義。1.試驗通過對腐植土中具降解纖維素能力的菌進行篩選,最終確定篩得放線菌一株,并編號NO.11,其在CMC-Na液體培養(yǎng)基中,內(nèi)切型-β-葡聚糖酶酶活為30.06U·mL-1,外切型-β-葡聚糖酶酶活為10.40U·mL-1,β-葡萄糖苷酶酶活為8.05U·mL-1,FPA酶酶活為 13.88U·mL-1。2.通過對菌株NO.11進行生理生化及分子生物學(xué)鑒定,最終確定該菌為暗灰鏈霉菌。3.通過正交實驗,得出菌株NO.11最適的產(chǎn)酶培養(yǎng)基配方為:0.2%的牛肉膏,2%的稻草粉,初始pH7.5,0.3%的KH2PO4,接種量為6%,發(fā)酵溫度為32℃,發(fā)酵時間為5 d。在此條件下菌株NO.11的CMC酶活力為55.28 U·mL-1,其產(chǎn)酶能力明顯提高。4.為獲得更高純度的纖維素酶,試驗通過硫酸銨分級鹽析、透析、葡聚糖凝膠G-100柱層析分離純化纖維素酶。試驗結(jié)果顯示,最終純化得到的酶液酶活為53.09U·mL-1,比活力為211.22 U·mg-1,純化倍數(shù)為1.90倍。通過SDS-PAGE可知,該放線菌所產(chǎn)CMC酶的分子量在65kDa左右,經(jīng)硫酸銨鹽析、透析、Sephadex G-100柱層析后,電泳檢測其可達到電泳純。5.在菌株NO.11所產(chǎn)內(nèi)切酶、外切酶、β-葡萄糖苷酶的酶促反應(yīng)中,其最適pH值為4.5;濾紙酶、外切酶及β-葡萄糖苷酶的最適反應(yīng)溫度均為50℃;內(nèi)切酶的最適反應(yīng)溫度為55℃;內(nèi)切酶在pH值4.0-6.5間其相對酶活在70%以上;在30-60℃的反應(yīng)溫度下,其相對酶活在70%以上;金屬離子Mg2+,Ca2+,Mn2+對各酶均有激活作用。Zn2+對外切酶有激活作用,但對內(nèi)切酶、β-葡萄糖苷酶都產(chǎn)生抑制作用。Fe3+對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，

本文編號：1543762