發(fā)布時(shí)間：2018-05-13 20:45

  本文選題：海水芽孢桿菌 + 發(fā)酵優(yōu)化　； 參考：《國家海洋局第三海洋研究所》2016年碩士論文

【摘要】：隨著養(yǎng)殖業(yè)規(guī)模持續(xù)擴(kuò)大,羽毛作為家禽的副產(chǎn)物的大量堆積不僅造成環(huán)境破壞、病原微生物滋生,還會造成角蛋白資源的嚴(yán)重浪費(fèi)。本研究采用條件溫和、工藝環(huán)保的微生物法降解羽毛角蛋白,利用液體發(fā)酵法探究菌株降解羽毛的最優(yōu)化條件,并對角蛋白酶基因進(jìn)行克隆,進(jìn)一步在大腸桿菌系統(tǒng)中進(jìn)行融合表達(dá),擬實(shí)現(xiàn)角蛋白酶的外源表達(dá)。1.以羽毛為唯一碳、氮源篩選到一株高效降解角蛋白的菌株海水芽孢桿菌Bacillus aquimaris JM7。對Bacillus aquimaris JM7以羽毛為底物的發(fā)酵條件進(jìn)行了單因素及正交試驗(yàn)優(yōu)化,實(shí)驗(yàn)結(jié)果顯示:Bacillus aquimaris JM7降解羽毛最適發(fā)酵條件為:培養(yǎng)基初始pH值為9.0;菌接種量4%(v/v);培養(yǎng)溫度40℃;搖床轉(zhuǎn)速150rpm;羽毛粉添加量為0.2%(m/v);額外添加氮源及蔗糖對菌株降解羽毛粉均有抑制作用。上述條件下培養(yǎng)30h后,羽毛粉降解率達(dá)74.5%,高于多數(shù)文獻(xiàn)報(bào)道水平。Bacillus aquimaris JM7降解羽毛主要產(chǎn)生谷氨酸、絲氨酸、脯氨酸等17種氨基酸。2.對角蛋白酶基因Ker02562進(jìn)行了克隆、表達(dá)和純化。采用PCR技術(shù)擴(kuò)增了角蛋白酶基因,其全長為963 bp,編碼了一個(gè)由320個(gè)氨基酸殘基組成、預(yù)計(jì)分子量為34.181kDa的蛋白質(zhì)。構(gòu)建了重組表達(dá)質(zhì)粒并轉(zhuǎn)化大腸桿菌BL21(DE3)進(jìn)行誘導(dǎo)表達(dá),SDS-PAGE分析表明,重組角蛋白酶E.coli-pCold I-Ker02562的分子量為38kDa左右(角蛋白酶34kDa,pCold I自身標(biāo)簽4kD左右),和預(yù)測分子量一致。3.對純化后的角蛋白酶酶學(xué)性質(zhì)研究結(jié)果表明:(1)酶的最適反應(yīng)溫度為40℃,在10-50℃條件下熱穩(wěn)定性良好;最適pH為7.0-10.0,該條件下酶能保持90%以上的活性;(2)金屬離子Ca2+,Fe2+,Sr2+對酶活具有促進(jìn)作用;(3)濃度為5mM的PMSF和1mM、5mM的EDTA對酶活有抑制作用,說明該酶為絲氨酸蛋白酶,其活性中心可能含有金屬離子;(4)有機(jī)溶劑DMSO、異丙醇和乙腈對酶活均有抑制作用,濃度越高,抑制作用越大。
[Abstract]:As the scale of aquaculture continues to expand a large number of feathers as by-products of poultry not only cause environmental damage pathogenic microorganisms breeding but also cause serious waste of keratin resources. In this study, a mild and environmentally friendly microbial method was used to degrade feather keratin, and liquid fermentation was used to explore the optimum conditions for degradation of feather, and the keratinase gene was cloned. Further fusion expression was carried out in Escherichia coli system to realize the extraneous expression of keratinase. A highly efficient keratin degrading strain, Bacillus aquimaris JM7, was isolated from the feather as the sole carbon source. The fermentation conditions of Bacillus aquimaris JM7 with feather as substrate were optimized by single factor and orthogonal experiment. The results showed that the optimum fermentation conditions were as follows: initial pH value of culture medium was 9. 0; inoculation amount was 4% / v / v; culture temperature was 40 鈩，

本文編號：1884733