高效降解羽毛的微白黄链霉菌Fea-10的分离鉴定及其角蛋白酶基因的异源表达

发布时间：2018-03-23 17:22

  本文选题：角蛋白酶　切入点：微白黄链霉菌Fea-10　出处：《西北农林科技大学》2017年硕士论文

【摘要】：角蛋白酶(keratinase)是一类可以降解顽固的角蛋白(如羽毛、羊毛、头发等)的蛋白酶,广泛存在于自然界中。角蛋白酶一般可以分类为丝氨酸蛋白酶和金属蛋白酶。由于角蛋白不能被普通的蛋白酶降解,且数量巨大,每年都会有数百万吨的含角蛋白废弃物产生,但是它们并不会在自然界中堆积,这种要是角蛋白降解微生物产生的角蛋白酶将这些废弃物分解,从而减轻了环境的负担。目前,已知细菌、真菌和放线菌中均有可以产生角蛋白酶的菌株,古细菌中也有可以产生角蛋白酶的报道。角蛋白降解微生物可以利用廉价的角质废弃物作为培养基质来产生角蛋白酶,使得角蛋白酶具有较低的生产成本,且角蛋白酶在各种工业领域具有很好的应用,具有很高的利用价值。本文在养鸡场堆积羽毛的土壤中,分离得到了一株角蛋白降解菌Fea-10。经过初步鉴定,Fea-10为微白黄链霉菌Streptomyces albidoflavus。Fea-10具有很高的角蛋白酶活性,可以完整的降解羽毛。通过已知的角蛋白酶的N端序列,在Fea-10基因组中找到两个可能的角蛋白酶基因gm2886和gm2888。这两个基因均由信号肽、前肽和成熟酶三个部分构成。将这两个基因的完整基因分别构建在pET15b-SUMO载体上,转入BL21(DE3)均不能表达;更换为pET28a载体和Rossta(DE3)宿主后,只有gm2888可以获得无活性的包涵体表达;将这两个基因去掉信号肽的酶原基因分别构建在pET22b载体上,转入Rossta(DE3),只有2886可以获得可溶性表达,但并没有角蛋白酶活性。考虑到可能链霉菌表达系统更适于链霉菌来源的角蛋白酶表达并折叠为有活性的成熟酶,使用链霉菌-大肠杆菌穿梭整合型质粒pSET152进行表达。表达宿主菌选择微弱角蛋白酶活的密旋链霉菌ACT12。构建gm2886带有原始启动子和红霉素启动子的表达载体和gm2888带有红霉素启动子的表达载体,分别接合转移导入ACT12。使用牛奶平板初筛接合子,并使用TSBY培养基发酵降解圈大的接合子,使用硫酸铵沉淀后,检测只有gm2886带红霉素的载体有角蛋白酶表达。使用Ni柱纯化重组蛋白,并测定其酶学性质。重组蛋白的最适温度为50℃,最适pH10.0,在40℃保温30 min后酶活仍有80%以上。金属离子Ca~(2+)、Mg~(2+)、K~+均抑制重组蛋白的活性;PMSF显著抑制重组蛋白的活性,重组蛋白为丝氨酸蛋白酶。重组蛋白对可溶的casein、Azo-casein、BSA和hemoglobin以及不可溶的羽毛和天青角蛋白均有活性。
[Abstract]:Keratinase is a kind of protease that degrades stubborn keratin (such as feathers, wool, hair, etc.). Keratin is widely distributed in nature. Keratin can be classified as serine protease and metalloproteinase. Because keratin can not be degraded by ordinary protease, and in large quantities, millions of tons of keratin waste are produced every year. But they don't pile up in nature, if keratinase, which is produced by keratin degradation microbes, breaks down these wastes, reducing the burden on the environment. Both fungi and actinomycetes have strains that produce keratinases, and there are reports of keratinases in ancient bacteria. Keratin degrading microbes can use cheap keratinized waste as a culture substrate to produce keratinases. Keratinase has a low production cost, and keratinase has a good application in various industrial fields, and has a high value of utilization. A keratin degrading strain Fea-10 was isolated. It was preliminarily identified that Streptomyces albidoflavus.Fea-10 had high keratinase activity and could completely degrade feathers. Two possible keratinase genes gm2886 and gm2888were found in the Fea-10 genome. The two genes were composed of signal peptide, prepeptide and mature enzyme. The complete genes of these two genes were constructed on pET15b-SUMO vector and transformed into BL21DE3). Only gm2888 could obtain inactive inclusion body expression after being replaced by pET28a vector and Rosstafen DE3) host, and the proenzyme genes which removed the signal peptide from the two genes were constructed on the pET22b vector respectively and transferred into pET22b vector, only 2886 of them could be expressed in a soluble way. Considering that Streptomyces expression systems may be more suitable for streptomyces to express and fold into active mature enzymes, Streptomycin-Escherichia coli shuttle integrated plasmid pSET152 was used to express the recombinant plasmid ACT12. Streptomyces sp. ACT12, which expressed the weak keratinase activity of the host strain, was used to construct the expression vector of gm2886 with the original promoter and erythromycin promoter and gm2888 with the expression of ACT12. The expression vector of erythromycin promoter, The conjugate transfer was introduced into ACT12. the conjugate was first screened with milk plate and fermented with TSBY medium to degrade the conjugate with large circle. After precipitation with ammonium sulfate, the conjugate was incubated with ammonium sulfate. The recombinant protein was purified by Ni column and its enzymatic properties were determined. The optimum temperature of recombinant protein was 50 鈩，

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