Brevibacillus parabrevis角蛋白酶的表征、异源表达及其分子改造

发布时间：2018-07-08 10:55

本文选题：角蛋白酶 + Brevibacillus　；参考：《江南大学》2017年博士论文

【摘要】：角蛋白酶是一类能特异性降解不溶性角蛋白底物的蛋白酶类,在制革、洗涤、医药等行业具有良好的应用前景,然而,角蛋白酶的应用仍受诸多因素限制,如酶活不够高、热稳定性差等问题,阻碍了角蛋白酶的工业化应用。为此,本论文以微生物角蛋白酶为研究对象,开展了产酶菌种选育、酶分离纯化、酶学特征探究、编码基因克隆表达、分子改造、酶解脱毛等一系列从理论到应用的研究工作。主要研究结果总结如下:(1)从皮革厂、禽类养殖场等富含角蛋白的环境中采集样品,以角蛋白为唯一碳源、氮源,分离筛选出高产角蛋白酶的微生物28株。由于低胶原活力的角蛋白酶在制革脱毛过程中不会对皮革质地造成损伤,本文结合胶原蛋白活力评价,筛选出2株具有高角蛋白酶活性和低胶原蛋白活性的菌株。通过形态学特征、生理与生化特性和分子生物学鉴定,将其分别命名为Acinetobacter sp.R-1和Brevibacillus parabrevis R-7。将上述2株细菌与研究室前期筛选获得的1株高产角蛋白酶的赤霉菌(Gibberella intermedia)一起用于羊皮脱毛效果的初步评价。结果显示,3株菌所产角蛋白酶均能完成羊皮的酶法脱毛,其中B.parabrevis脱毛效率最高,可在7 h内完成脱毛过程。因而,从产酶水平、脱毛性能以及胶原活力等综合考虑,选择B.parabrevis用于后续研究。(2)本文进一步对B.parabrevis所产角蛋白酶进行了分离纯化,并研究其酶学特性。将B.parabrevis发酵液上清依次经过硫酸铵分级沉淀、DEAE阴离子交换层析,纯化得到电泳纯的B.parabrevis角蛋白酶,结合SDS-PAGE和MALDI-TOF/TOF MS方法分析确定其相对分子量为28 kDa。角蛋白酶的最适pH和温度分别为8.0和60°C;在pH6.0-9.0和20-40°C条件下温育1 h后,残余酶活仍高于80%。Na+、Ca2+等金属离子对角蛋白酶有较强的促进作用,而Zn2+、Mn2+、Fe2+、Cu2+和Co2+则对角蛋白酶活性表现出显著的抑制效果。Tween 40、Tween 80和Triton X-100等表面活性剂对角蛋白酶的活性具有明显促进作用;而PMSF和EDTA则对酶活完全抑制,初步推测该酶属于丝氨酸金属蛋白酶类。底物特异性分析表明,该角蛋白酶对I型胶原蛋白无催化水解活性,这与前期的研究结果是一致的;以角蛋白为底物时,该酶的Km值和Vmax分别为15.67 mg?m L-1及1666.67 U?mg-1?min-1。(3)B.parabrevis角蛋白酶编码基因的克隆和异源表达。以该菌株的基因组DNA为模板,通过PCR扩增获得角蛋白酶编码基因(kerbp),其开放阅读框全长1152 bp,编码383个氨基酸,由31个氨基酸的信号肽序列、77个氨基酸的前肽序列和275个氨基酸的成熟肽序列所组成。将目的基因转入大肠杆菌宿主细胞,成功构建产角蛋白酶的重组菌E.coli BL21(DE3)/pET-22b(+)-kerbp,利用镍离子亲和柱对重组酶进行分离纯化,获得电泳纯的重组角蛋白酶,该酶分子量与野生酶基本一致。通过序列分析预测该酶的活性中心,发现Asp-32、His-64和Ser-221这3个氨基酸残基位点高度保守,采用重叠延伸PCR技术和酶活检测证实这3个保守位点为该角蛋白酶的催化活性中心,为后续改造研究奠定了基础。对重组酶的应用研究结果显示,该酶可以独立完成羊皮脱毛,进一步选择脂肪酶、淀粉酶与角蛋白酶进行复配脱毛,结果表明脂肪酶和淀粉酶分别与角蛋白酶复配使用均可以提高脱毛效率,对脱毛具有协同作用,可以节约蛋白酶的用量。(4)为进一步改善角蛋白酶的应用性能,采用计算机辅助手段对该酶热稳定性进行分子改造。利用SWISS-MODEL在线软件对角蛋白酶进行同源建模,获得酶分子的三维同源模型。通过对B-factor值计算、分子动力学模拟和同源序列比对,对3-D结构进行分析,预测影响酶热稳定性的关键氨基酸位点,并对其进行定点突变改造。通过对单点突变株的比酶活测定,获得了正突变株T218S、S236C和N181D,突变酶在60°C的半衰期相比野生酶分别提升3.05倍、1.18倍和1倍,T50也分别提高5.4°C、4°C和2°C。对这三个位点进一步组合突变,实验结果显示三个位点的组合突变对改善角蛋白酶的热稳定性并无协同效应,组合突变株中仅有N181D-T218S和N181D-S236C稳定性具有提升,其T50分别提高5.1°C和2.9°C,在60°C下的半衰期分别为野生酶的4.09倍和1.54倍。对突变酶热稳定性提高的原因进行深入分析,发现突变酶相比野生酶氢键数量增多,其中S236C突变酶除了氢键增加外,还形成1个盐桥,增加了侧链刚性;通过对蛋白质表面电荷的分析发现,N181D、S236C、N181D-T218S突变酶与野生酶相比在突变位点局部区域内表面电荷明显减少,结构更加致密。
[Abstract]:Keratin is a class of proteases that can specifically degrade insoluble keratin substrates. It has a good application prospect in leather making, washing, medicine and other industries. However, the application of keratin is still limited by many factors, such as low enzyme activity and poor thermal stability. Biological angle protease is the research object. A series of research work on the selection of enzyme producing strain, enzyme separation and purification, enzymology characteristics inquiry, coding gene cloning and expression, molecular transformation and enzymatic depilation are carried out. The main research results are summarized as follows: (1) collecting samples from the environment of keratin rich in leather factories, poultry farms and so on. 28 strains of high yield keratin were isolated and screened with keratin as the sole carbon source and nitrogen source. Due to the low collagen activity of keratin in the process of tannery and hair removal, the leather texture was not damaged. In this paper, 2 strains with high egg white enzyme activity and low collagen activity were screened through the evaluation of collagen activity. The morphological characteristics, physiological and biochemical characteristics and molecular biological identification were named as Acinetobacter sp.R-1 and Brevibacillus parabrevis R-7. respectively. A preliminary evaluation of the above 2 strains of bacteria and 1 strains of high yield keratin (Gibberella intermedia) obtained from the early stage of the study room was used to evaluate the hairs of the sheep skin. The keratin produced by 3 strains of bacteria can all complete the enzymatic hair removal of the sheepskin, in which the B.parabrevis depilatory efficiency is the highest, and the hair removal process can be completed within 7 h. Therefore, from the comprehensive consideration of the enzyme production level, the hair removal performance and the collagen activity, B.parabrevis is selected for the follow-up study. (2) the keratin produced by B.parabrevis has been further carried out in this paper. The B.parabrevis fermentation liquid supernatant was sequentially precipitated by ammonium sulfate precipitation, DEAE anion exchange chromatography was used to purify the electrophoretic B.parabrevis keratin. The optimum pH and temperature of its relative molecular weight of 28 kDa. keratin were determined by SDS-PAGE and MALDI-TOF/TOF MS method, respectively, and the temperature was 8., respectively. 0 and 60 degrees C; after incubating 1 h under the condition of pH6.0-9.0 and 20-40 C, the residual enzyme activity is still higher than 80%.Na+, Ca2+ and other metal ions have a strong promoting effect on the diagproteinase, while Zn2+, Mn2+, Fe2+, Cu2+ and Co2+ show significant inhibitory effect on the activity of the proteinase 40. The enzyme activity was obviously promoted, while the enzyme activity was completely suppressed by PMSF and EDTA. It was preliminarily speculated that the enzyme belonged to the serine metalloproteinase. The substrate specificity analysis showed that the keratin did not catalyze the hydrolysis of I type collagen, which was consistent with the previous research results. When keratin was used as the substrate, the Km value and Vmax of the enzyme were 1, respectively. Cloning and heterologous expression of 5.67 mg? M L-1 and 1666.67 U? Mg-1? Min-1. (3) B.parabrevis keratin gene. Using the genome DNA of the strain as a template, the keratin encoding gene (kerbp) is obtained by PCR amplification. Its open reading frame is 1152 BP, encoding 383 amino acids, 31 amino acid signal peptide sequences, 77 amino acids. The sequence of the propeptide and the mature peptide of 275 amino acids was formed. The target gene was transferred into the host cell of Escherichia coli, and the recombinant E.coli BL21 (DE3) /pET-22b (+) -kerbp of the keratin was successfully constructed. The recombinant protease was purified by the nickel ion affinity column to obtain the recombinant protease of the electrostroke. The molecular weight of the enzyme and the wild enzyme base were obtained. The 3 amino acid residues in Asp-32, His-64 and Ser-221 were highly conserved by sequence analysis. The 3 conserved sites were proved to be the catalytic active center of the protease by overlapping extended PCR and enzyme activity detection. The application of the recombinant enzyme to the subsequent modification was studied. The results showed that the enzyme could complete the hair removal of the sheep's skin independently, the lipase was further selected, the amylase and the keratin were compound and depilatory. The results showed that the use of lipase and amylase in the compound with keratin could improve the hair removal efficiency, and had synergistic effect on hair removal, and could save the amount of protease. (4) further improvement of horns eggs The application performance of white enzyme, using computer aided method to reconstruct the enzyme's thermal stability. Using SWISS-MODEL online software to model the homology of the protease, obtain the three-dimensional homologous model of the enzyme molecule. By calculating the B-factor value, the molecular dynamics simulation and the homologous sequence alignment, the structure of the 3-D is analyzed and the influence enzyme is predicted. The key amino acid loci of thermal stability were reformed at fixed point mutation. The positive mutant T218S, S236C and N181D were obtained by measuring the specific enzyme activity of single point mutants. The half-life of the mutant enzyme in 60 degree C was 3.05 times, 1.18 times and 1 times higher than that of the wild enzyme, and T50 was also improved by 5.4 degree C, 4 degree C and 2 degree C. to these three loci. Combined mutation, the experimental results showed that the combination mutation of three sites had no synergistic effect on the improvement of thermal stability of keratin. The only N181D-T218S and N181D-S236C stability in the combined mutants increased, and the T50 increased by 5.1 C and 2.9 degrees C respectively. The half-life of the mutant was 4.09 and 1.54 times that of the wild enzyme, respectively. The reason for the qualitative improvement was analyzed, and the number of hydrogen bonds of the mutant enzyme was increased compared to the wild enzyme. In addition to the increase of the hydrogen bond, the S236C mutant also formed 1 salt bridges and increased the side chain rigidity. By analyzing the surface charge of the protein, the N181D, S236C, and N181D-T218S mutase were compared with the wild enzyme at the local region of the mutation site. The surface charge is obviously reduced and the structure is more compact.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q55

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