本文選題：角蛋白酶 + Brevibacillus�。� 參考：《江南大學(xué)》2017年博士論文

【摘要】：角蛋白酶是一類能特異性降解不溶性角蛋白底物的蛋白酶類,在制革、洗滌、醫(yī)藥等行業(yè)具有良好的應(yīng)用前景,然而,角蛋白酶的應(yīng)用仍受諸多因素限制,如酶活不夠高、熱穩(wěn)定性差等問(wèn)題,阻礙了角蛋白酶的工業(yè)化應(yīng)用。為此,本論文以微生物角蛋白酶為研究對(duì)象,開展了產(chǎn)酶菌種選育、酶分離純化、酶學(xué)特征探究、編碼基因克隆表達(dá)、分子改造、酶解脫毛等一系列從理論到應(yīng)用的研究工作。主要研究結(jié)果總結(jié)如下:(1)從皮革廠、禽類養(yǎng)殖場(chǎng)等富含角蛋白的環(huán)境中采集樣品,以角蛋白為唯一碳源、氮源,分離篩選出高產(chǎn)角蛋白酶的微生物28株。由于低膠原活力的角蛋白酶在制革脫毛過(guò)程中不會(huì)對(duì)皮革質(zhì)地造成損傷,本文結(jié)合膠原蛋白活力評(píng)價(jià),篩選出2株具有高角蛋白酶活性和低膠原蛋白活性的菌株。通過(guò)形態(tài)學(xué)特征、生理與生化特性和分子生物學(xué)鑒定,將其分別命名為Acinetobacter sp.R-1和Brevibacillus parabrevis R-7。將上述2株細(xì)菌與研究室前期篩選獲得的1株高產(chǎn)角蛋白酶的赤霉菌(Gibberella intermedia)一起用于羊皮脫毛效果的初步評(píng)價(jià)。結(jié)果顯示,3株菌所產(chǎn)角蛋白酶均能完成羊皮的酶法脫毛,其中B.parabrevis脫毛效率最高,可在7 h內(nèi)完成脫毛過(guò)程。因而,從產(chǎn)酶水平、脫毛性能以及膠原活力等綜合考慮,選擇B.parabrevis用于后續(xù)研究。(2)本文進(jìn)一步對(duì)B.parabrevis所產(chǎn)角蛋白酶進(jìn)行了分離純化,并研究其酶學(xué)特性。將B.parabrevis發(fā)酵液上清依次經(jīng)過(guò)硫酸銨分級(jí)沉淀、DEAE陰離子交換層析,純化得到電泳純的B.parabrevis角蛋白酶,結(jié)合SDS-PAGE和MALDI-TOF/TOF MS方法分析確定其相對(duì)分子量為28 kDa。角蛋白酶的最適pH和溫度分別為8.0和60°C;在pH6.0-9.0和20-40°C條件下溫育1 h后,殘余酶活仍高于80%。Na+、Ca2+等金屬離子對(duì)角蛋白酶有較強(qiáng)的促進(jìn)作用,而Zn2+、Mn2+、Fe2+、Cu2+和Co2+則對(duì)角蛋白酶活性表現(xiàn)出顯著的抑制效果。Tween 40、Tween 80和Triton X-100等表面活性劑對(duì)角蛋白酶的活性具有明顯促進(jìn)作用;而PMSF和EDTA則對(duì)酶活完全抑制,初步推測(cè)該酶屬于絲氨酸金屬蛋白酶類。底物特異性分析表明,該角蛋白酶對(duì)I型膠原蛋白無(wú)催化水解活性,這與前期的研究結(jié)果是一致的;以角蛋白為底物時(shí),該酶的Km值和Vmax分別為15.67 mg?m L-1及1666.67 U?mg-1?min-1。(3)B.parabrevis角蛋白酶編碼基因的克隆和異源表達(dá)。以該菌株的基因組DNA為模板,通過(guò)PCR擴(kuò)增獲得角蛋白酶編碼基因(kerbp),其開放閱讀框全長(zhǎng)1152 bp,編碼383個(gè)氨基酸,由31個(gè)氨基酸的信號(hào)肽序列、77個(gè)氨基酸的前肽序列和275個(gè)氨基酸的成熟肽序列所組成。將目的基因轉(zhuǎn)入大腸桿菌宿主細(xì)胞,成功構(gòu)建產(chǎn)角蛋白酶的重組菌E.coli BL21(DE3)/pET-22b(+)-kerbp,利用鎳離子親和柱對(duì)重組酶進(jìn)行分離純化,獲得電泳純的重組角蛋白酶,該酶分子量與野生酶基本一致。通過(guò)序列分析預(yù)測(cè)該酶的活性中心,發(fā)現(xiàn)Asp-32、His-64和Ser-221這3個(gè)氨基酸殘基位點(diǎn)高度保守,采用重疊延伸PCR技術(shù)和酶活檢測(cè)證實(shí)這3個(gè)保守位點(diǎn)為該角蛋白酶的催化活性中心,為后續(xù)改造研究奠定了基礎(chǔ)。對(duì)重組酶的應(yīng)用研究結(jié)果顯示,該酶可以獨(dú)立完成羊皮脫毛,進(jìn)一步選擇脂肪酶、淀粉酶與角蛋白酶進(jìn)行復(fù)配脫毛,結(jié)果表明脂肪酶和淀粉酶分別與角蛋白酶復(fù)配使用均可以提高脫毛效率,對(duì)脫毛具有協(xié)同作用,可以節(jié)約蛋白酶的用量。(4)為進(jìn)一步改善角蛋白酶的應(yīng)用性能,采用計(jì)算機(jī)輔助手段對(duì)該酶熱穩(wěn)定性進(jìn)行分子改造。利用SWISS-MODEL在線軟件對(duì)角蛋白酶進(jìn)行同源建模,獲得酶分子的三維同源模型。通過(guò)對(duì)B-factor值計(jì)算、分子動(dòng)力學(xué)模擬和同源序列比對(duì),對(duì)3-D結(jié)構(gòu)進(jìn)行分析,預(yù)測(cè)影響酶熱穩(wěn)定性的關(guān)鍵氨基酸位點(diǎn),并對(duì)其進(jìn)行定點(diǎn)突變改造。通過(guò)對(duì)單點(diǎn)突變株的比酶活測(cè)定,獲得了正突變株T218S、S236C和N181D,突變酶在60°C的半衰期相比野生酶分別提升3.05倍、1.18倍和1倍,T50也分別提高5.4°C、4°C和2°C。對(duì)這三個(gè)位點(diǎn)進(jìn)一步組合突變,實(shí)驗(yàn)結(jié)果顯示三個(gè)位點(diǎn)的組合突變對(duì)改善角蛋白酶的熱穩(wěn)定性并無(wú)協(xié)同效應(yīng),組合突變株中僅有N181D-T218S和N181D-S236C穩(wěn)定性具有提升,其T50分別提高5.1°C和2.9°C,在60°C下的半衰期分別為野生酶的4.09倍和1.54倍。對(duì)突變酶熱穩(wěn)定性提高的原因進(jìn)行深入分析,發(fā)現(xiàn)突變酶相比野生酶氫鍵數(shù)量增多,其中S236C突變酶除了氫鍵增加外,還形成1個(gè)鹽橋,增加了側(cè)鏈剛性;通過(guò)對(duì)蛋白質(zhì)表面電荷的分析發(fā)現(xiàn),N181D、S236C、N181D-T218S突變酶與野生酶相比在突變位點(diǎn)局部區(qū)域內(nèi)表面電荷明顯減少,結(jié)構(gòu)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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q55

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