地衣芽胞杆菌AprE蛋白酶基因的克隆表达及解析

发布时间：2018-07-26 19:10

【摘要】：地衣芽胞杆菌碱性蛋白酶是工业上重要的蛋白酶类,进一步提高其在碱性条件下的活力可改善其在洗涤剂和化妆品工业方面的应用价值。地衣芽胞杆菌是蛋白酶最重要的生产菌株之一,具有非常明显的工业应用优势,但是其蛋白酶系仍不清晰,进一步解析其蛋白酶系,对制造更符合工业需求的工程菌有促进作用。本研究通过对菌库内的菌种筛选获得一种在碱性条件下酶活水平显著提高的地衣芽胞杆菌B186,在对该菌和工业菌株2709的WprA、Epr及AprE三个蛋白酶进行表达的前提下,发现AprE是地衣芽胞杆菌的主要胞外蛋白酶之一,重点对该酶进行分析。从成熟的工业菌株2709中获得蛋白酶基因aprE2709,从地衣芽胞杆菌B186扩增出蛋白酶AprE209的基因。在枯草芽胞杆菌WB600中对二者的编码基因进行了成功克隆与表达,进一步导入地衣芽胞杆菌中,获得了重组菌Z113 (pHY-aprE209),Z113 (pHY-aprE2709)。重组酶AprE209的最适作用pH为11.0,最适作用温度为50 ℃,在30～37 ℃下和pH 12.0时仍具有很高的活力。重组酶AprE2709的最适作用pH和温度为10.0和50 ℃,在40～50 ℃下保持相对较好的稳定性。进一步通过生物信息学的手段对AprE209和AprE2709进行对比分析,对AprE209耐碱机制进行了初步解析。AprE209中活性位点His139周围比AprE2709多了一个氢键,AprE209中Lys270比AprE2709的Glu270周围多了两个氢键,这可能是其在碱性条件下仍具有良好的活性和稳定性的原因。
[Abstract]:Bacillus licheniformis alkaline protease is an important protease in industry. Further improving its activity under alkaline conditions can improve its application value in detergents and cosmetics industry. Bacillus licheniformis is one of the most important producing strains of protease. It can promote the manufacture of engineering bacteria that meet the needs of industry. In this study, a Bacillus licheniformis B1866 was obtained by screening the bacteria in the library. The three proteases, WprAgne Epr and AprE, were expressed by Bacillus licheniformis and the industrial strain 2709. AprE was found to be one of the main extracellular proteases of Bacillus licheniformis. The protease gene aprE2709 was obtained from mature industrial strain 2709 and the protease AprE209 gene was amplified from Bacillus licheniformis B186. The encoding genes were cloned and expressed successfully in Bacillus subtilis WB600. The recombinant strain Z113 (pHY-aprE209) Z113 (pHY-aprE2709) was further introduced into Bacillus licheniformis. The optimum pH and temperature of the recombinant enzyme AprE209 were 11.0 and 50 鈩，

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