【摘要】：地衣芽胞桿菌堿性蛋白酶是工業(yè)上重要的蛋白酶類,進(jìn)一步提高其在堿性條件下的活力可改善其在洗滌劑和化妝品工業(yè)方面的應(yīng)用價(jià)值。地衣芽胞桿菌是蛋白酶最重要的生產(chǎn)菌株之一,具有非常明顯的工業(yè)應(yīng)用優(yōu)勢,但是其蛋白酶系仍不清晰,進(jìn)一步解析其蛋白酶系,對制造更符合工業(yè)需求的工程菌有促進(jìn)作用。本研究通過對菌庫內(nèi)的菌種篩選獲得一種在堿性條件下酶活水平顯著提高的地衣芽胞桿菌B186,在對該菌和工業(yè)菌株2709的WprA、Epr及AprE三個(gè)蛋白酶進(jìn)行表達(dá)的前提下,發(fā)現(xiàn)AprE是地衣芽胞桿菌的主要胞外蛋白酶之一,重點(diǎn)對該酶進(jìn)行分析。從成熟的工業(yè)菌株2709中獲得蛋白酶基因aprE2709,從地衣芽胞桿菌B186擴(kuò)增出蛋白酶AprE209的基因。在枯草芽胞桿菌WB600中對二者的編碼基因進(jìn)行了成功克隆與表達(dá),進(jìn)一步導(dǎo)入地衣芽胞桿菌中,獲得了重組菌Z113 (pHY-aprE209),Z113 (pHY-aprE2709)。重組酶AprE209的最適作用pH為11.0,最適作用溫度為50 ℃,在30～37 ℃下和pH 12.0時(shí)仍具有很高的活力。重組酶AprE2709的最適作用pH和溫度為10.0和50 ℃,在40～50 ℃下保持相對較好的穩(wěn)定性。進(jìn)一步通過生物信息學(xué)的手段對AprE209和AprE2709進(jìn)行對比分析,對AprE209耐堿機(jī)制進(jìn)行了初步解析。AprE209中活性位點(diǎn)His139周圍比AprE2709多了一個(gè)氫鍵,AprE209中Lys270比AprE2709的Glu270周圍多了兩個(gè)氫鍵,這可能是其在堿性條件下仍具有良好的活性和穩(wěn)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，

本文編號(hào)：2147010