本文選題：脂肪酶 + 畢赤酵母異源表達(dá)��； 參考：《天津科技大學(xué)》2016年碩士論文

【摘要】：脂肪酶是一類可催化甘油三酯水解生成甘油及長(zhǎng)鏈脂肪酸的水解酶。來(lái)源于耶氏解酯酵母的胞外脂肪酶LipY2,由于其在較低pH下仍具有較高的穩(wěn)定性且活性不受膽鹽的抑制,因此可作為口服類替代藥物治療胰腺炎引起的胰酶分泌不足等疾病。然而該酶在口服過(guò)程中,常因其易被胰蛋白酶降解而降低療效。故本研究擬通過(guò)蛋白質(zhì)工程手段對(duì)LipY2進(jìn)行分子改造,以提高其對(duì)胰蛋白酶的抗性。本研究將肪酶基因lipY2克隆至分泌型質(zhì)粒pPIC9K上,并在畢赤酵母GS115中實(shí)現(xiàn)了異源表達(dá)。搖瓶發(fā)酵條件下,甲醇誘導(dǎo)培養(yǎng)60 h后重組脂肪酶LipY2的表達(dá)量最高達(dá)到1033 U/mL。同時(shí)根據(jù)脂肪酶LipY2的胰蛋白酶酶解質(zhì)譜分析以及脂肪酶的三維結(jié)構(gòu)分析,利用序列比對(duì)及同源建模相結(jié)合的手段,確定了 LiPY2中易被胰蛋白酶降解的位點(diǎn)。并針對(duì)這些位點(diǎn),對(duì)lipY2基因進(jìn)行了定點(diǎn)改造,構(gòu)建了 7個(gè)突變體,分別為M36、M63、M215以及組合突變體M36-63、M36-215、M63-215和M36-63-215。分別研究了不同位點(diǎn)的改變,對(duì)LiPY2的酶活、抗胰蛋白酶水解穩(wěn)定性及酸穩(wěn)定性等酶學(xué)性質(zhì)的影響,結(jié)果如下:與LipY2相比,M36(突變位點(diǎn)為K36、K39)的最適pH下降了一個(gè)單位,在相同濃度胰蛋白酶處理下的半衰期提高了 8.1%;M63(突變位點(diǎn)為R63)的最適pH也下降了一個(gè)單位,在相同濃度胰蛋白酶處理下的半衰期提高了 16.6%;M36-63(突變位點(diǎn)為K36、K39、R63)最適pH同樣下降了一個(gè)單位,在相同濃度胰蛋白酶處理下的半衰期提高了 22.3%。突變體M36、M63和M36-63的酶活力與LipY2基本相同,但在較低pH 1～3范圍內(nèi)的穩(wěn)定性以及對(duì)胰蛋白酶的抗性都有所提高,尤其以M36-63的效果最為顯著。而M215(突變位點(diǎn)為K215、K218、R220)及 M36-215(突變位點(diǎn)為 K36、K39、K215、K218、R220)、M63-215 突變位點(diǎn)為(R63、K215、K218、R220)和 M36-63-215(突變位點(diǎn)為 K36、K39、R63、K215、K218、R220)的酶活力卻非常低。
[Abstract]:Lipase is a kind of hydrolase which can catalyze the hydrolysis of triglycerides to glycerol and long chain fatty acids. LipY2, an extracellular lipase derived from yeast-lyase yeast, can be used as an oral substitute for the treatment of pancreatitis due to its high stability and activity not inhibited by bile salt. However, during oral administration, the enzyme often reduces its efficacy because of its easy degradation by trypsin. Therefore, in order to improve the resistance to trypsin, LipY2 was modified by protein engineering. In this study, lipY2 was cloned into secretory plasmid pPIC9K and heterologous expression was achieved in Pichia pastoris GS115. Under the condition of shaking flask fermentation, the expression of lipase LipY2 was up to 1033 U / mL after 60 h of methanol induction and culture. On the basis of lipase lipY2 trypsin mass spectrometry and lipase three-dimensional structure analysis, sequence alignment and homology modeling were used to determine the sites of LipY2 easily degraded by trypsin. Aiming at these sites, lipY2 gene was modified by site-directed transformation, and seven mutants, M36-633-215 and M36-63-215M36-63-215 and M36-63-215respectively, were constructed. The effects of different sites on the enzyme activity, hydrolysis stability and acid stability of LiPY2 were studied. The results were as follows: compared with LipY2, the optimum pH of M36 (mutant site K36 K39) was reduced by one unit. At the same concentration of trypsin, the optimal pH value of M63 (mutant site R63) was increased by one unit, and the half life increased by 8. 1% at the same concentration of trypsin. At the same concentration of trypsin, the half-life increased by 16.6C M36-63 (the mutation site was K36K39R63) and the optimum pH decreased by one unit, and the half-life increased by 22.3g under the same concentration of trypsin. The enzyme activities of M36-63 and M36-63 were basically the same as those of LipY2, but the stability and trypsin resistance of M36-63 were increased in the range of lower pH 1 ~ (3), especially the effect of M36-63. The enzyme activities of M215 (K215K218R220) and M36-215 (K36K39K215K218K220M23-215) and M36-63-215 (K36K39K39K215K215K218R220) and M36-63-215M36-63-215 (K36K39K39K215K215K218R220) were very low.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q55

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉文山;徐莉;趙鶴云;楊江科;閆云君;;利用a凝集素在釀酒酵母表面展示解脂耶氏酵母脂肪酶Lip2[J];微生物學(xué)報(bào);2008年11期

2 汪小鋒;王俊;楊江科;閆云君;;微生物發(fā)酵生產(chǎn)脂肪酶的研究進(jìn)展[J];生物技術(shù)通報(bào);2008年04期

3 趙鶴云;黃瑛;楊江科;徐莉;閆云君;;解脂耶氏酵母表達(dá)系統(tǒng)研究進(jìn)展[J];生物加工過(guò)程;2008年03期

4 黃瑛;黎潔;閆云君;;固定化脂肪酶催化棉籽油酯交換制備生物柴油新工藝[J];中國(guó)油脂;2008年02期

5 閻金勇;楊江科;徐莉;閆云君;;白地霉Y162脂肪酶基因克隆及其在畢赤酵母中的高效表達(dá)[J];微生物學(xué)報(bào);2008年02期

6 單保明;夏力;賈小平;項(xiàng)曙光;;生物柴油研究進(jìn)展[J];山東化工;2008年01期

7 張中義;吳新俠;;脂肪酶的研究進(jìn)展[J];食品與藥品;2007年12期

8 舒正玉;楊江科;徐莉;閆云君;;黑曲霉脂肪酶基因的克隆及其在畢赤酵母中的表達(dá)[J];武漢大學(xué)學(xué)報(bào)(理學(xué)版);2007年02期

9 閆云君;舒正玉;楊江科;;細(xì)菌脂肪酶的結(jié)構(gòu)和功能研究進(jìn)展[J];食品與生物技術(shù)學(xué)報(bào);2006年04期

10 袁彩,林琳,施巧琴,吳松剛;擴(kuò)展青霉堿性脂肪酶基因在畢赤酵母中的高效表達(dá)[J];生物工程學(xué)報(bào);2003年02期

，

本文編號(hào)：1987160