本文選題：耐熱堿性脂肪酶　切入點(diǎn)：里氏木霉　出處：《浙江大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：嗜熱踝節(jié)菌脂肪酶(Talaromyces thermophilus lipase, TTL)具有優(yōu)異的耐熱、耐堿性能,在造紙、廢紙脫墨、含酶洗滌劑生產(chǎn)、生物柴油制備、手性化合物拆分等領(lǐng)域有重要的應(yīng)用前景。但嗜熱踝節(jié)菌自身產(chǎn)酶水平較低,難以實(shí)現(xiàn)規(guī)�；a(chǎn)。本文對(duì)嗜熱踝節(jié)菌脂肪酶基因進(jìn)行了密碼子優(yōu)化,實(shí)現(xiàn)了該基因在畢赤酵母和里氏木霉中的重組與表達(dá),論文取得的主要研究結(jié)果如下：根據(jù)畢赤酵母表達(dá)系統(tǒng)的偏好性對(duì)嗜熱踝節(jié)菌脂肪酶基因進(jìn)行了密碼子優(yōu)化,將優(yōu)化后的基因連接到pPIC9K載體的AOX1啟動(dòng)子和a信號(hào)序列之后,構(gòu)建了重組表達(dá)載體pPIC9K-TTL。電擊將此載體轉(zhuǎn)入畢赤酵母基因組中,并通過(guò)G418抗性篩選得到了220株轉(zhuǎn)化子,PCR技術(shù)檢測(cè)表明外源脂肪酶基因已穩(wěn)定整合到重組畢赤酵母的基因組中。對(duì)重組轉(zhuǎn)化子進(jìn)行搖瓶產(chǎn)酶實(shí)驗(yàn),在1.5%甲醇誘導(dǎo)、初始畢赤酵母濃度為3.5×108細(xì)胞/mL、發(fā)酵液初始pH值6.0的條件下,脂肪酶活力可達(dá)到156 IU/mL。SDS-PAGE結(jié)果顯示：重組子發(fā)酵液在39 kDa處有一條明顯的蛋白質(zhì)條帶,與嗜熱踝節(jié)菌脂肪酶分子量一致。實(shí)驗(yàn)表明：嗜熱踝節(jié)菌脂肪酶基因已在畢赤酵母細(xì)胞中得到了成功表達(dá)和胞外分泌。對(duì)嗜熱踝節(jié)菌脂肪酶基因在里氏木霉中的異源表達(dá)進(jìn)行了研究,為外源基因在里氏木霉中的表達(dá)構(gòu)建了高效的轉(zhuǎn)化體系：在原生質(zhì)體介導(dǎo)轉(zhuǎn)化時(shí),將預(yù)處理8h后的里氏木霉孢子用10mg/mL蝸牛酶酶解1h,調(diào)整原生質(zhì)體濃度為5×108細(xì)胞/mL,在PEG6000介導(dǎo)下進(jìn)行外源基因轉(zhuǎn)化可得到較高轉(zhuǎn)化效率；農(nóng)桿菌介導(dǎo)外源基因轉(zhuǎn)化時(shí),選用OD600為0.8的農(nóng)桿菌EHA105和預(yù)萌發(fā)3h的里氏木霉孢子混合均勻,在添加200 μM乙酰丁香酮的誘導(dǎo)培養(yǎng)基上于24℃、pH 5.3環(huán)境下進(jìn)行共培養(yǎng),此條件下可得到較高轉(zhuǎn)化效率。相比原生質(zhì)體轉(zhuǎn)化,農(nóng)桿菌介導(dǎo)轉(zhuǎn)化的操作周期更短,效率更高,轉(zhuǎn)化子更穩(wěn)定,轉(zhuǎn)化子的異源蛋白表達(dá)水平也較高,是將外源基因轉(zhuǎn)入里氏木霉基因組中的有效手段。針對(duì)里氏木霉表達(dá)系統(tǒng)的密碼子偏好性對(duì)ttl基因進(jìn)行優(yōu)化,將密碼子優(yōu)化后的基因插入到含有里氏木霉cbhl啟動(dòng)子、信號(hào)肽和終止子的表達(dá)盒中,以此構(gòu)建含有潮霉素抗性標(biāo)記的雙元載體pCB-H-PstT。采用原生質(zhì)體介導(dǎo)或農(nóng)桿菌介導(dǎo)轉(zhuǎn)化將重組載體轉(zhuǎn)入里氏木霉細(xì)胞中,通過(guò)兩步法篩選方案得到重組里氏木霉轉(zhuǎn)化子。搖瓶發(fā)酵72h時(shí)重組子的脂肪酶活力可達(dá)241 IU/mL。 PCR和SDS-PAGE檢測(cè)中可發(fā)現(xiàn)明顯的ttl基因和相應(yīng)的堿性脂肪酶蛋白條帶,這表明外源ttl基因已在里氏木霉中得到成功表達(dá)和胞外分泌。為了克服里氏木霉隨機(jī)轉(zhuǎn)化的不確定性并進(jìn)一步提高脂肪酶的表達(dá)水平,對(duì)嗜熱踝節(jié)菌脂肪酶基因在里氏木霉中的定向整合表達(dá)進(jìn)行了研究。以新鮮里氏木霉菌絲的基因組DNA為模板,通過(guò)特異性引物擴(kuò)增出cbhl基因的左翼(1.4kb)和右翼序列(1.5kb),將此同源臂和脂肪酶基因表達(dá)盒PstT進(jìn)行連接(總長(zhǎng)約6 kb),構(gòu)建含有潮霉素抗性標(biāo)記的定向整合表達(dá)載體pCB-H-LER.將此重組質(zhì)粒轉(zhuǎn)入里氏木霉基因組中得到里氏木霉定向整合轉(zhuǎn)化子。在乳糖濃度為3%、酵母粉濃度為0.9%、pH值為5.5的條件下,轉(zhuǎn)化子的脂肪酶活力可達(dá)375 IU/mL,沒(méi)有檢測(cè)到纖維二糖水解酶活性。SDS-PAGE分析表明轉(zhuǎn)化子的發(fā)酵液可檢測(cè)到相應(yīng)的堿性脂肪酶蛋白條帶,而沒(méi)有纖維二糖水解酶條帶。這表明外源脂肪酶基因已經(jīng)定向整合到里氏木霉cbhl基因位點(diǎn)上,并抑制了里氏木霉自身纖維二糖水解酶的表達(dá),從而提高了異源脂肪酶的表達(dá)水平。酶學(xué)性質(zhì)研究結(jié)果表明：來(lái)源于里氏木霉和畢赤酵母的重組脂肪酶的酶學(xué)性質(zhì)較為相似,重組畢赤酵母脂肪酶在pH 8.0到10.5條件下具有明顯的催化活性,在pH 9.5催化活性最高；在40℃至70℃條件下催化活性明顯,其最適反應(yīng)溫度為60℃；它對(duì)高溫和強(qiáng)堿環(huán)境表現(xiàn)出了較強(qiáng)的耐受性,在60℃、pH 11的條件下處理一個(gè)小時(shí)后,仍可維持最高酶活力70%以上。其催化穩(wěn)定性良好,可抵抗脂肪酸聚集引起的變性。Ca2+對(duì)脂肪酶有輕微的促進(jìn)作用,Na+和K+對(duì)TTL的影響不大,Mn2+、Fe2+、Cu2+和Zn2+則有較強(qiáng)的抑制作用。此外,TTL對(duì)乙醇、丁醇、甲醇和異丙醇表現(xiàn)出了較強(qiáng)的耐受性,但其活力受到丙酮的明顯抑制。表面活性劑對(duì)TTL有一定的抑制作用,其中Tween20的抑制效果最為明顯。本研究成功地實(shí)現(xiàn)了嗜熱踝節(jié)菌脂肪酶基因在畢赤酵母和里氏木霉中的克隆與表達(dá),為耐熱堿性脂肪酶的進(jìn)一步規(guī)�；a(chǎn)奠定了基礎(chǔ),相關(guān)結(jié)果具有重要的學(xué)術(shù)價(jià)值和工業(yè)應(yīng)用前景。
[Abstract]:Thermophilic bacteria lipase (Talaromyces thermophilus ankle section lipase, TTL) has excellent heat resistance, alkali resistance, in the paper, deinking, enzyme containing detergent production, preparation of biodiesel, the resolution of chiral compounds has important application prospect. But the thermophilic bacteria from the ankles enzyme level is low, it is difficult to achieve the scale of production. The codon optimization of thermophilic bacteria lipase gene section this paper realizes heat ankle, recombination and expression of the gene in Pichia pastoris and Trichoderma reesei. The main results are as follows: according to the Pichia pastoris expression system of preference of thermophilic lipase gene from the heat of the ankle joint codon optimization, after the optimized gene linked to the pPIC9K vector of the AOX1 promoter and a signal sequence, the recombinant expression vector pPIC9K-TTL. was constructed this shock vector into genome of Pichia pastoris, and the anti G418 Of the 220 transformants were screened, PCR assay indicated that exogenous lipase gene has been integrated into the genome of the recombinant Pichia pastoris. The recombinant transformants were subjected to enzyme production in shake flask experiments, induced by 1.5% methanol, the initial yeast concentration of 3.5 * 108 cells /mL, initial pH value of the fermented liquor 6. Next, the lipase activity reached 156 IU/mL.SDS-PAGE showed that the recombinant fermentation liquid has an obvious protein band at 39 kDa, and the thermophilic bacteria lipase molecular weight consistent with ankle joint. The experimental results show that the thermophilic lipase gene from hot section in Bi Chijiao ankle mother cells expressed successfully and exocytosis. The study on heterologous lipase gene from thermophilic section heat ankle in Trichoderma reesei, construct efficient transformation system for heterologous gene expression in Trichoderma reesei in protoplast mediated transformation, the pretreatment of 8h Trichoderma spores solution 1H 10mg/mL snail enzyme, adjust the protoplast concentration of 5 /mL * 108 cells, exogenous gene transformation can get higher conversion efficiency in PEG6000 mediated; Agrobacterium mediated gene transformation, OD600 was selected as 0.8 Agrobacterium EHA105 and pre 3H Richter adorable Trichoderma spores mixed evenly, with the addition of 200 M acetosyringone induction medium at 24 DEG C, were cultured under pH 5.3, this condition can be obtained with high conversion efficiency. Compared with protoplast transformation, Agrobacterium mediated transformation of the operating cycle shorter, more efficient, more stable transformants. Transformants expressing heterologous protein levels were also higher, is the effective means of exogenous gene into the genome of Trichoderma reesei. The expression of TTL gene codon optimized system, the codon optimized genes are inserted into the water A Trichoderma reesei cbhl promoter and signal peptide expression cassette terminator, binary vector pCB-H-PstT. constructs containing hygromycin resistance marker by protoplast mediated and Agrobacterium mediated transformation of the recombinant vector into trichodermareesei cells, through the two step screening scheme to obtain the recombinant Trichoderma reesei transformant the activity of lipase fermentation. Up to 72h of recombinant 241 IU/mL. PCR and SDS-PAGE detection can be found in distinct TTL genes and corresponding alkaline lipase protein bands, indicating that exogenous TTL gene has been successfully expressed and secreted in Trichoderma reesei. In order to overcome the uncertainty and further random transformation to improve the level of expression of lipase, expression of lipase gene from thermophilic directional integration section heat ankle in Trichoderma reesei. The genome of Trichoderma reesei DNA fresh mycelium as template, The specific primers of cbhl gene (1.4kb) and the left wing sequences (1.5kb), the homologous arm and connected lipase gene expression cassette PstT (total length of about 6 KB), directional construction containing hygromycin resistance marker integration expression vector pCB-H-LER. the recombinant plasmid was transferred into trichodermareesei directional integration the transformant of Trichoderma reesei genome. The lactose concentration was 3%, the yeast concentration was 0.9%, pH value is 5.5, up to 375 IU/mL lipase activity of transformants, did not detect the two cellobiohydrolases.SDS-PAGE activity analysis showed that the fermentation liquid of transformants can be detected by the corresponding alkaline lipase protein band. Without the two cellobiohydrolases bands. This indicated that the exogenous gene has been integrated into the directional lipase of Trichoderma reesei cbhl gene, and inhibit the expression of Trichoderma reesei its two cellobiohydrolases, so as to improve the The heterologous lipase expression level. Characterization results show that the enzymatic properties of recombinant lipase from Trichoderma and yeast Pichia pastoris is similar to that of recombinant Pichia pastoris lipase have obvious catalytic activity in pH 8 to 10.5 pH in the 9.5 conditions, the highest catalytic activity; at 40 to 70 DEG C under the condition of the catalytic activity obviously, the optimal reaction temperature is 60 DEG C; it is of high temperature and strong alkali environment showed strong tolerance, at 60 degrees, one hour pH treatment under the condition of 11, still maintained the highest enzyme activity more than 70%. Its good catalytic stability, can resist fatty acid aggregation caused by degeneration of.Ca2+ have a slight effect on the lipase, Na+ and K+ have little effect on TTL, Mn2+, Fe2+, Cu2+ and Zn2+ have inhibitory effect on TTL. In addition, ethanol, butanol, methanol and isopropanol showed strong tolerance, but its live Inhibit power by acetone. Surfactants on TTL inhibited, the inhibition effect of Tween20 was most obvious. This study succeeded in cloning and expression of lipase gene from thermophilic section heat ankle in Pichia pastoris and Trichoderma reesei, laid the foundation for further large-scale production of thermostable alkaline lipase, related results with academic value and industrial application prospect.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q55;Q78

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本文編號(hào)：1596068