一种耐热碱性脂肪酶基因的克隆与表达

发布时间：2018-03-11 02:03

本文选题：耐热碱性脂肪酶　切入点：里氏木霉　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：嗜热踝节菌脂肪酶(Talaromyces thermophilus lipase, TTL)具有优异的耐热、耐碱性能,在造纸、废纸脱墨、含酶洗涤剂生产、生物柴油制备、手性化合物拆分等领域有重要的应用前景。但嗜热踝节菌自身产酶水平较低,难以实现规模化生产。本文对嗜热踝节菌脂肪酶基因进行了密码子优化,实现了该基因在毕赤酵母和里氏木霉中的重组与表达,论文取得的主要研究结果如下：根据毕赤酵母表达系统的偏好性对嗜热踝节菌脂肪酶基因进行了密码子优化,将优化后的基因连接到pPIC9K载体的AOX1启动子和a信号序列之后,构建了重组表达载体pPIC9K-TTL。电击将此载体转入毕赤酵母基因组中,并通过G418抗性筛选得到了220株转化子,PCR技术检测表明外源脂肪酶基因已稳定整合到重组毕赤酵母的基因组中。对重组转化子进行摇瓶产酶实验,在1.5%甲醇诱导、初始毕赤酵母浓度为3.5×108细胞/mL、发酵液初始pH值6.0的条件下,脂肪酶活力可达到156 IU/mL。SDS-PAGE结果显示：重组子发酵液在39 kDa处有一条明显的蛋白质条带,与嗜热踝节菌脂肪酶分子量一致。实验表明：嗜热踝节菌脂肪酶基因已在毕赤酵母细胞中得到了成功表达和胞外分泌。对嗜热踝节菌脂肪酶基因在里氏木霉中的异源表达进行了研究,为外源基因在里氏木霉中的表达构建了高效的转化体系：在原生质体介导转化时,将预处理8h后的里氏木霉孢子用10mg/mL蜗牛酶酶解1h,调整原生质体浓度为5×108细胞/mL,在PEG6000介导下进行外源基因转化可得到较高转化效率；农杆菌介导外源基因转化时,选用OD600为0.8的农杆菌EHA105和预萌发3h的里氏木霉孢子混合均匀,在添加200 μM乙酰丁香酮的诱导培养基上于24℃、pH 5.3环境下进行共培养,此条件下可得到较高转化效率。相比原生质体转化,农杆菌介导转化的操作周期更短,效率更高,转化子更稳定,转化子的异源蛋白表达水平也较高,是将外源基因转入里氏木霉基因组中的有效手段。针对里氏木霉表达系统的密码子偏好性对ttl基因进行优化,将密码子优化后的基因插入到含有里氏木霉cbhl启动子、信号肽和终止子的表达盒中,以此构建含有潮霉素抗性标记的双元载体pCB-H-PstT。采用原生质体介导或农杆菌介导转化将重组载体转入里氏木霉细胞中,通过两步法筛选方案得到重组里氏木霉转化子。摇瓶发酵72h时重组子的脂肪酶活力可达241 IU/mL。 PCR和SDS-PAGE检测中可发现明显的ttl基因和相应的碱性脂肪酶蛋白条带,这表明外源ttl基因已在里氏木霉中得到成功表达和胞外分泌。为了克服里氏木霉随机转化的不确定性并进一步提高脂肪酶的表达水平,对嗜热踝节菌脂肪酶基因在里氏木霉中的定向整合表达进行了研究。以新鲜里氏木霉菌丝的基因组DNA为模板,通过特异性引物扩增出cbhl基因的左翼(1.4kb)和右翼序列(1.5kb),将此同源臂和脂肪酶基因表达盒PstT进行连接(总长约6 kb),构建含有潮霉素抗性标记的定向整合表达载体pCB-H-LER.将此重组质粒转入里氏木霉基因组中得到里氏木霉定向整合转化子。在乳糖浓度为3%、酵母粉浓度为0.9%、pH值为5.5的条件下,转化子的脂肪酶活力可达375 IU/mL,没有检测到纤维二糖水解酶活性。SDS-PAGE分析表明转化子的发酵液可检测到相应的碱性脂肪酶蛋白条带,而没有纤维二糖水解酶条带。这表明外源脂肪酶基因已经定向整合到里氏木霉cbhl基因位点上,并抑制了里氏木霉自身纤维二糖水解酶的表达,从而提高了异源脂肪酶的表达水平。酶学性质研究结果表明：来源于里氏木霉和毕赤酵母的重组脂肪酶的酶学性质较为相似,重组毕赤酵母脂肪酶在pH 8.0到10.5条件下具有明显的催化活性,在pH 9.5催化活性最高；在40℃至70℃条件下催化活性明显,其最适反应温度为60℃；它对高温和强碱环境表现出了较强的耐受性,在60℃、pH 11的条件下处理一个小时后,仍可维持最高酶活力70%以上。其催化稳定性良好,可抵抗脂肪酸聚集引起的变性。Ca2+对脂肪酶有轻微的促进作用,Na+和K+对TTL的影响不大,Mn2+、Fe2+、Cu2+和Zn2+则有较强的抑制作用。此外,TTL对乙醇、丁醇、甲醇和异丙醇表现出了较强的耐受性,但其活力受到丙酮的明显抑制。表面活性剂对TTL有一定的抑制作用,其中Tween20的抑制效果最为明显。本研究成功地实现了嗜热踝节菌脂肪酶基因在毕赤酵母和里氏木霉中的克隆与表达,为耐热碱性脂肪酶的进一步规模化生产奠定了基础,相关结果具有重要的学术价值和工业应用前景。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q55;Q78

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