【作者】 夏颖；

【导师】 夏黎明；

【作者基本信息】 浙江大学 ， 化学工程与技术， 2016， 硕士

【摘要】 作为纤维素复合酶系的重要组成部分,内切-β-葡聚糖酶在可再生纤维素资源的生物转化与利用、棉织物生物整理、制浆造纸以及轻工食品等领域中应用广泛。目前常用的纤维素酶生产菌种里氏木霉(Trichoderma reesei)所产的内切-p-葡聚糖酶仅适用于酸性反应体系,其应用范围受到了很大的限制。采用基因工程技术,构建新型内切-p-葡聚糖酶高产菌株,对于纤维素酶制剂的工业化应用具有重要的推动作用。刺糙青霉(Penicillium echinulatum)可产生一种耐热、耐碱的内切-p-葡聚糖酶,具有重要的工业应用价值,但野生型刺糙青霉的产酶水平很低,难以实现规模化生产。本文以刺糙青霉的内切-p-葡聚糖酶基因(Egll)为研究对象,首先对其进行密码子优化,获得符合里氏木霉密码子偏好的新型内切-p-葡聚糖酶基因Eglln,该基因全长1175 bp,编码含387个氨基酸的成熟肽。之后将该基因置于里氏木霉强启动子Pcbhl(纤维二糖水解酶Ⅰ)及其信号肽和终止子Tcbhl(维二糖水解酶Ⅰ)之间,以pCAMBIA1300为载体骨架构建重组质粒pCB-PET,该质粒同时含有目的基因Eglln和潮霉素筛选标记。采用根瘤农杆菌介导转化技术将重组质粒pCB-PET导入里氏木霉的分生孢子中,在潮霉素抗性平板上筛选获得226个阳性转化子,进一步通过复筛获得5个优良的重组转化子。将5个转化子重复传代培养10个批次后,分别提取染色体DNA进行PCR验证,均可检测到目的基因Eglln条带,说明该基因已稳定地整合到里氏木霉基因组中。采用SDS-PAGE蛋白电泳对转化子培养液进行检测,获得了与目的基因表达产物相符的蛋白条带(约41 kDa),表明Eglln已在重组里氏木霉中成功实现了胞外表达。重组转化子在30℃,摇瓶转速200 r/min条件下培养48 h,取发酵上清液在pH8.0,60℃条件下检测,内切-p-葡聚糖酶活力可高达382.6 U/ml,为宿主菌株的22.5倍。采用分批发酵工艺,在2 m3发酵罐中对重组转化子T1进行产酶试验,其碱性内切-p-葡聚糖酶活力在96 h可达到1070 U/ml,为摇瓶条件下的2.8倍。表明发酵罐条件下溶氧、搅拌的改善有利于目的基因在重组转化子中的高效表达。酶学性质研究结果表明：该酶耐热性能较好,在70℃以下性能稳定,其最适催化温度为60℃；该酶在pH 5.0-10.5范围内稳定性较好、具有明显的催化活性,其最适pH为8.0,属于碱性纤维素酶。Ca2+对重组内切-p-葡聚糖酶有明显的激活作用,而Hg2+、Mg2+、Al3+、zn3+、Cu2+.和Fe3+在一定程度减弱酶的催化作用,其中Hg2+可以完全抑制重组内切-p-葡聚糖酶活性。本文成功地实现了刺糙青霉内切-p-葡聚糖酶基因在里氏木霉中的重组与胞外表达,有关研究结果在里氏木霉纤维素酶的定向进化以及内切-p-葡聚糖酶的工业化应用方面具有重要的促进作用。更多还原

【Abstract】 As the major cellulase component, exo-β-glucanase has great value in textile industry and pulp bleaching industry. However, the endo-β-glucanase produced by Trichoderma reesei only applies to acidic system, which limits its further applications in above-mentioned areas. In this study, recombinant DNA technology is adopted to constructing the novel endo-β-glucanase producing strain. This study plays an important role in promoting industrial applications of cellulaseIn this study, a novel endo-β-glucanase (EGL1) secreted by mutant Penicillium echinulatum was optimized according to the codon bias of Trichoderma reesei, obtaining the new gene Eglln. The length of Eglln is 1175 bp, encoding 387 amino acid residues. The codon-optimized gene(Eglln) was inserted between Trichoderma reesei strong promoter Pcbh1 (including secreting signal peptide sequence) and terminator, further ligated to pCAMBIA1300 vector to construct a recombination plasmid pCB-PET with hygromycin B resistance marker.The plasmid pCB-PET was transformed into the conidium of T. reesei ZU-02 via an optimized Agrobacterium tumefaciens mediated transformation. 226 positive transformants were screened by hygromycin B, and 5 fast growth transformants were obtained by further rescreening on the culture plates using carboxymethylcellulose as sole carbon source. The chromosomal DNA from 5 recombinant transformants were extracted separately after subculture for 10 generations. It was further confirmed that the Eglln gene had been integrated into the chromosomal DNA of T. reesei by PCR detection. An obvious protein band (approximately 41 kDa) corresponding to the expressing product of target gene Eglln was detected in fermentation broth of recombinant T. reesei by SDS-PAGE which proved that the Eglln gene had extracellularly expressed successfully.Enzyme production by the 5 recombinant transformants was performed in shaking flasks. After 48 h fermentation at 30℃ under agitation (200 r/min), endo-β-glucanase activity in the culture broth was measured at pH 8.0,60 ℃. The activity produced by transformants reached 382.6 U/ml, which was 22.5-fold as high as that of the host strain. The batch fermentation experiment of the transformant T1 was carried out in a 2 m3 fermenter. The recombinant endo-β-glucanase activity reached 1070 U/ml after 96 h fermentation, which was 2.8-fold as high as that under the shaking flask condition.The study on enzymatic properties showed that the recombinant EGL1N exhibited high thermostability. It was stable below 70℃ and its optimal temperature for reaction was 60 ℃. The enzyme also presented good hydrolytic activity and stability over a broad pH range (5.0-10.5) and an optimum pH value of 8.0. The CMCase activity was strongly stimulated when Ca2+ was employed. The CMCase activity was completely inhibited by Hg2+ and an effective inhibition was also observed with Mg2+ 、Al3+、Zn3+, Cu2+ and Fe3+. These characteristics of the cellulase will be of great importance to modern industry.This study showed that the optimized Penicillium echinulatum endo-β-glucanase gene had been successfully transformed and expressed in T. reesei. The research result acts as a significant role in the directed evolution of cellulase producing strains and related industrial applications as well.更多还原