嗜热真菌新的热稳定纤维素酶的克
发布时间：2019-06-27 18:57
【摘要】：人类进入21世纪后,整个社会面临着石油和矿石等不可再生能源的日益紧缺及自然环境等方面越来越严峻的挑战,寻找可以代替石油和矿石的可再生资源已迫在眉睫。木质纤维素生物质作为地球上一种非常重要的可再生能源,在自然界中不仅分布最为广泛,而且其多糖的含量最为丰富,是自然界中碳源的唯一可再生形式。利用生物酶法降解纤维素是目前利用纤维素的有效手段,通过纤维素水解酶将大分子纤维素长链分解为寡糖从而高效绿色地利用生物燃料,对于降低温室气体排放和解决能源危机有着巨大的积极影响。在木质纤维素生物质的酶法转化过程中,活性高稳定性强的纤维素水解酶是减少成本、提高酶解效率的关键,因此,新型高效纤维素水解酶已经得到国内外专家的广泛关注。嗜热毁丝菌(Myceliophthora thermophila)作为一种自然界中常见的嗜热真菌,能够在较高温度条件下生长繁殖,其生长环境的温度可以达到45℃-50℃的高温。嗜热毁丝菌作为第一个完成全基因组测序的丝状真菌,近年来作为发现新型纤维素酶类的重要材料被研究报道,其中包括三大类纤维素酶:β-葡聚糖酶(EC 3.2.1.21)、内切纤维素酶(EC 3.1.1.4)和外切纤维素酶(EC 3.2.1.74;EC 3.2.1.91)。本研究在以微晶纤维素作为唯一碳源的培养基和以葡萄糖为唯一碳源的培养基上,相同条件下培养嗜热毁丝菌并提取菌丝RNA,利用RNA-seq测序技术,经过筛选后发现了两个高水平表达的未知功能基因cel1和cel2,并对其基因表达量进行RT-PCR和Q-PCR验证。利用RT-PCR技术克隆得到这两个基因的全长序列,提交GenBank进行注册,登录号分别为KM099282和KM099283。两个基因cel1和cel2的开放阅读框分别编码Cel1和Cel2蛋白,其氨基酸序列长度分别为211和203 aa,并且均包含一条预测的信号肽序列,推测为外分泌蛋白。Blast结果显示Cel1和Cel2同属于DUF4360超家族蛋白。成熟蛋白的预测分子量分别为20.7和19.8 kDa,通过在线糖基化位点软件分析氨基酸序列,结果显示Cel1和Cel2两个蛋白均有糖基化位点,推测为糖蛋白。利用基因重组的方法,将两个基因cel1和cel2分别与真核表达载体pPIC9K相连,构建质粒pPIC9K/cel1和pPIC9K/cel2,利用电击转化的方法分别将两个重组质粒成功转化到毕赤酵母GS115细胞中。转化完毕后,于MD/MM培养基平板上筛选His+Mut+表型的酵母转化子。筛选出的转化子经过PCR验证和抗生素G418筛选得到多拷贝的整合子后,将整合子进行甲醇诱导培养。通过对各整合子目的蛋白的表达量进行检测,分别筛选得到菌株gs-mt-cel1和gs-mt-cel2,作为工程菌株进行保存。在甲醇的诱导下,对工程菌株gs-mt-cel1和gs-mt-cel2进行发酵,相应的重组蛋白得到了高效表达,蛋白的表达量分别为4.4和3.8mg/ml。同时对表达的重组蛋白利用histraptmff柱进行纯化,sds-page检测纯化后的蛋白分子量分别为73.8和77.5kda,糖染色结果显示其均为糖蛋白。对两个蛋白cel1和cel2进行性质研究,酶谱检测发现其均可以水解纤维素底物,并且cel2还可有效的水解木聚糖。利用薄层层析tlc和配有脉冲电流检测器的高效阴离子交换色谱hpaec-pad,分别研究cel1和cel2对纤维寡糖、羧甲基纤维素的水解产物,发现其水解产物大多为聚合度较低的寡糖,符合纤维素降解酶的水解特性;对cel2作用木聚寡糖、木聚糖的水解产物测定,发现可将木聚多糖降解为聚合度较低的木聚寡糖,具有木聚糖降解酶活性。对cel1和cel2酶活性质进行研究,发现两个酶的最适反应ph均为5.0,最适反应温度均为60℃,70℃处理1小时后仍有60%以上的酶活,说明cel1和cel2蛋白热稳定性较高。对cel1和cel2酶的底物特异性进行测定,发现cel1和cel2对cmc-na、磷酸膨胀纤维素、脱脂棉、滤纸、微晶纤维素等纤维素类底物均具有良好的水解活性,cel2对木聚糖也有较高的水解活性,但是两个酶对几丁质和果胶质没有明显作用。分别利用去糖基化酶和化学方法tmsf对cel1和cel2去糖基化,对得到去糖基化后的蛋白进行测定,发现其分子量降低到预测值大小。利用去糖基化酶对cel1和cel2进行去糖基化后分别生成decel1和decel2,利用realtimeq-pcr测定四个蛋白的溶解曲线中溶解温度tm值的变化,发现decel1的tm比cel1降低2.5℃,decel2的tm比cel2降低3.2℃,说明糖基化与酶的热稳定有关。在cel1和cel2中均发现两个保守的天冬氨酸位点d59和d84,cel2中具有高度保守的谷氨酸位点e66和e96,序列分析后推测这些保守位点可能对cel1和cel2的水解活性起重要作用。将序列中的天冬氨酸(d)位点突变为天冬酰胺(n),将谷氨酸(e)位点突变为谷氨酰胺(q)后,得到六个突变酶cel1-d59n、cel1-d84n、cel2-d59n、cel2-e66q、cel2-d84n和cel2-e96q。对突变酶进行活性测定,发现cel1-d59n、cel1-d84n、cel2-d59n和cel2-d84n失去了对纤维素底物的水解能力,说明天冬氨酸位点d59和d84对cel1和cel2酶水解纤维素底物有着极其重要的作用;cel2-e66q和cel2-e96q失去了对木聚糖的水解能力,说明谷氨酸位点e66和e96是cel2对木聚糖水解活性的关键位点。本次研究首次发现并证明了duf4360超家族的两个未知功能蛋白cel1具有热稳定纤维素降解酶活性;Cel2具有热稳定双功能酶活性,可有效降解纤维素和木聚糖。新的木质纤维素水解酶的发现,不仅增加了人们对水解酶家族的认识,同时对木质素转化生物燃料提供了有利的资源,为进一步工业生产奠定基础。
[Abstract]:After the 21st century, the whole society is facing more and more serious challenges such as oil and ore and other non-renewable energy, and it is urgent to find renewable resources that can replace oil and ore. The lignocellulose biomass, as a very important renewable energy in the earth, is not only the most widely distributed in nature, but also the content of its polysaccharide is the most abundant, it is the only reproducible form of the carbon source in nature. The biological enzyme-degrading cellulose is an effective method for utilizing cellulose, and the long-chain decomposition of the large-molecular cellulose into the oligosaccharide by the cellulose hydrolase can effectively and effectively use the biological fuel, and has a great positive effect on reducing the greenhouse gas emission and solving the energy crisis. In the process of the enzymatic conversion of the lignocellulose biomass, the high-stability cellulose hydrolase is the key to reduce the cost and improve the efficiency of the enzymolysis. Therefore, the novel high-efficiency cellulose hydrolase has been widely concerned by the experts at home and abroad. Myceliopatha thermophila, as a common thermophile in nature, can grow and propagate under high temperature conditions, and the temperature of the growth environment can reach the high temperature of 45-50 DEG C. As the first filamentous fungus to complete the whole-genome sequencing, the thermophiles have been reported in recent years as an important material for the discovery of a new type of cellulase, including three major types of cellulases: a-glucanase (EC 3.2. 1.21), Endoglucanase (EC 3.1. 1.4) and exonuclease (EC 3.2. 1.74; EC 3.2. 1.91). In this study, under the same conditions, the culture medium of microcrystalline cellulose as the sole carbon source and the culture medium with glucose as the sole carbon source were used to culture and extract the Mycelium and extract the mycelium RNA. Two high-level expression of the unknown functional genes celer1 and cel2 were detected by RNA-seq sequencing. And carrying out RT-PCR and Q-PCR verification on the gene expression quantity. The full length sequence of these two genes was cloned by RT-PCR and the accession number was KM099282 and KM099283, respectively. The open reading frames of the two genes celer1 and celer2 respectively encode the Cel1 and Cel2 proteins, the amino acid sequence lengths of which are 211 and 203aa, respectively, and each contain a predicted signal peptide sequence, which is presumed to be an outer secretion protein. The Blast results show that Cel1 and Cel2 are in the same family as the DUF4360 superfamily. The predicted molecular weight of mature protein is 20.7 and 19.8 kDa, respectively, and the amino acid sequence is analyzed by the on-line glycosylation site software. The results show that both of the two proteins of Cel1 and Cel2 have glycosylation sites and are presumed to be glycoproteins. Two genes celer1 and celer2 were respectively connected to the eukaryotic expression vector pPIC9K to construct the plasmid pPIC9K/ celer1 and pPIC9K/ celer2, and the two recombinant plasmids were successfully transformed into the Pichia pastoris GS115 cells by using the method of electric shock transformation. After the transformation, a yeast transformant of His + Mut + phenotype was selected on the MD/ MM medium plate. After the screened transformants were subjected to PCR verification and the antibiotic G418 was screened for multiple copies of the integrin, the whole of the transformants were subjected to methanol induction culture. The strain gs-mt-cel1 and gs-mt-cel2 were respectively screened and stored as engineering strain by the detection of the expression levels of all the integrin target proteins. Under the induction of methanol, the engineering strain gs-mt-cel1 and gs-mt-cel2 were fermented, and the corresponding recombinant protein was expressed in high efficiency, and the expression of the protein was 4.4 and 3.8 mg/ ml, respectively. The average molecular weight of the purified protein was 73.8 and 77.5 kda, respectively. The properties of two proteins, cel1 and cel2, were studied, and the enzyme spectrum was found to be able to hydrolyze the cellulose substrate, and the cel2 could also be used to hydrolyze the xylan. By using the thin layer chromatography (tlc) and the high-efficiency anion exchange chromatography (hpaec-pad) equipped with the pulse current detector, the hydrolysis products of cel1 and cel2 on the fiber-oligosaccharides and methyl cellulose were respectively studied. It was found that the degradation of the polysaccharose to the lower degree of polymerization of the xylo-oligosaccharides with the activity of the xylan-degrading enzyme. The activity of cel1 and cel2 was studied. The optimum reaction ph of two enzymes was 5.0, the optimum reaction temperature was 60 鈩，
本文编号：2507053
[Abstract]:After the 21st century, the whole society is facing more and more serious challenges such as oil and ore and other non-renewable energy, and it is urgent to find renewable resources that can replace oil and ore. The lignocellulose biomass, as a very important renewable energy in the earth, is not only the most widely distributed in nature, but also the content of its polysaccharide is the most abundant, it is the only reproducible form of the carbon source in nature. The biological enzyme-degrading cellulose is an effective method for utilizing cellulose, and the long-chain decomposition of the large-molecular cellulose into the oligosaccharide by the cellulose hydrolase can effectively and effectively use the biological fuel, and has a great positive effect on reducing the greenhouse gas emission and solving the energy crisis. In the process of the enzymatic conversion of the lignocellulose biomass, the high-stability cellulose hydrolase is the key to reduce the cost and improve the efficiency of the enzymolysis. Therefore, the novel high-efficiency cellulose hydrolase has been widely concerned by the experts at home and abroad. Myceliopatha thermophila, as a common thermophile in nature, can grow and propagate under high temperature conditions, and the temperature of the growth environment can reach the high temperature of 45-50 DEG C. As the first filamentous fungus to complete the whole-genome sequencing, the thermophiles have been reported in recent years as an important material for the discovery of a new type of cellulase, including three major types of cellulases: a-glucanase (EC 3.2. 1.21), Endoglucanase (EC 3.1. 1.4) and exonuclease (EC 3.2. 1.74; EC 3.2. 1.91). In this study, under the same conditions, the culture medium of microcrystalline cellulose as the sole carbon source and the culture medium with glucose as the sole carbon source were used to culture and extract the Mycelium and extract the mycelium RNA. Two high-level expression of the unknown functional genes celer1 and cel2 were detected by RNA-seq sequencing. And carrying out RT-PCR and Q-PCR verification on the gene expression quantity. The full length sequence of these two genes was cloned by RT-PCR and the accession number was KM099282 and KM099283, respectively. The open reading frames of the two genes celer1 and celer2 respectively encode the Cel1 and Cel2 proteins, the amino acid sequence lengths of which are 211 and 203aa, respectively, and each contain a predicted signal peptide sequence, which is presumed to be an outer secretion protein. The Blast results show that Cel1 and Cel2 are in the same family as the DUF4360 superfamily. The predicted molecular weight of mature protein is 20.7 and 19.8 kDa, respectively, and the amino acid sequence is analyzed by the on-line glycosylation site software. The results show that both of the two proteins of Cel1 and Cel2 have glycosylation sites and are presumed to be glycoproteins. Two genes celer1 and celer2 were respectively connected to the eukaryotic expression vector pPIC9K to construct the plasmid pPIC9K/ celer1 and pPIC9K/ celer2, and the two recombinant plasmids were successfully transformed into the Pichia pastoris GS115 cells by using the method of electric shock transformation. After the transformation, a yeast transformant of His + Mut + phenotype was selected on the MD/ MM medium plate. After the screened transformants were subjected to PCR verification and the antibiotic G418 was screened for multiple copies of the integrin, the whole of the transformants were subjected to methanol induction culture. The strain gs-mt-cel1 and gs-mt-cel2 were respectively screened and stored as engineering strain by the detection of the expression levels of all the integrin target proteins. Under the induction of methanol, the engineering strain gs-mt-cel1 and gs-mt-cel2 were fermented, and the corresponding recombinant protein was expressed in high efficiency, and the expression of the protein was 4.4 and 3.8 mg/ ml, respectively. The average molecular weight of the purified protein was 73.8 and 77.5 kda, respectively. The properties of two proteins, cel1 and cel2, were studied, and the enzyme spectrum was found to be able to hydrolyze the cellulose substrate, and the cel2 could also be used to hydrolyze the xylan. By using the thin layer chromatography (tlc) and the high-efficiency anion exchange chromatography (hpaec-pad) equipped with the pulse current detector, the hydrolysis products of cel1 and cel2 on the fiber-oligosaccharides and methyl cellulose were respectively studied. It was found that the degradation of the polysaccharose to the lower degree of polymerization of the xylo-oligosaccharides with the activity of the xylan-degrading enzyme. The activity of cel1 and cel2 was studied. The optimum reaction ph of two enzymes was 5.0, the optimum reaction temperature was 60 鈩，
本文编号：2507053
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