嗜熱真菌新的熱穩(wěn)定纖維素酶的克
發(fā)布時(shí)間：2019-06-27 18:57
【摘要】：人類進(jìn)入21世紀(jì)后,整個(gè)社會(huì)面臨著石油和礦石等不可再生能源的日益緊缺及自然環(huán)境等方面越來越嚴(yán)峻的挑戰(zhàn),尋找可以代替石油和礦石的可再生資源已迫在眉睫。木質(zhì)纖維素生物質(zhì)作為地球上一種非常重要的可再生能源,在自然界中不僅分布最為廣泛,而且其多糖的含量最為豐富,是自然界中碳源的唯一可再生形式。利用生物酶法降解纖維素是目前利用纖維素的有效手段,通過纖維素水解酶將大分子纖維素長(zhǎng)鏈分解為寡糖從而高效綠色地利用生物燃料,對(duì)于降低溫室氣體排放和解決能源危機(jī)有著巨大的積極影響。在木質(zhì)纖維素生物質(zhì)的酶法轉(zhuǎn)化過程中,活性高穩(wěn)定性強(qiáng)的纖維素水解酶是減少成本、提高酶解效率的關(guān)鍵,因此,新型高效纖維素水解酶已經(jīng)得到國(guó)內(nèi)外專家的廣泛關(guān)注。嗜熱毀絲菌(Myceliophthora thermophila)作為一種自然界中常見的嗜熱真菌,能夠在較高溫度條件下生長(zhǎng)繁殖,其生長(zhǎng)環(huán)境的溫度可以達(dá)到45℃-50℃的高溫。嗜熱毀絲菌作為第一個(gè)完成全基因組測(cè)序的絲狀真菌,近年來作為發(fā)現(xiàn)新型纖維素酶類的重要材料被研究報(bào)道,其中包括三大類纖維素酶:β-葡聚糖酶(EC 3.2.1.21)、內(nèi)切纖維素酶(EC 3.1.1.4)和外切纖維素酶(EC 3.2.1.74;EC 3.2.1.91)。本研究在以微晶纖維素作為唯一碳源的培養(yǎng)基和以葡萄糖為唯一碳源的培養(yǎng)基上,相同條件下培養(yǎng)嗜熱毀絲菌并提取菌絲RNA,利用RNA-seq測(cè)序技術(shù),經(jīng)過篩選后發(fā)現(xiàn)了兩個(gè)高水平表達(dá)的未知功能基因cel1和cel2,并對(duì)其基因表達(dá)量進(jìn)行RT-PCR和Q-PCR驗(yàn)證。利用RT-PCR技術(shù)克隆得到這兩個(gè)基因的全長(zhǎng)序列,提交GenBank進(jìn)行注冊(cè),登錄號(hào)分別為KM099282和KM099283。兩個(gè)基因cel1和cel2的開放閱讀框分別編碼Cel1和Cel2蛋白,其氨基酸序列長(zhǎng)度分別為211和203 aa,并且均包含一條預(yù)測(cè)的信號(hào)肽序列,推測(cè)為外分泌蛋白。Blast結(jié)果顯示Cel1和Cel2同屬于DUF4360超家族蛋白。成熟蛋白的預(yù)測(cè)分子量分別為20.7和19.8 kDa,通過在線糖基化位點(diǎn)軟件分析氨基酸序列,結(jié)果顯示Cel1和Cel2兩個(gè)蛋白均有糖基化位點(diǎn),推測(cè)為糖蛋白。利用基因重組的方法,將兩個(gè)基因cel1和cel2分別與真核表達(dá)載體pPIC9K相連,構(gòu)建質(zhì)粒pPIC9K/cel1和pPIC9K/cel2,利用電擊轉(zhuǎn)化的方法分別將兩個(gè)重組質(zhì)粒成功轉(zhuǎn)化到畢赤酵母GS115細(xì)胞中。轉(zhuǎn)化完畢后,于MD/MM培養(yǎng)基平板上篩選His+Mut+表型的酵母轉(zhuǎn)化子。篩選出的轉(zhuǎn)化子經(jīng)過PCR驗(yàn)證和抗生素G418篩選得到多拷貝的整合子后,將整合子進(jìn)行甲醇誘導(dǎo)培養(yǎng)。通過對(duì)各整合子目的蛋白的表達(dá)量進(jìn)行檢測(cè),分別篩選得到菌株gs-mt-cel1和gs-mt-cel2,作為工程菌株進(jìn)行保存。在甲醇的誘導(dǎo)下,對(duì)工程菌株gs-mt-cel1和gs-mt-cel2進(jìn)行發(fā)酵,相應(yīng)的重組蛋白得到了高效表達(dá),蛋白的表達(dá)量分別為4.4和3.8mg/ml。同時(shí)對(duì)表達(dá)的重組蛋白利用histraptmff柱進(jìn)行純化,sds-page檢測(cè)純化后的蛋白分子量分別為73.8和77.5kda,糖染色結(jié)果顯示其均為糖蛋白。對(duì)兩個(gè)蛋白cel1和cel2進(jìn)行性質(zhì)研究,酶譜檢測(cè)發(fā)現(xiàn)其均可以水解纖維素底物,并且cel2還可有效的水解木聚糖。利用薄層層析tlc和配有脈沖電流檢測(cè)器的高效陰離子交換色譜hpaec-pad,分別研究cel1和cel2對(duì)纖維寡糖、羧甲基纖維素的水解產(chǎn)物,發(fā)現(xiàn)其水解產(chǎn)物大多為聚合度較低的寡糖,符合纖維素降解酶的水解特性;對(duì)cel2作用木聚寡糖、木聚糖的水解產(chǎn)物測(cè)定,發(fā)現(xiàn)可將木聚多糖降解為聚合度較低的木聚寡糖,具有木聚糖降解酶活性。對(duì)cel1和cel2酶活性質(zhì)進(jìn)行研究,發(fā)現(xiàn)兩個(gè)酶的最適反應(yīng)ph均為5.0,最適反應(yīng)溫度均為60℃,70℃處理1小時(shí)后仍有60%以上的酶活,說明cel1和cel2蛋白熱穩(wěn)定性較高。對(duì)cel1和cel2酶的底物特異性進(jìn)行測(cè)定,發(fā)現(xiàn)cel1和cel2對(duì)cmc-na、磷酸膨脹纖維素、脫脂棉、濾紙、微晶纖維素等纖維素類底物均具有良好的水解活性,cel2對(duì)木聚糖也有較高的水解活性,但是兩個(gè)酶對(duì)幾丁質(zhì)和果膠質(zhì)沒有明顯作用。分別利用去糖基化酶和化學(xué)方法tmsf對(duì)cel1和cel2去糖基化,對(duì)得到去糖基化后的蛋白進(jìn)行測(cè)定,發(fā)現(xiàn)其分子量降低到預(yù)測(cè)值大小。利用去糖基化酶對(duì)cel1和cel2進(jìn)行去糖基化后分別生成decel1和decel2,利用realtimeq-pcr測(cè)定四個(gè)蛋白的溶解曲線中溶解溫度tm值的變化,發(fā)現(xiàn)decel1的tm比cel1降低2.5℃,decel2的tm比cel2降低3.2℃,說明糖基化與酶的熱穩(wěn)定有關(guān)。在cel1和cel2中均發(fā)現(xiàn)兩個(gè)保守的天冬氨酸位點(diǎn)d59和d84,cel2中具有高度保守的谷氨酸位點(diǎn)e66和e96,序列分析后推測(cè)這些保守位點(diǎn)可能對(duì)cel1和cel2的水解活性起重要作用。將序列中的天冬氨酸(d)位點(diǎn)突變?yōu)樘於０?n),將谷氨酸(e)位點(diǎn)突變?yōu)楣劝滨０?q)后,得到六個(gè)突變酶cel1-d59n、cel1-d84n、cel2-d59n、cel2-e66q、cel2-d84n和cel2-e96q。對(duì)突變酶進(jìn)行活性測(cè)定,發(fā)現(xiàn)cel1-d59n、cel1-d84n、cel2-d59n和cel2-d84n失去了對(duì)纖維素底物的水解能力,說明天冬氨酸位點(diǎn)d59和d84對(duì)cel1和cel2酶水解纖維素底物有著極其重要的作用;cel2-e66q和cel2-e96q失去了對(duì)木聚糖的水解能力,說明谷氨酸位點(diǎn)e66和e96是cel2對(duì)木聚糖水解活性的關(guān)鍵位點(diǎn)。本次研究首次發(fā)現(xiàn)并證明了duf4360超家族的兩個(gè)未知功能蛋白cel1具有熱穩(wěn)定纖維素降解酶活性;Cel2具有熱穩(wěn)定雙功能酶活性,可有效降解纖維素和木聚糖。新的木質(zhì)纖維素水解酶的發(fā)現(xiàn),不僅增加了人們對(duì)水解酶家族的認(rèn)識(shí),同時(shí)對(duì)木質(zhì)素轉(zhuǎn)化生物燃料提供了有利的資源,為進(jìn)一步工業(yè)生產(chǎn)奠定基礎(chǔ)。
[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，
本文編號(hào)：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，
本文編號(hào)：2507053
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