菊粉内切酶和蔗糖酶基因的高效重组表达及其在水解菊粉中的应用
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摘要
菊粉酶(Inulinase)是一种呋喃果糖基水解酶，它靶向作用于菊粉中的β-2,1糖苷键并将其水解为果糖和葡萄糖。菊粉酶可以分为菊粉外切酶和菊粉内切酶。菊粉外切酶可以从菊粉分子的非还原末端催化水解下β-D-呋喃果糖残基，其底物可为菊粉、蔗糖和果聚糖；菊粉内切酶可以水解菊粉内部的β-2,1糖苷键使其变成菊糖三糖、菊糖四糖和菊糖五糖为主的菊粉寡糖。蔗糖酶(Invertase)是一种β-呋喃果糖苷酶，它可以催化蔗糖水解为果糖和葡萄糖，广泛存在于细菌、酵母、丝状真菌、高等植物和许多动物细胞中。其中，酒精酵母的蔗糖酶已被深入研究，并广泛应用于食品和发酵工业。酒精酵母的蔗糖酶主要是由SUC2基因编码的。
本研究首先将节杆菌属Arthrobacter sp. S37来源的菊粉内切酶基因EnIA在酵母菌解脂亚罗维亚中表达，并研究了重组菊粉内切酶的性质及水解菊粉的产物。将菊粉内切酶基因EnIA连接到表达质粒pINA1317上，在Yarrowia lipolytica Po1h中进行分泌表达。重组转化子1317-EnIA生产的重组菊粉内切酶的酶活力和比活力大小分别为16.7U/mL和93.4U/mg，表观分子量大小为78.9kDa。重组酶的最适pH值为4.0，在pH2.0-8.0范围内，重组酶可以保持较高的活性。在40℃以下，重组酶有很好的稳定性，其最适作用温度为50℃。Li+离子对重组酶活性有激活作用。重组菊粉内切酶水解菊粉的主要产物为菊粉二糖。当把重组菊粉内切酶和菊粉外切酶混合水解菊粉时，混合物的菊粉酶活力大于两者菊粉酶相加之和，表现出协同作用。
本实验室保藏的一株高产酒精酵母Saccharomyces sp. W0菌株可以缓慢地糖化菊粉产生单糖，利用菊粉发酵生产少量的酒精。将菊粉内切酶基因EnIA在酒精酵母W0菌株中表达，提高W0菌株的菊粉酶活力和酒精产量。把菊粉内切酶基因EnIA连接到酒精酵母表达载体pMIDSC31（δ序列插入型）和pMIRSC31（rDNA序列插入型）上，整合到W0菌株的基因组DNA中。δ序列插入型重组转化子D5的菊粉酶活力高于rDNA序列插入型重组转化子R1。重组转化子D5的菊粉酶活力达8.6U/mL。在5-L发酵罐体系中，转化子D5以菊粉含量为30%（w/v）的培养基进行酒精发酵，酒精产量为13.6%（v/v,20℃）。转化子D5同步糖化生料菊芋粉，在1-L发酵体系中，酒精产量达10.1%（v/v,20℃）。
W0菌株具有很低的菊粉酶活性，根据氨基酸序列的多重比对分析结果推测可能与蔗糖酶基因SUC2相关。蔗糖酶基因SUC2与酒精酵母具有菊粉酶活力可以发酵菊粉生产酒精之间的关系还鲜有报道。本研究将SUC2基因完全敲除并将原始SUC2基因在敲除菌株中超表达，根据敲除和超表达之后的实验结果解释蔗糖酶基因SUC2与W0菌株具有菊粉酶活性是否相关。蔗糖酶基因SUC2被敲除后，完全敲除菌株W4失去蔗糖酶和菊粉酶活力，不能在以蔗糖或菊粉为唯一碳源的培养基中正常生长，可以在以果糖为唯一碳源的培养基中生长。在敲除菌株W4中超表达蔗糖酶SUC2基因，超表达转化子SUC2-1的SUC2基因表达量为W0菌株的113.6倍，蔗糖酶和菊粉酶活力分别是W0菌株的2.53倍和6.60倍。SUC2基因超表达转化子水解菊粉的产物是单糖，可以利用30%（w/v）的菊粉发酵培养基生产13.4%（v/v,20℃）的酒精。在1-L发酵体系中，超表达转化子SUC2-1同步糖化生料菊芋粉的酒精产量达10.9%（v/v,20℃）。本实验首次证明W0菌株的SUC2基因与其胞外和胞内菊粉酶活性有关，也证明了在W0菌株中超表达蔗糖酶的技术也可应用到酒精发酵工业上。
Inulinases are fructofuranosyl hydrolases that target on the β-2,1linkage ofinulin and hydrolyze it into fructose and glucose. They can be divided intoexo-inulinase and endo-inulinase. The exo-inulinase catalyzes the removal of theterminal fructose residues from the non-reducing end of the inulin molecule while theendo-inulinase hydrolyzes the internal linkages in inulin to yield inulotriose,inulotetraose, and inulopentaose. Invertase is the beta-fructofuranosidase thatcatalyzes the irreversible hydrolysis of sucrose into glucose and frucose. It is widelypresent in bacteria, yeasts, fungi, higher plants and in some animal cells. Especially,invertase from Saccharomyces cerevisiae has been intensively investigated andapplied to food and fermentation industries. The SUC2gene encodes the maininvertase activity.
In the study, the endo-inulinase gene (EnIA) from Arthrobacter sp. S37wasligated into the expression vector pINA1317and over-expressed in Yarrowialipolytica Po1h. It was found that the endo-inulinase activity and specificendo-inulinase activity produced by the transformant1317-EnIA were16.7U/mL and93.4U/mg, respectively. The recombinant EnIA was purified and characterized. Themolecular weight of the purified rEnIA was78.9kDa. The optimal pH andtemperature of the purified rEnIA were4.0and50℃, respectively. The purifiedrEnIA was stable in the temperature range of4-40℃and in the pH range of2.0-8.0.The activity of rEnIA was greatly stimulated in the presence of Li+. The purifiedrEnIA could actively convert inulin into disaccharides. Moreover, the mixture ofrEnIA and exo-inulinase has a higher inulinase activity than the sum of rEnIA andexo-inulinase activity. It suggested that expressing both the endo-inulinase andexo-inulinase gene in the same host would get the recombinants with higher inulinaseactivity.
The high ethanol producing yeast Saccharomyces sp. strain W0, which ispreserved in our lab could ferment inulin and produce low concentration of ethanolfrom inulin hydrolysate. After the endo-inulinase gene from Arthrobacter sp. S37wasligated the expression vectors pMIDSC31and pMIRSC31, the endo-inulinase genewas inserted into the chromosomal DNA of Saccharomyces sp. W0. It was found thatthe inulinase activity of the recombinant yeast D5in which the endo-inulinase genewas inserted into the delta sequence was higher than that of the recombinant yeast R1in which the endo-inulinase gene was inserted into18S rDNA sequence. More ethanolfrom inulin was produced by the recombinant yeast D5than by the recombinant yeastR1. But Saccharomyces sp. W0produced the lowest inulinase activity andconcentration of ethanol. During the5-L fermentation, the recombinant yeast D5could produce13.6mL of ethanol per100mL of the fermented medium from30%(w/v) inulin. The recombinant yeast D5could actively convert the unsterilized mealof Jerusalem artichoke tubers, yielding10.1mL of ethanol per100mL of thefermented medium.
The Saccharomyces sp. W0has the low inulinase activity. However, little isknown about relationship between the invertase gene (SUC2) and inulinase activityand ethanol production from inulin in strain W0. Therefore, in this study, the SUC2gene was disrupted in Saccharomyces sp. W0and the native SUC2gene wasover-expressed in the disruptant. Then, the relationship between the invertase geneand inulinase activity and ethanol production was examined. After the SUC2gene inSaccharomyces sp. W0was removed, the disruptant W4obtained could not produceany extracellular invertase and inulinase activities, could not grow in the mediacontaining sucrose and inulin, but could grow in the medium containing fructose.However, after the SUC2gene was over-expressed in the disruptant W4, therecombinant yeast strain SUC2-1obtained could produce much higher extracellularinvertase and inulinase activities than Saccharomyces sp. W0and the transcriptionallevel of the SUC2gene in the recombinant yeast strain SUC2-1was also much higherthan that in Saccharomyces sp. W0. This is the first time to report that the SUC2genein Saccharomyces sp. W0is closely related to both extracellular invertase and inulinase activities. The invertase over-produced by the recombinant yeast strainSUC2-1could actively convert inulin into monosaccharides. The recombinant yeaststrain SUC2-1over-expressing the SUC2gene could produce over13.4%(v/v)ethanol from30%(w/v) inulin, suggesting that Saccharomyces sp. W0over-producing invertase also can be applied to ethanol fermentation industry.
本研究首先将节杆菌属Arthrobacter sp. S37来源的菊粉内切酶基因EnIA在酵母菌解脂亚罗维亚中表达，并研究了重组菊粉内切酶的性质及水解菊粉的产物。将菊粉内切酶基因EnIA连接到表达质粒pINA1317上，在Yarrowia lipolytica Po1h中进行分泌表达。重组转化子1317-EnIA生产的重组菊粉内切酶的酶活力和比活力大小分别为16.7U/mL和93.4U/mg，表观分子量大小为78.9kDa。重组酶的最适pH值为4.0，在pH2.0-8.0范围内，重组酶可以保持较高的活性。在40℃以下，重组酶有很好的稳定性，其最适作用温度为50℃。Li+离子对重组酶活性有激活作用。重组菊粉内切酶水解菊粉的主要产物为菊粉二糖。当把重组菊粉内切酶和菊粉外切酶混合水解菊粉时，混合物的菊粉酶活力大于两者菊粉酶相加之和，表现出协同作用。
本实验室保藏的一株高产酒精酵母Saccharomyces sp. W0菌株可以缓慢地糖化菊粉产生单糖，利用菊粉发酵生产少量的酒精。将菊粉内切酶基因EnIA在酒精酵母W0菌株中表达，提高W0菌株的菊粉酶活力和酒精产量。把菊粉内切酶基因EnIA连接到酒精酵母表达载体pMIDSC31（δ序列插入型）和pMIRSC31（rDNA序列插入型）上，整合到W0菌株的基因组DNA中。δ序列插入型重组转化子D5的菊粉酶活力高于rDNA序列插入型重组转化子R1。重组转化子D5的菊粉酶活力达8.6U/mL。在5-L发酵罐体系中，转化子D5以菊粉含量为30%（w/v）的培养基进行酒精发酵，酒精产量为13.6%（v/v,20℃）。转化子D5同步糖化生料菊芋粉，在1-L发酵体系中，酒精产量达10.1%（v/v,20℃）。
W0菌株具有很低的菊粉酶活性，根据氨基酸序列的多重比对分析结果推测可能与蔗糖酶基因SUC2相关。蔗糖酶基因SUC2与酒精酵母具有菊粉酶活力可以发酵菊粉生产酒精之间的关系还鲜有报道。本研究将SUC2基因完全敲除并将原始SUC2基因在敲除菌株中超表达，根据敲除和超表达之后的实验结果解释蔗糖酶基因SUC2与W0菌株具有菊粉酶活性是否相关。蔗糖酶基因SUC2被敲除后，完全敲除菌株W4失去蔗糖酶和菊粉酶活力，不能在以蔗糖或菊粉为唯一碳源的培养基中正常生长，可以在以果糖为唯一碳源的培养基中生长。在敲除菌株W4中超表达蔗糖酶SUC2基因，超表达转化子SUC2-1的SUC2基因表达量为W0菌株的113.6倍，蔗糖酶和菊粉酶活力分别是W0菌株的2.53倍和6.60倍。SUC2基因超表达转化子水解菊粉的产物是单糖，可以利用30%（w/v）的菊粉发酵培养基生产13.4%（v/v,20℃）的酒精。在1-L发酵体系中，超表达转化子SUC2-1同步糖化生料菊芋粉的酒精产量达10.9%（v/v,20℃）。本实验首次证明W0菌株的SUC2基因与其胞外和胞内菊粉酶活性有关，也证明了在W0菌株中超表达蔗糖酶的技术也可应用到酒精发酵工业上。
Inulinases are fructofuranosyl hydrolases that target on the β-2,1linkage ofinulin and hydrolyze it into fructose and glucose. They can be divided intoexo-inulinase and endo-inulinase. The exo-inulinase catalyzes the removal of theterminal fructose residues from the non-reducing end of the inulin molecule while theendo-inulinase hydrolyzes the internal linkages in inulin to yield inulotriose,inulotetraose, and inulopentaose. Invertase is the beta-fructofuranosidase thatcatalyzes the irreversible hydrolysis of sucrose into glucose and frucose. It is widelypresent in bacteria, yeasts, fungi, higher plants and in some animal cells. Especially,invertase from Saccharomyces cerevisiae has been intensively investigated andapplied to food and fermentation industries. The SUC2gene encodes the maininvertase activity.
In the study, the endo-inulinase gene (EnIA) from Arthrobacter sp. S37wasligated into the expression vector pINA1317and over-expressed in Yarrowialipolytica Po1h. It was found that the endo-inulinase activity and specificendo-inulinase activity produced by the transformant1317-EnIA were16.7U/mL and93.4U/mg, respectively. The recombinant EnIA was purified and characterized. Themolecular weight of the purified rEnIA was78.9kDa. The optimal pH andtemperature of the purified rEnIA were4.0and50℃, respectively. The purifiedrEnIA was stable in the temperature range of4-40℃and in the pH range of2.0-8.0.The activity of rEnIA was greatly stimulated in the presence of Li+. The purifiedrEnIA could actively convert inulin into disaccharides. Moreover, the mixture ofrEnIA and exo-inulinase has a higher inulinase activity than the sum of rEnIA andexo-inulinase activity. It suggested that expressing both the endo-inulinase andexo-inulinase gene in the same host would get the recombinants with higher inulinaseactivity.
The high ethanol producing yeast Saccharomyces sp. strain W0, which ispreserved in our lab could ferment inulin and produce low concentration of ethanolfrom inulin hydrolysate. After the endo-inulinase gene from Arthrobacter sp. S37wasligated the expression vectors pMIDSC31and pMIRSC31, the endo-inulinase genewas inserted into the chromosomal DNA of Saccharomyces sp. W0. It was found thatthe inulinase activity of the recombinant yeast D5in which the endo-inulinase genewas inserted into the delta sequence was higher than that of the recombinant yeast R1in which the endo-inulinase gene was inserted into18S rDNA sequence. More ethanolfrom inulin was produced by the recombinant yeast D5than by the recombinant yeastR1. But Saccharomyces sp. W0produced the lowest inulinase activity andconcentration of ethanol. During the5-L fermentation, the recombinant yeast D5could produce13.6mL of ethanol per100mL of the fermented medium from30%(w/v) inulin. The recombinant yeast D5could actively convert the unsterilized mealof Jerusalem artichoke tubers, yielding10.1mL of ethanol per100mL of thefermented medium.
The Saccharomyces sp. W0has the low inulinase activity. However, little isknown about relationship between the invertase gene (SUC2) and inulinase activityand ethanol production from inulin in strain W0. Therefore, in this study, the SUC2gene was disrupted in Saccharomyces sp. W0and the native SUC2gene wasover-expressed in the disruptant. Then, the relationship between the invertase geneand inulinase activity and ethanol production was examined. After the SUC2gene inSaccharomyces sp. W0was removed, the disruptant W4obtained could not produceany extracellular invertase and inulinase activities, could not grow in the mediacontaining sucrose and inulin, but could grow in the medium containing fructose.However, after the SUC2gene was over-expressed in the disruptant W4, therecombinant yeast strain SUC2-1obtained could produce much higher extracellularinvertase and inulinase activities than Saccharomyces sp. W0and the transcriptionallevel of the SUC2gene in the recombinant yeast strain SUC2-1was also much higherthan that in Saccharomyces sp. W0. This is the first time to report that the SUC2genein Saccharomyces sp. W0is closely related to both extracellular invertase and inulinase activities. The invertase over-produced by the recombinant yeast strainSUC2-1could actively convert inulin into monosaccharides. The recombinant yeaststrain SUC2-1over-expressing the SUC2gene could produce over13.4%(v/v)ethanol from30%(w/v) inulin, suggesting that Saccharomyces sp. W0over-producing invertase also can be applied to ethanol fermentation industry.
引文
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