Caldicellulosiruptor besciiDSM嗜热果胶酶系特征及结构域功能研究
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摘要
果胶物质是半乳糖醛酸以α-1,4糖苷键聚合而成的多糖链，广泛存在于植物初生细胞壁中。果胶酶是一系列以水解果胶物质为底物的酶类的总称，在高等植物和微生物中分布甚广。植物在成熟过程中，果胶酶使植物细胞壁更紧致松软，对植物组织腐败再生平衡也起到了重要作用。
果胶酶按其作用方式的不同可分为两大类，即酯酶和解聚酶，而解聚酶又分为水解酶和裂解酶。水解酶是通过引入水分子水解糖苷键，而裂解酶是通过反式消去作用，断裂糖苷键。果胶酶是目前工业领域研究的热点，可以应用于各种行业，在果汁制造和造纸工业中的应用最为常见，近些年，逐渐开始应用于酿酒、饲料制造、纺织行业中。
我们以来源于嗜热菌Caldicellulosiruptor bescii DSM6725的果胶酶系（4株果胶酶）为研究对象，重点对重组果胶水解酶CbPelA、果胶裂解酶CbPelC的基本性质进行表征。并设计突变体，研究CbPelC中结构域之间的关系。
本课题组从Caldicellulosiruptor bescii DSM6725中获得四个果胶酶基因CbPelA（E.C.3.2.1.67）、CbPelB、CbPelC（E.C.4.2.2.2）及CbPelD（E.C.4.2.2.9）。通过设计引物扩增CbPelA-D，连接到载体pET-20b(+)上，并分别转入E. coliorigami (DE3)、E. coli BL21(DE3)感受态，构建了Caldicellulosiruptor besciiDSM6725果胶酶系的原核表达体系。经Blsat序列比对分析果胶水解酶CbPelA（E.C.3.2.1.67）、果胶裂解酶CbPelC（E.C.4.2.2.2），与已报道的同家族果胶酶氨基酸序列同源性分别最高为46%和54%，都具有新型果胶酶基因。采用热处理和镍离子亲和层析法对CbPelA、CbPelC进行纯化，分别得到约50kD，46kD大小的蛋白条带，与理论预测结果一致。酶学实验表明，该两株梅的最适温度分别为72℃，70℃，最适pH分别为5.2和9.5。通过DNS法和235nm最大吸收法分别检测该两株果胶酶的活力，由于两株酶的作用机理不同，检测手段也不同，CbPelA只能通过DNS还原糖检测法来测定活力，CbPelC可以通过不饱和双键在235nm存在最大吸收法，来测定其活力，这也证实了CbPelC的裂解酶作用机理。活力测定CbPelA最大反应速率为386.8U/mg-1，而CbPelC的催化结构域比活力达到3000U mg-1以上。迄今为止，CbPelC及其催化结构域CbPelC-CD的活力远高于已报道的其它第三家族的果胶酶。两株酶都具有良好的热稳定性，半衰期分别长达14h，10h。薄层层析和粘度系数下降实验证实前者为外切酶，后者为内切酶。底物特异性研究实验显示，随着底物酯化度的升高，酶的活力逐渐降低。底物酯化度20-34%时，CbPelC的活力与以多聚半乳糖醛酸为底物时没有明显差异。两株果胶酶活性只特异性针对多聚半乳糖醛酸，对CMC、Avicel、葡聚糖、木聚糖、壳聚糖、可溶性淀粉等其他多糖类皆没有活力。一价离子Na+、K+、NH4+对酶促反应影响较小；大多数二价金属离子Mg2+、Zn2+、Ca2+、Ba2+、Co2+、Ni2+都对CbPelA的酶促反应有不同程度的抑制，其中Cu2+，Cd2+完全抑制CbPelA酶活力。CbPelC酶表现为Ca2+完全依赖性，没有Ca2+存在时，CbPelC没有活力，Ca2+浓度0.8mM-1.0mM时，酶活力达到最大值。
CbPelC、CbPelD的结构中含有一个未知功能结构域，并且没有类似报道，我们以CbPelC为例，通过设计两个突变体，探讨野生型CbPelC和突变体CbPelC-CA、CbPelC-CD三者之间的关系。实验结果表明，该未知功能结构域与钙离子结合、与可溶底物多聚半乳糖醛酸链的结合及与体外其他多糖的结合均无关系，而在结合不可溶生物质底物时，具有重要的作用。
综上所述，本课题成功获得Caldicellulosiruptor bescii DSM6725果胶酶系的四株新型嗜热果胶酶，其中CbPelA、CbPelC具有较高的酶活力和优异的热稳定性，突变体的设计有助于我们更深入的理解其结构域的功能和催化机理。这项工作对今后继续探究有效利用生物质能源具有重要的意义，为其产业化应用奠定了基础。
Pectic substances mainly consist of the polymerization of galacturonans with α-1,4glycosidic bond and the carboxyl groups of galacturonic acid are partially esterifiedby methyl groups. Pectinolytic enzymes are related enzymes,targeting specificlinkages of the pectin as substances，and widely distributed in higher plants andmicroorganisms.Pectinolytic enzymes,which made the cell wall tighter and softer,arehelpful in the plant tissue manipulation and storage process of plant tissue, while theyalso played an important role in maintaining the balance of regeneration corruption inplant tissue.In addition, Pectinolytic enzymes also played a leading role in the plantselongation and fruits corruption.
According to the pattern of action, pectinolytic enzymes are classified intodepolymerases and pectin methylesterase, Depolymerases act on pectic substances bytwo different mechanisms, hydrolysis, in which they catalyze the hydrolytic cleavagewith the introduction of water across the oxygen bridge and trans-elimination lysis, inwhich they break the glycosidic bond by a trans-elimination reaction without anyparticipation of water molecule. Pectinolytic enzymes are important emergingenzymes, widely used in the food industry for the fruit juice extraction andclarification and wine brewing.They are also used in feed processing, environmentalprotection, plant disease resistance and other aspects.Pectin lyases were found inrecent years to be important enzyme cleaner mainly used for pulp bleaching andtextile biorefining industry, replacing traditional textile refining.It has the advantageto resolve environmental and energy issues through enzymatic refining methods, interms of both quality and environmental protection,which can not be compared withthe advantages of traditional crafts. With the continuous emergence of new uses ofpectin lyase, the enzyme become a hot research topic in recent years.
A polygalacturonase CbPelA（E.C.3.2.1.67）and a pectate lyase（E.C.4.2.2.2）Caldicellulosiruptor besciiDSM6725were made to be our research object and the basic properties were characterized.They were studied through the enzyme activity,thermal stability,substrate specificity and structures.
Two predicted pectinolytic enzymes genes CbPelA（E.C.3.2.1.67）and CbPelC（E.C.4.2.2.2） from Caldicellulosiruptor bescii DSM6725were successfullycloned and expressed in Escherichia coli. Polygalacturonase CbPelA (EC3.2.1.67)and pectin lyase CbPelC (EC4.2.2.2) belong to the family of pectinolyticenzymes,After Blsat sequence analysis pectinolytic enzymes reported the highestamino acid sequence homology46%and54%of the clones were initially identifiedwith novel pectinase genes. CbPelA and CbPelC were connected to the vectorpET-20b(+) by designing primers, and were transferred to E. coli origami (DE3) andE. coli BL21(DE3), respectively. Prokaryotic expression system of two strains of theenzymes were constructed. The recombinant CbPelA and CbPelC were purified withthermal treatment and Ni-NTA agarose column. The molecular mass of purifiedenzyme was revealed about50kD and46kD, which was agreed with the predictedmolecular weight of the mature protein. The results of enzymology experimentsshowed that the optimal temperature of CbPelA and CbPelC were72℃,70℃, theoptimum pH were5.2and9.5respectively. Pectinase activities were assayed by therate of increase in number of reducing groups and the increase in absorbance at235nm.The mechanisms of the action of the enzymes were different,the detectionmethods were different.The activitiy of CbPelA can only be determined by the DNSreducing sugar assay.The activitiy of CbPelC can be determined by the increase inabsorbance at235nm due to formation of the4:5double bonds produced at thenon-reducing ends of the unsaturated products,and the cleavage of mechanism ofCbPelC was confirmed by the methods. The Vmaxand Kmof CbPelA were detected tobe386.8U mg-1and0.31mg ml-1, respectively. while the specific activity of CbPelCreached more than3000U mg-1. CbPelA and CbPelC presented high thermal stabilitywith the half life of14h and10h at70oC.It was comfirmed that CbPelA was anexopolygalacturonase and CbPelC was an endo-pectate lyase by the results ofThin-layer chromatography and Viscosity reduction measurements. The recombinantshowed maximum activity on polygalacturonic acid with no activity on othersubstrates like on CMC,Avicel,glucan,xylan,chitosan and soluble starch,etc. Theeffect of methylation degrees of pectin on activity was determined.The activity ofCbPelA and CbPelC decreased with the degree of methyl-esterification. The activity of CbPelA towards PGA was over5-fold than the high degree of esterification ofpectin. The activity of CbPelC on20-34%methyl esterfied pectin had no significantdifference with the activity towards PGA. The result for the influences of metal ionsof CbPelA indicated that Monovalent cations Na+,K+,NH4+had no obvious effect onenzyme activity, while most of divalent metal cations Mg2+,Zn2+,Ca2+,Ba2+, Co2+,Ni2+inhibited the enzyme activity and the activity of CbPelA was completely lost whenCu2+and Cd2+were added.CbPelC structure comprised a domain of unknownfunction, The domain was believed to be a CBM by sequence analysis and mutantsdesign,which may be related to the binding of insoluble substrate. Catalytic andsubstrate binding mechanism were also discussed by crystal structure.
In summary, two novel enzymes from the anaerobic thermophilicCaldicellulosiruptor bescii were characterized in detail. They have high enzymeactivities and excellent thermal stability. CbPelA and CbPelC demonstratedoutstanding molecular properties, therefore have the potential application in bioenergyand food industry. This work has important significance for the future to continue toexplore the effective use of biomass energy for industrial applications of thefoundation.
果胶酶按其作用方式的不同可分为两大类，即酯酶和解聚酶，而解聚酶又分为水解酶和裂解酶。水解酶是通过引入水分子水解糖苷键，而裂解酶是通过反式消去作用，断裂糖苷键。果胶酶是目前工业领域研究的热点，可以应用于各种行业，在果汁制造和造纸工业中的应用最为常见，近些年，逐渐开始应用于酿酒、饲料制造、纺织行业中。
我们以来源于嗜热菌Caldicellulosiruptor bescii DSM6725的果胶酶系（4株果胶酶）为研究对象，重点对重组果胶水解酶CbPelA、果胶裂解酶CbPelC的基本性质进行表征。并设计突变体，研究CbPelC中结构域之间的关系。
本课题组从Caldicellulosiruptor bescii DSM6725中获得四个果胶酶基因CbPelA（E.C.3.2.1.67）、CbPelB、CbPelC（E.C.4.2.2.2）及CbPelD（E.C.4.2.2.9）。通过设计引物扩增CbPelA-D，连接到载体pET-20b(+)上，并分别转入E. coliorigami (DE3)、E. coli BL21(DE3)感受态，构建了Caldicellulosiruptor besciiDSM6725果胶酶系的原核表达体系。经Blsat序列比对分析果胶水解酶CbPelA（E.C.3.2.1.67）、果胶裂解酶CbPelC（E.C.4.2.2.2），与已报道的同家族果胶酶氨基酸序列同源性分别最高为46%和54%，都具有新型果胶酶基因。采用热处理和镍离子亲和层析法对CbPelA、CbPelC进行纯化，分别得到约50kD，46kD大小的蛋白条带，与理论预测结果一致。酶学实验表明，该两株梅的最适温度分别为72℃，70℃，最适pH分别为5.2和9.5。通过DNS法和235nm最大吸收法分别检测该两株果胶酶的活力，由于两株酶的作用机理不同，检测手段也不同，CbPelA只能通过DNS还原糖检测法来测定活力，CbPelC可以通过不饱和双键在235nm存在最大吸收法，来测定其活力，这也证实了CbPelC的裂解酶作用机理。活力测定CbPelA最大反应速率为386.8U/mg-1，而CbPelC的催化结构域比活力达到3000U mg-1以上。迄今为止，CbPelC及其催化结构域CbPelC-CD的活力远高于已报道的其它第三家族的果胶酶。两株酶都具有良好的热稳定性，半衰期分别长达14h，10h。薄层层析和粘度系数下降实验证实前者为外切酶，后者为内切酶。底物特异性研究实验显示，随着底物酯化度的升高，酶的活力逐渐降低。底物酯化度20-34%时，CbPelC的活力与以多聚半乳糖醛酸为底物时没有明显差异。两株果胶酶活性只特异性针对多聚半乳糖醛酸，对CMC、Avicel、葡聚糖、木聚糖、壳聚糖、可溶性淀粉等其他多糖类皆没有活力。一价离子Na+、K+、NH4+对酶促反应影响较小；大多数二价金属离子Mg2+、Zn2+、Ca2+、Ba2+、Co2+、Ni2+都对CbPelA的酶促反应有不同程度的抑制，其中Cu2+，Cd2+完全抑制CbPelA酶活力。CbPelC酶表现为Ca2+完全依赖性，没有Ca2+存在时，CbPelC没有活力，Ca2+浓度0.8mM-1.0mM时，酶活力达到最大值。
CbPelC、CbPelD的结构中含有一个未知功能结构域，并且没有类似报道，我们以CbPelC为例，通过设计两个突变体，探讨野生型CbPelC和突变体CbPelC-CA、CbPelC-CD三者之间的关系。实验结果表明，该未知功能结构域与钙离子结合、与可溶底物多聚半乳糖醛酸链的结合及与体外其他多糖的结合均无关系，而在结合不可溶生物质底物时，具有重要的作用。
综上所述，本课题成功获得Caldicellulosiruptor bescii DSM6725果胶酶系的四株新型嗜热果胶酶，其中CbPelA、CbPelC具有较高的酶活力和优异的热稳定性，突变体的设计有助于我们更深入的理解其结构域的功能和催化机理。这项工作对今后继续探究有效利用生物质能源具有重要的意义，为其产业化应用奠定了基础。
Pectic substances mainly consist of the polymerization of galacturonans with α-1,4glycosidic bond and the carboxyl groups of galacturonic acid are partially esterifiedby methyl groups. Pectinolytic enzymes are related enzymes,targeting specificlinkages of the pectin as substances，and widely distributed in higher plants andmicroorganisms.Pectinolytic enzymes,which made the cell wall tighter and softer,arehelpful in the plant tissue manipulation and storage process of plant tissue, while theyalso played an important role in maintaining the balance of regeneration corruption inplant tissue.In addition, Pectinolytic enzymes also played a leading role in the plantselongation and fruits corruption.
According to the pattern of action, pectinolytic enzymes are classified intodepolymerases and pectin methylesterase, Depolymerases act on pectic substances bytwo different mechanisms, hydrolysis, in which they catalyze the hydrolytic cleavagewith the introduction of water across the oxygen bridge and trans-elimination lysis, inwhich they break the glycosidic bond by a trans-elimination reaction without anyparticipation of water molecule. Pectinolytic enzymes are important emergingenzymes, widely used in the food industry for the fruit juice extraction andclarification and wine brewing.They are also used in feed processing, environmentalprotection, plant disease resistance and other aspects.Pectin lyases were found inrecent years to be important enzyme cleaner mainly used for pulp bleaching andtextile biorefining industry, replacing traditional textile refining.It has the advantageto resolve environmental and energy issues through enzymatic refining methods, interms of both quality and environmental protection,which can not be compared withthe advantages of traditional crafts. With the continuous emergence of new uses ofpectin lyase, the enzyme become a hot research topic in recent years.
A polygalacturonase CbPelA（E.C.3.2.1.67）and a pectate lyase（E.C.4.2.2.2）Caldicellulosiruptor besciiDSM6725were made to be our research object and the basic properties were characterized.They were studied through the enzyme activity,thermal stability,substrate specificity and structures.
Two predicted pectinolytic enzymes genes CbPelA（E.C.3.2.1.67）and CbPelC（E.C.4.2.2.2） from Caldicellulosiruptor bescii DSM6725were successfullycloned and expressed in Escherichia coli. Polygalacturonase CbPelA (EC3.2.1.67)and pectin lyase CbPelC (EC4.2.2.2) belong to the family of pectinolyticenzymes,After Blsat sequence analysis pectinolytic enzymes reported the highestamino acid sequence homology46%and54%of the clones were initially identifiedwith novel pectinase genes. CbPelA and CbPelC were connected to the vectorpET-20b(+) by designing primers, and were transferred to E. coli origami (DE3) andE. coli BL21(DE3), respectively. Prokaryotic expression system of two strains of theenzymes were constructed. The recombinant CbPelA and CbPelC were purified withthermal treatment and Ni-NTA agarose column. The molecular mass of purifiedenzyme was revealed about50kD and46kD, which was agreed with the predictedmolecular weight of the mature protein. The results of enzymology experimentsshowed that the optimal temperature of CbPelA and CbPelC were72℃,70℃, theoptimum pH were5.2and9.5respectively. Pectinase activities were assayed by therate of increase in number of reducing groups and the increase in absorbance at235nm.The mechanisms of the action of the enzymes were different,the detectionmethods were different.The activitiy of CbPelA can only be determined by the DNSreducing sugar assay.The activitiy of CbPelC can be determined by the increase inabsorbance at235nm due to formation of the4:5double bonds produced at thenon-reducing ends of the unsaturated products,and the cleavage of mechanism ofCbPelC was confirmed by the methods. The Vmaxand Kmof CbPelA were detected tobe386.8U mg-1and0.31mg ml-1, respectively. while the specific activity of CbPelCreached more than3000U mg-1. CbPelA and CbPelC presented high thermal stabilitywith the half life of14h and10h at70oC.It was comfirmed that CbPelA was anexopolygalacturonase and CbPelC was an endo-pectate lyase by the results ofThin-layer chromatography and Viscosity reduction measurements. The recombinantshowed maximum activity on polygalacturonic acid with no activity on othersubstrates like on CMC,Avicel,glucan,xylan,chitosan and soluble starch,etc. Theeffect of methylation degrees of pectin on activity was determined.The activity ofCbPelA and CbPelC decreased with the degree of methyl-esterification. The activity of CbPelA towards PGA was over5-fold than the high degree of esterification ofpectin. The activity of CbPelC on20-34%methyl esterfied pectin had no significantdifference with the activity towards PGA. The result for the influences of metal ionsof CbPelA indicated that Monovalent cations Na+,K+,NH4+had no obvious effect onenzyme activity, while most of divalent metal cations Mg2+,Zn2+,Ca2+,Ba2+, Co2+,Ni2+inhibited the enzyme activity and the activity of CbPelA was completely lost whenCu2+and Cd2+were added.CbPelC structure comprised a domain of unknownfunction, The domain was believed to be a CBM by sequence analysis and mutantsdesign,which may be related to the binding of insoluble substrate. Catalytic andsubstrate binding mechanism were also discussed by crystal structure.
In summary, two novel enzymes from the anaerobic thermophilicCaldicellulosiruptor bescii were characterized in detail. They have high enzymeactivities and excellent thermal stability. CbPelA and CbPelC demonstratedoutstanding molecular properties, therefore have the potential application in bioenergyand food industry. This work has important significance for the future to continue toexplore the effective use of biomass energy for industrial applications of thefoundation.
引文
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