Enzymatic Properties And Functional Studies Of Thermostable Pectinases From Caldicellulosiruptor Bescii

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.