Screening And Study Of A Thermophilic Xylan Hydrolase System Of The Genus Caldicellulosiruptor

Agroforestry waste is mainly composed of cellulose,hemicellulose,lignin and other macromolecular compounds.Xylan as the component in the highest content of hemicellulose,the degradation of xylan can reduce the environmental pollution caused by burning,and also realized the waste resource utilization.Natural xylan structure often containing side chains with different substituents like a-L-arabinofuranosyl,acetyl,feruloyl and 4-O-methyl-D-glucuronopyranosyl.The complete hydrolysis of xylan requires a large variety of cooperatively acting enzymes like endo-1,4-xylanases,?-1,4-xylosidases,a-L-arabinofuranosidases,acetyl xylan esterases and ferulic acid esterases.In this paper,based on genome annotation,protein blast,and protein structural prediction,we got a thermostable acetyl xylan esterases Axe7 from the genome of the thermophilic anaerobic bacteria Caldicellulosiruptor sp.F32 and two ?-1,4-xylosidases CsXyl39 A and CsXyl39 B in the same genus Caldicellulosiruptor saccharolyticus.After gene cloning,plasmid construction,protein expression and purification in E.coli,the recombinant protein were produced.The properties of enzymes were determined by different methods and the practical applications was also explored.Axe7,which is expressed by Caldicellulosiruptor sp.F32,has the advantages of high optimum temperature,good thermostability,high catalytic efficiency and so on.When4-Methylumbelliferyl-acetate was used as the substrate,the optimum pH and temperature was 6.5-7.0 and 85°C respectively.Under the optimum conditions,the half life of Axe7 was more than 4 hours and the enzyme activity was inhibited by different metal ions.The kinetics parameters showed that Axe7 had a better catalytic efficiency on 4-MUA.Many excellent properties made Axe7 as a potential enzyme for the biorefinery industry.?-1,4-xylosidases,CsXyl39 A and CsXyl39 B obtained from Caldicellulosiruptor saccharolyticus,were located in the same gene cluster of C.saccharolyticus,but the sequence similarity was very low.Enzymatic properties showed that these two enzymes were thermostable and had a similar optimum temperature and pH.CsXyl39 A had strong tolerance to organic solvents,while CsXyl39 B had a higher ability to tolerate xylose and glucose.In the practical application research,after immobilized by macroporous resin,the organic solvent tolerance of CsXyl39 A had been further improved but the thermostability was not significantly changed.The enzyme can also be used in the hydrolysis of notoginsenoside R1,reaction products were determined by HPLC to generate ginsenoside Rg1 which had better pharmacological action.However,CsXyl39 B had good synergistic effect with ?-1,4 xylanase,and both of them could obviously improved the hydrolysis efficiency of xylan.These excellent properties made these two?-1,4-xylosidases as an industrially potential enzymes.