Recombinant Expression And Characterization Of Three Novel Xylosidases

Xylosidases can hydrolyze non-reducing terminal residues of xylooligosaccharides and xyloglycoside substrates to generate xylose.There are widely used in energy industry,food industry and medical industry.Xylosidases are the key component of hemicellulase system.They can be divided into?-xylosidases and?-xylosidases.?-xylosidases are often used in the structure modification and analysis of xyloglucan.?-xylosidases can be used not only for promoting xylanases to degrade xylan,but also for structural modification and analysis of corresponding carbohydrate compounds.Although there are a large number of microorganisms producing xylosidases in nature,but whose enzyme activity and enzymology properties are not so ideal.Therefore,to continue to excavate xylosidases with excellent properties not only can enrich the xylosidases repository,as well as for the use of hemicellulose and structure analysis of carbohydrate compounds to provide effective tool.Based on this,the genomes of early laboratory screening Paenibacillus polymyxa and Bacteroides ovatus that degrade hemicellulose polysaccharides were used as templates,successful restructured and expressed three xylosidases,and studied the enzymology properties of the recombinant enzyme.The main research results are as follows:1.In this study,the xylosidase gene ppxyl6a of glycoside hydrolase family 31?GH31?from Paenibacillus polymyxa,the GH31 xylosidase gene bogh31b and GH43 xylosidase gene boxyl4b from Bacteroides ovatus were cloned.Three new xylosidases PpXyl6A,BoGH31B and BoXyl4B have successfully realized the soluble expression in the prokaryotic host E.coli BL21?DE3?.After the purification by nickel column,the target proteins with high purity can be obtained,whose molecular weight was 86.7 kDa,88.0 kDa and 37.0 kDa,respectively.2.The properties of recombinant xylosidases were studied.At pH 6.0-10.0 range and under the condition of 30 ? has high activity and good stability.The optimal pH of PpXyl6A was 8.0,the optimal temperature was 40 ?.BoGH31B and BoXyl4B optimum pH was 6.0and 8.5,respectively.The optimum reaction temperatures were 40?and 35?,respectively.Fe³⁺,Hg²⁺,Cu²⁺and EDTA almost completely inhibited the activity of the three xylosidases.Na⁺,K⁺,low-concentration Li⁺,Mg²⁺and DTT had significant promoting effects on BoGH31B activity.Taking pNP?Xyl as substrate,the K_m and V_maxax of PpXyl6A pure enzymes were 4.2±0.1 mM and 1.7±0.1 mol/min/mg,the K_m and V_maxax of BoGH31B pure enzymes were2.4±0.1 mM and 3.5±0.2 mol/min/mg,respectively.The pure enzyme K_m and V_maxax of BoXyl4B were 1.1±0.2 mM and 7.5±0.2 mol/min/mg using pNP?Xyl as substrate.Compared with the reported?-xylosidase,BoXyl4B showed a stronger substrate affinity.3.The substrate selectivity of recombinant xylosidases were further studied by using a series of pNP-glycosides,oligosaccharides,ginsenosides containing different oligosaccharides,and polysaccharides.The results showed that PpXyl6A was a selective exo-?-1,6-xylosidase,which could hydrolyze isoprimeverose and xyloglucan oligosaccharides whose?-xylose at non-reducing end.Therefore,it can be used to study xyloglucooligosaccharides in hemicellulose structure.BoGH31B can only hydrolyze pNP?Xyl and has the activity of exo-?-xylosidase.BoXyl4B is a bifunctional enzyme that has both?-1,4-xylosidase and?-arabinosidase activity,characterized by the activity of?-1,4-xylosidase activity,Act on the beta 1,4 glycosidic bond of xylooligosaccharide and alpha 1,5 glycosidic bond of arabinooligosaccharides to form xylose and arabinose,respectively.4.The synergistic degradation of substrate by recombinant enzymes was studied.?-xylosidase PpXyl6A could promote the complete degradation of xyloglucooligosaccharide by synergistic effect of?-fucosidase,?-galactosidase and xyloglucanase.BoGH31B acts on xyloglucooligosaccharide in combination with the above three enzymes,and there is no xylose formation.BoXyl4B can effectively promote the degradation of xylan by xylanase.When BoXyl4B and xylanase,arabinosidase interact with wheat arabinoxylan,significantly increased the amount of reducing sugar released.Therefore,BoXyl4B can be applied to the efficient conversion of hemicellulose and structure analysis of xylose compounds.To sum up,three xylosidases were prepared in this paper and their functions were characterized.The results not only have important significance to promote the study of hemicellulose degradation,but also provide a new tool enzyme for the analysis of the structure of carbohydrate compounds.