Study On α-L-arabinofuranosidase Of Fecal Microbial Metagenomics

Xylan is the main component of plant hemicellulose,and the degradation of xylan in lignocellulose is the key link to obtain value-added by-products such as oligosaccharides,xylitol and furfural.However,xylan structure is complex and complete degradation requires the participation of various hemicellulose degradation enzymes.α-L-arabinofuranosidases is an important hemicellulase that catalyzes the cleavage of α-(1,2),α-(1,3)or α-(1,5)-arabinofuranose bonds in xylan to assist in the complete hydrolysis of xylan,which is mainly used in the food industry,bioethanol and pharmaceuticals.Therefore,the search for α-L-arabinofuranosidases with better ethanol and temperature tolerance is crucial for their applications.In this study,novelα-L-arabinofuranosidases genes were mined from fecal microbial metagenomics,cloned,heterologously expressed and investigated for their enzymatic properties,synergistic xylanase degradation of xylan and modification of their thermal stability.It is summarized as follows:(1)Cloning and expression of α-L-arabinofuranosidases genes from the metagenomics of fecal microorganismsThe α-L-arabinofuranosidases genes AbfNC2b＿38 and AbfNC2b＿54,with full lengths of 1509 bp and 1506 bp,encoding 502 and 501 amino acids,respectively,belonging to the 51 family of glycoside hydrolases,were obtained from the fecal microbial metagenomics.The recombinant enzymes AbfNC2b＿38 and AbfNC2b＿54were obtained by induction of expression and purification after transferring into E.coli BL21(DE3).(2)Characterization of recombinant enzymes AbfNC2b＿38 and AbfNC2b＿54The enzymatic properties showed that the optimum temperature and p H of the recombinant enzymes were 55 ℃ and p H 6.0,and the relative enzyme activities were maintained above 80% at 30-50 ℃ for 1 h.The relative enzyme activities of AbfNC2b＿38 were between 113%-93% at p H 6.0-9.0 for 1 h,and AbfNC2b＿54 only maintained above 50% at p H 5.0-7.0.The relative enzyme activity of recombinant enzyme AbfNC2b＿38 remained above 65% after 1 h of treatment with 5%-30% ethanol,while that of AbfNC2b＿54 decreased with the increase of ethanol concentration.(3)Study on the degradation of xylan by recombinant enzyme with xylanaseUnder pH 5.0 and 50 ℃,the recombinant α-L-arabinofuranosidases and commercial xylanase showed the highest synergistic degradation rate of xylan.1.21 for AbfNC2b＿38 and 2.23 mg/m L for reducing sugar yield,and 1.19 for AbfNC2b＿54 and2.03 mg/m L for reducing sugar yield.Compared with commercial α-Larabinofuranosidase,the recombinant enzyme in this study was more efficient in synergizing with xylanase to degrade xylan.(4)Modification of thermal stability of recombinant enzyme AbfNC2b＿38A total of 12 mutants with significantly improved thermal stability were obtained by thermal stability modification of recombinant enzyme AbfNC2b＿38 based on Bfactor factor.Among them,mutants S16 R,G61H,G61 Y,A215E and A215 V tolerated at 60 ℃ for 1 h with relative enzyme activity maintained at 70% and above,while the wild type relative enzyme activity was only about 15%;mutants S16 R,G61H,G61 Y and A215 E tolerated at 65 ℃ for 1 h with relative enzyme activity maintained at 50%and above,while the wild type lost its activity.