| Along with the energy crisis around the world,the researches on conversion of biomass to biofuels and chemical products are attracting more and more attentions.Biorefinery of xylan,which is the main component of hemicellulose in lignocellulose,has also attracted much attention.In present work,a fungal strain FC2-2 produing xylanase was isolated from soil samples.The crude xylanase produced by FC2-2 was characterized and the solid cultivation medium for xylanse production was optimized.A xylanase AfxynA was purified from FC2-2,and the gene encoding AfxynA was cloned and expressed in Escherichia.coli.Three amnio acid residues of AfxynA were found to be involved in xylanase activity as well as formation of enzymatic hydrolysate.The fungal strain FC2-2 isolated from soil was identified as Aspergillus fumigatus by molecular identification as well as morphologic observation.The optimum pH of the crude xylanase produced by FC2-2 was 5.5,and the optimum temperature was 60 ?.The crude enzyme could convert birchwood xylan and sugarcane bagasse xylan into mainly xylobiose.When xylobiose was produed in a range of substrate concentration,the maximum yield of xylobiose was 54.7%,at the substrate concentration of 50 g/L.When the substrate concentration was fixed at 10 g/L,the production of xylobiose varied along with the dose of xylanase,and the maximum yield of xylobiose was 70%,at the xylanase dose of 200 U/g substarte.During solid cultivation of fungal strain FC2-2,the optimum carbon source for xylanase production was sugarcane bagasse mixed with wheat bran,at the ratio of 8:2.The optimum condition of xylanase prodution was:Nitrogen source was urea;time was 3 days;temperature was 28 ?;the pH was 7.0;the optimum ratio of solid to liquid was 1:1,whereas the inoculum size and the solid size had little influence on xylanase production.One xylanase AfxynA produced by FC2-2 was purified and characterized.The purified xylanase AfxynA was about 21.0 kDa.The optimum pH of AfxynA was 5.5,and the optimum temperature was 65 ?.AfxynA remained stable at the pH range of 3.0 to 10.0,and the half-life of AfxynA was 70 h at 50 ?.AfxynA could hydrolyze birchwood xylan,beechwood xylan and bagasse xylan,but not other polysaccharides,such as mannan,methycellulose,CMC,Avicel,and hydroethylcellulose.The Km of rAfxynA was 2.93 ± 0.15 mg/ml,and Vmax was 2346 ± 42.5 U/mg protein.When AfxynA was used in hydrolysis of xylooligosaccharides as well as birchwood xylan and sugarcane bagasse xylan,the main produt were xylobiose,and AfxynA could not hydrolyze xylobiose.These results indicated that AfxynA possessing both xylanase activity and transglycosylation activity.The N-terminal sequence of AfxynA was determined to be AGTPSSTG The analysis of amino acid sequence indicated that AfxynA belongs to glycosyl hydrolase family 11.The xylariase gene AfxynA was cloned into pET30a(+)and expressed in E.coli.The size of recombinant xylanase rAfxynA was about 21.0 kDa,and the optimum pH and temperature was 7.0 and 50 ?,respectively.Recombinant xylanase rAfxynA could hydrolyze birchwood xylan,beechwood xylan and bagasse xylan,but not other polysaccharides,such as mannan,methycellulose,carboxymethylcellulose,Avicel,and hydroethylcellulose.The Km of rAfxynA was 2.55 ? 0.10 mg/ml,and Vmax was 2563 ± 115.3 U/mg protein.rAfxynA colud convert to xylan and xylooligosaccharides larger than xylobiose to mainly xylobiose,and rAfxynA could not hydrolyze xylobiose.In order to find out the amnio acid residues required in enzymatic reactions,three amino acid residues,Asn70,Arg121 and Gln135,were site-directed mutated into Ala.The mutant enzymes N70A,R121A,and Q135A showed reduced enzyme activity in comparison with the native enzyme.More interestingly,the hydrolysis product of birchwood xylan by these mutant xylanases showed less proportion of xylobiose than that by the native enzyme.These results indicated that three amino acid residues were involved in xylanase activity and formation of xylobiose as the major production enzymatic hydrolysate. |
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