Recombination Expression And Enzymatic Characterization Of Acetate Xylan Esterase In Volvariella Volvacea

The genes encoding acetyl xylan esterase (AXE) has been cloned and sequenced from the edible mushroom V. volvacea. In order to achieve high-level expression in Pichia pastoris, the Codon was optimized according to the codon preference of P. pastoris. We synthesized twenty-six overlapping oligonucleotides and constructed the full-length AXE gene by PCR procedure. The full-length cDNA of AXE (GenBank No. DQ888226) consisted of 1363 bp encoding 349 amino acids with a calculated mass of 39,990 Da. The recombinant gene expressed in yeast Pchia pastoris, and we studied the influence of the different culture conditions to the activity of recombinant acetyl xylan esterase, which include the expression condition: two medium (BMMH and BMMY), different concentration of YNB ,cell density and initial pH.The expression condition optima for reAXE are: medium BMMY, 100%YNB,inocula(OD₆₀₀=30), initial pH6.0, 1.0%(V/V) methanol concentration. The recombinant protein was purified in a one-step procedure by affinity chromatography using Ni-NTA Agarose gel,and SDS-PAGE analysis revealed that the purified reAXE migrated as two bands with molecular masses of 48 kDa and 66 kDa, after treatment by endoglycosidase H (endo H), only one protein band with molecular masses of 45 kDa was observed. As determined by a 2-min assay with 4-methylumbelliferyl acetate as the substrate, the optimal activity of acetyl xylan esterases occurred at pH 8.0 and 50℃, and the enzyme had a Km of 307.69μM and a Vmax of 769.23IU/μM,respectively when 4-methylumbelliferyl acetate was used as the substrate. Hydrolysis assays with different acetylated substrates showed that the enzyme is specific for deacetylating the O-acetyl group of polysaccharides: acetyl xylan, tetra-acetate xylose andα-naphthyl acetate. The highest specific activity recorded for tetra-acetate xylose (52450 IU/μmol).α-Naphthyl acetate was a poor substrate for the enzyme and was hydrolyzed at only 1.8% of the rate observed with tetra-acetate xylose.Binding assay with different substrates showed the binding character of the enzyme,reAXE also exhibited a capacity to bind to avicel(the residual reAXE activities in the supernatant decreased to 77.3%) and H4PO3 acid swollen cellulose(the residual reAXE activities in the supernatant decreased to 12.0%). No significant adsorption of reAXE to insoluble oat spelt xylan(the residual reAXE activities in the supernatant decreased to 92.2%) was observed.