Molecular characterization of a new acetyl xylan esterase (AXEII) from edible straw mushroom Volvariella volvacea with both de-O-acetylation and de-N-acetylation activity.
A new Volvariella volvacea gene encoding a carbohydrate esterase (CE) family 4 acetyl xylan esterase (AXE) (designated as VvaxeII) was cloned and characterized. The coded polypeptide had 253 amino acid residues, with the first 19 serving as a secretion signal peptide. The VvaxeII transcript levels were high when the fungus was grown on oat spelt xylan, cellobiose, microcrystalline cellulose, carboxymethyl-cellulose, lactose, galactose, and chitin from crab as carbon sources. The recombinant VvAXEII produced by expression of VvaxeII in Pichia pastoris exhibited activity toward acetylated oat spelt xylan and various chitinous substrates, but was totally inactive against artificial aromatic acetates such as beta-nitrophenyl, 4-methylumbelliferyl, and alpha-naphthyl acetates. Enzyme-catalyzed hydrolysis was maximal at pH 7.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 1.42 mg mL(-1) and a V(max) value of 833 IU micromol(-1) protein using glycol chitin as a substrate. The recombinant VvAXEII requires activation by bivalent cations such as Co2+ and Mg2+. Interestingly, the recombinant VvAXEII showed no deacetylation activity to fully acetylated monosaccharides such as xylose tetraacetate.
Liu
Xiufeng
X
Department of Biological Engineering, Nanjing Forestry University, Nanjing, Jiangsu, China.
Ding
Shaojun
S
D000115:Acetylesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002686:Chitin / Q000031:analogs & derivatives / Q000378:metabolism; D003001:Cloning, Molecular; D003035:Cobalt / Q000494:pharmacology; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000494:pharmacology; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020869:Gene Expression Profiling; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D055367:Volvariella / Q000201:enzymology* / Q000235:genetics
D004271:DNA, Fungal; D020536:Enzyme Activators; D005656:Fungal Proteins; D021382:Protein Sorting Signals; D002686:Chitin; D003035:Cobalt; C031610:glycolchitin; D000115:Acetylesterase; C081851:acetylxylan esterase; D008274:Magnesium