Characterization of xyn10J, a novel family 10 xylanase from a compost metagenomic library.
A gene encoding an extracellular xylanase was cloned from a compost metagenomic library. The xylanase gene, xyn10J, was 1,137 bp in length and was predicted to encode a protein of 378 amino acid residues with a putative signal peptide of 27 amino acid residues. The molecular mass of the mature Xyn10J was calculated to be 39,882 Da with a pI of 6.09. Xyn10J had a motif GVKVHFTEMDI characteristic of most members of glycosyl hydrolase family 10. The amino acid sequence of Xyn10J showed 60.0% identity to that of XynH, a xylanase from an uncultured soil bacterium and 55% identity to XylC of Cellvibrio mixtus. Site-directed mutagenesis of the expected active site based on the sequence analysis indicated that an aspartic acid residue (Asp207), in addition to the identified catalytic residues Glu165 and Glu270, plays a crucial role for the catalytic activity. The purified Xyn10J had a mass of about 40 kDa and was optimally active at pH 7.0 and 40 °C. Xyn10J hydrolyzed beechwood xylan > birchwood xylan > oat spelt xylan > arabinoxylan. Xyn10J hydrolyzed xylotetraose and xylohexaose exclusively to xylobiose, xylopentaose, and xylotriose mainly to xylobiose with transglycosylation activity. The saccharification of reed (Phragmites communis) powder by commercial enzymes was significantly increased by the addition of a small amount of Xyn10J to the commercial preparation. Xyn10J is the first xylanase screened directly from a compost metagenomic library, and the enzyme has the potential to be used in the conversion of biomass to fermentable sugars for biofuel production.
Department of Agricultural Chemistry, Sunchon National University, Suncheon, Republic of Korea.
Yu Seok
YS
Jeong
Han Beur
HB
Na
Sung Kyum
SK
Kim
Yong Ho
YH
Kim
Eun Ju
EJ
Kwon
Jungho
J
Kim
Han Dae
HD
Yun
Jung-Kul
JK
Lee
Hoon
H
Kim
D000595:Amino Acid Sequence; D018533:Biomass; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019143:Evolution, Molecular; D005285:Fermentation; D015723:Gene Library; D006031:Glycosylation; D056186:Metagenomics; D008969:Molecular Sequence Data; D010802:Phylogeny; D012988:Soil Microbiology
D002482:Cellulose; D043364:Endo-1,4-beta Xylanases