Cloning and enzymatic characterization of a xylanase gene from a soil-derived metagenomic library with an efficient approach.
Screening interesting biocatalysts directly from soil samples is a more convenient and applicable approach than conventional cultivation-dependent ones. In our present work, a soil-derived metagenomic library containing 24,000 transformants was constructed with an efficient strategy for cloning xylanase genes. A gene encoding the enzyme (XynH) able to hydrolyze xylan was obtained. Similarity analysis revealed that this enzyme is a new member in the family 10 of xylanases. The molecular mass of XynH purified from Escherichia coli was estimated to be 39 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. It was found to display the maximal activity at lower temperature, under weakly alkaline conditions, different from most of xylanases. The K(m) and V(max) values of XynH with birchwood xylan as substrate are 7.5 mg/ml and 190 micromol min(-1 )mg(-1), respectively. It is greatly interesting to note that the activity of XynH was not reduced significantly by Mn(2+), Zn(2+), Co(2+), Ag(+), and Cu(2+), even at the concentration of 5 mM, which strongly inhibits most of the other xylanases studied previously.
Hu
Yong
Y
The College of Life Sciences, Hubei University, Wuhan 430062, China.
Zhang
Guimin
G
Li
Aiying
A
Chen
Jing
J
Ma
Lixin
L
D003001:Cloning, Molecular / Q000379:methods*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015698:Genomic Library; D007700:Kinetics; D008969:Molecular Sequence Data; D012988:Soil Microbiology
D043364:Endo-1,4-beta Xylanases