Nucleotide sequence of the Clostridium stercorarium xynB gene encoding an extremely thermostable xylanase, and characterization of the translated product.
The nucleotides of the xynB gene of Clostridium stercorarium F-9 were sequenced. The structural gene consists of an open reading frame of 1161 bp encoding a xylanase (XynB) in family F of 387 amino acids with a molecular weight of 44,377. The molecular weight of the enzyme purified from a recombinant Escherichia coli was around 41,000, smaller than the predicted value, on SDS-polyacrylamide gel electrophoresis due to the lack of 32 amino acids at the N-terminus. Intact XynB with a molecular weight of around 43,000 was immunologically detected in the total cell proteins of a recombinant E. coli and C. stercorarium F-9. The purified XynB was active toward xylan, carboxymethylcellulose, p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-D-cellobioside. The pH optimum was 7.0 and it was quite stable over the pH range of 5 to 12 at 4 degrees C. This enzyme was optimally active at 80 degrees C and retained about 50% of the original activity even after incubation at 100 degrees C for 10 min.
Fukumura
M
M
Faculty of Bioresources, Mie University, Tsu, Japan.
Sakka
K
K
Shimada
K
K
Ohmiya
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005960:Glucosides / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry; D006027:Glycosides / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009598:Nitrophenylgalactosides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis / Q000235:genetics; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry
D005960:Glucosides; D006027:Glycosides; C008646:4-nitrophenyl beta-D-xyloside; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; C034170:4-nitrophenyl beta-cellobioside; D006026:Glycoside Hydrolases; D014995:Xylosidases; C034169:cellobiosidase; D001617:beta-Glucosidase; D043325:Xylan Endo-1,3-beta-Xylosidase; C026579:exo-1,4-beta-D-xylosidase