The processing of high-molecular-weight xylanase (XynE, 110 kDa) from Aeromonas caviae ME-1 to 60-kDa xylanase (XynE60) in Escherichia coli and purification and characterization of XynE60.
A xylanase gene (xynE) encoding XynE (110 kDa) was cloned from a lambda phage genomic library of Aeromonas caviae ME-1 which is a multiple-xylanase-producing bacterium. Upon nucleotide sequence analysis, we found that xynE comprises 2823 by and encodes a protein of 941 amino acid residues (104,153 Da), which was similar to endo-beta-1,4-xylanases which are categorized to glycosyl hydrolase family 10. An Escherichia coli transformant that harbored pXED30 carrying xynE produced 110-, 84-, 72-, and 66-kDa xylanases in the cell-free extract, and 72- and 66-kDa xylanases in the culture supernatant. We purified the 66-kDa xylanase to electrophoretic homogeneity from a culture supernatant by a series of column chromatographies. The calculated molecular mass of the purified xylanase determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was 60,154.50 Da, and the xylanase was designated XynE60. Analysis of the N-terminal 10 amino acid residues and the determined molecular mass of XynE60 revealed that XynE60 is a product processed at the Gly26-Gly27, and Thr565-Ala566 sites of XynE by proteolytic cleavage. XynE60 showed optimal activity at 55 degrees C and pH 8.0, and was stable below 45 degrees C and at pH 7.0-8.5. The K(m) and V(max) of XynE60 were calculated to be 8.1 mg/ml and 6897 nkat/mg, respectively.
Liu
Chen Jian
CJ
United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Suzuki
Tohru
T
Hirata
Satoru
S
Kawai
Keiichi
K