Dipeptidyl peptidase IV from Xanthomonas maltophilia: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.
The dipeptidyl peptidase IV [EC 3.4.14.5] gene of Xanthomonas maltophilia, expressed in Escherichia coli, was cloned by the shotgun method. Nucleotide sequence analysis revealed an open reading frame of 2,223 bp, coding for a protein of 741 amino acids with a predicted molecular weight of 82,080. The expressed enzyme was extracted with SDS, and the solubilized enzyme was purified about 1,030-fold on columns of Toyopearl HW65C, DEAE-Toyopearl twice, and hydroxyapatite, with an activity recovery of 50%. The enzyme hydrolyzed a proline-containing peptide at the penultimate position, and was inhibited by diisopropyl phosphofluoridate. The enzyme was most active at pH 8.5, and was stable between at pH 7.0 and 9.0. The molecular weight of the purified enzyme was estimated to be 83,000 and 165,000 by SDS-PAGE and gel filtration, respectively.
Kabashima
T
T
School of Pharmaceutical Sciences, Nagasaki University. t-kabashima@cc.nagasaki-u.ac.jp
Ito
K
K
Yoshimoto
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011487:Protein Conformation; D015183:Restriction Mapping; D013379:Substrate Specificity; D014974:Xanthomonas
D018819:Dipeptidyl Peptidase 4