An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis.
We provide evidence that chitinase A from Vibrio carchariae acts as an endochitinase. The chitinase A gene isolated from V. carchariae genome encodes 850 amino acids expressing a 95-kDa precursor. Peptide masses of the native enzyme identified from MALDI-TOF or nanoESIMS were identical with the putative amino acid sequence translated from the corresponding nucleotide sequence. The enzyme has a highly conserved catalytic TIM-barrel region as previously described for Serratia marcescens ChiA. The Mr of the native chitinase A was determined to be 62,698, suggesting that the C-terminal proteolytic cleavage site was located between R597 and K598. The DNA fragment that encodes the processed enzyme was subsequently cloned and expressed in Escherichia coli. The expressed protein exhibited chitinase activity on gel activity assay. Analysis of chitin hydrolysis using HPLC/ESI-MS confirmed the endo characteristics of the enzyme.
Suginta
Wipa
W
School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. wipa@ccs.sut.ac.th
Vongsuwan
Archara
A
Songsiriritthigul
Chomphunuch
C
Prinz
Heino
H
Estibeiro
Peter
P
Duncan
Rory R
RR
Svasti
Jisnuson
J
Fothergill-Gilmore
Linda A
LA
D000595:Amino Acid Sequence; D002686:Chitin / Q000737:chemistry* / Q000378:metabolism*; D002688:Chitinases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D020539:Sequence Analysis, Protein / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D002686:Chitin; D002688:Chitinases; C094590:chitinase ChiA