Cloning of the 52-kDa chitinase gene from Serratia marcescens KCTC2172 and its proteolytic cleavage into an active 35-kDa enzyme.
A chitinase gene (pCHI52) encoding the 52-kDa chitinase was isolated from a Serratia marcescens KCTC2172 cosmid library. This chitinase gene consists of 2526 bp with an open reading frame that encodes 485 amino acids. Escherichia coli harboring the pCHI52 gene secreted not only a 52-kDa but also a 35-kDa chitinase into the culture supernatant. We purified both 52-kDa and 35-kDa chitinases using a chitin affinity column and Sephacryl-S-300 gel filtration chromatography. We determined that the 17 N-terminal amino acid sequences of the 52-kDa and the 35-kDa chitinase are identical. Furthermore, a protease obtained from S. marcescens KCTC2172 cleaved the 52-kDa chitinase into the 35-kDa protein with chitinase activity. These results suggest that the 35-kDa chitinase derives from the 52-kDa chitinase by post-translational proteolytic modification. The optimal reaction temperature of 45 degrees C and the optimal pH of 5.5 were identical for both enzymes. The specific activities of the 52-kDa and 35-kDa chitinases on natural swollen chitin were 67 mumol min-1 mg-1 and 60 mumol min-1 mg-1, respectively.
Gal
S W
SW
Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, South Korea.
Choi
J Y
JY
Kim
C Y
CY
Cheong
Y H
YH
Choi
Y J
YJ
Lee
S Y
SY
Bahk
J D
JD
Cho
M J
MJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002688:Chitinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D017353:Gene Deletion; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012706:Serratia marcescens / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D007527:Isoenzymes; D002688:Chitinases; D010450:Endopeptidases