Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells.
Bacillus licheniformis CBFOS-03 is a chitinase producing bacteria isolated from oyster (Crassostrea gigas) shell waste. We have cloned and expressed the chi18B gene of B. licheniformis CBFOS-03, which encodes a glycohydrolase family 18 chitinase (GH18). Chi18B is a predicted 598 amino acid protein that consists of a catalytic domain (GH18), a fibronectin type III domain (Fn3), and a chitin binding domain (CBD). Purified Chi18B showed optimum chitinase activity at pH 9 and 55 degrees C, and activity was stimulated with 25 mM Mn2+. In kinetic analysis, Chi18B showed Km values of 9.07 +/- 0.65 microM and 129.27 +/- 0.38 microM with the substrates 4-methylumbelliferyl-N-N'-diacetylchitobiose and alpha-chitin, respectively. Studies of C-terminal deletion constructs revealed that the GH18 domain with one amino acid in C-terminal region was sufficient for chitinase activity; however, fusions of full length and CBD-deleted constructs to green florescent protein (GFP) and yellow florescent protein (YFP) suggest that the C-terminus is supposedly important in binding to shell powder. Full length Chi18B with GFP showed green fluorescence with oyster shell powder, but GH18+Fn3 with GFP did not. Similarly, full length Chi18B with YFP showed yellow fluorescence with clam (Chamelea gallina) shell and disk abalone (Haliotis discus) shell powder, but GH18+Fn3 with YFP construct did not. So, the CBD domain of Chi18B appears to play an important role in binding of oyster and other marine shells. It is likely to be used as a probe to identify the presence of chitin in marine shells like oyster shell, clam shell, and disk abalone shell using fusions of Chi18B with fluorescent proteins.
Islam
Shah Asraful
SA
Division of Applied Life Science (BK21 Program), Gyeongsang National University, Chinju 660701, Republic of Korea.
Cho
Kye Man
KM
Hong
Sun Joo
SJ
Math
Renukaradhya K
RK
Kim
Jong Min
JM
Yun
Myoung Geun
MG
Cho
Ji Joong
JJ
Heo
Jae Young
JY
Lee
Young Han
YH
Kim
Hoon
H
Yun
Han Dae
HD
D000818:Animals; D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001709:Biotechnology / Q000379:methods*; D020134:Catalytic Domain; D002686:Chitin / Q000032:analysis* / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D005353:Fibronectins / Q000378:metabolism; D007700:Kinetics; D008386:Marine Biology; D008974:Mollusca / Q000737:chemistry*; D010125:Ostreidae / Q000737:chemistry / Q000201:enzymology; D011485:Protein Binding
D005353:Fibronectins; D002686:Chitin; D002688:Chitinases