Purification and characterization of three thermostable endochitinases of a noble Bacillus strain, MH-1, isolated from chitin-containing compost.
A thermophilic and actinic bacterium strain, MH-1, which produced three different endochitinases in its culture fluid was isolated from chitin-containing compost. The microorganism did not grow in any of the usual media for actinomyces but only in colloidal chitin supplemented with yeast extract and (2, 6-O-dimethyl)-beta-cyclodextrin. Compost extract enhanced its growth. In spite of the formation of branched mycelia, other properties of the strain, such as the formation of endospores, the presence of meso-diaminopimelic acid in the cell wall, the percent G+C of DNA (55%), and the partial 16S ribosomal DNA sequence, indicated that strain MH-1 should belong to the genus Bacillus. Three isoforms of endochitinase (L, M, and S) were purified to homogeneity and characterized from Bacillus sp. strain MH-1. They had different molecular masses (71, 62, and 53 kDa), pIs (5.3, 4.8, and 4.7), and N-terminal amino acid sequences. Chitinases L, M, and S showed relatively high temperature optima (75, 65, and 75 degreesC) and stabilities and showed pH optima in an acidic range (pH 6.5, 5.5, and 5.5, respectively). When reacted with acetylchitohexaose [(GlcNAc)6], chitinases L and S produced (GlcNAc)2 at the highest rate while chitinase M produced (GlcNAc)3 at the highest rate. None of the three chitinases hydrolyzed (GlcNAc)2. Chitinase L produced (GlcNAc)2 and (GlcNAc)3 in most abundance from 66 and 11% partially acetylated chitosan. The p-nitrophenol (pNP)-releasing activity of chitinase L was highest toward pNP-(GlcNAc)2, and those of chitinases M and S were highest toward pNP-(GlcNAc)3. All three enzymes were inert to pNP-GlcNAc. AgCl, HgCl2, and (GlcNAc)2 inhibited the activities of all three enzymes, while MnCl2 and CaCl2 slightly activated all of the enzymes.
Sakai
K
K
Department of Applied Chemistry, Faculty of Engineering, Oita University, Oita 870-1192, Japan. sakai@cc.oita-u.ac.jp
Yokota
A
A
Kurokawa
H
H
Wakayama
M
M
Moriguchi
M
M
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification* / Q000201:enzymology* / Q000302:isolation & purification / Q000648:ultrastructure; D001673:Biodegradation, Environmental; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003470:Culture Media; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D010802:Phylogeny; D012037:Refuse Disposal; D013696:Temperature
D003470:Culture Media; D002686:Chitin; D002688:Chitinases