title: Highly thermostable and surfactant-activated chitinase from a subseafloor bacterium, Laceyella putida.
abstract: A novel chitinase (LpChiA) was purified to homogeneity from a culture of Laceyella putida JAM FM3001. LpChiA hydrolyzed colloidal chitin optimally at a pH of 4 in an acetate buffer and temperature of 75 ºC. The enzyme was remarkably stable to incubation at 70 ºC up to 1 h at pH 5.2, and its activity half-life was 3 days. The molecular mass of the enzyme was around 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and around 75 kDa by gel filtration, suggesting it is a homodimer. The enzyme activity was enhanced about 60% when pre-incubated with anionic, cationic, and nonionic surfactants. The gene for LpChiA was cloned by PCR and sequenced. The nucleotide sequence of the gene consisted of 1,683 bp encoding 560 amino acids. The N-terminal and internal amino acid sequences of the purified LpChiA from L. putida suggested that the mature enzyme was composed of 384 amino acids after cleaving its 176 N-terminal amino acids and dimerized to express its activity. The deduced amino acid sequence of the mature enzyme showed the highest similarity to chitinase of Laceyella sacchari with 79% identity.
authors.affiliation: Graduate School of Engineering, Toyo University, 2100 Kujirai, Kawagoe, Saitama, 350-8585, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Shibasaki
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Uchimura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Miura
authors.forename: Tohru
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Ron
authors.initials: R
authors.lastname: Usami
authors.forename: Koki
authors.initials: K
authors.lastname: Horikoshi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D008386:Marine Biology; D008970:Molecular Weight; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013501:Surface-Active Agents; D002688:Chitinases