title: A broad pH range and processive chitinase from a metagenome library.
abstract: Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N'-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.
authors.affiliation: Departmento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: S S
authors.initials: SS
authors.lastname: Thimoteo
authors.affiliation: Departmento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.|Agência de Inovação, Instituto de Tecnologia do Paraná - Tecpar, Curitiba, PR, Brasil.
authors.forename: A
authors.initials: A
authors.lastname: Glogauer
authors.affiliation: Departmento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.|Instituto Carlos Chagas, Fiocruz, Curitiba, PR, Brasil.
authors.forename: H
authors.initials: H
authors.lastname: Faoro
authors.affiliation: Departmento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: E M
authors.initials: EM
authors.lastname: de Souza
authors.affiliation: Departmento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: L F
authors.initials: LF
authors.lastname: Huergo
authors.affiliation: Institute for Biology and Biotechnology of Plants, WWU Münster University, Münster, Germany.
authors.forename: B M
authors.initials: BM
authors.lastname: Moerschbacher
authors.affiliation: Departmento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.
authors.forename: F O
authors.initials: FO
authors.lastname: Pedrosa
mesh_terms: D002686:Chitin / Q000737:chemistry / Q000235:genetics*; D002688:Chitinases / Q000737:chemistry / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli; D015870:Gene Expression / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D054892:Metagenome / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D002686:Chitin; D002688:Chitinases