title: Comparison of chitinolytic enzymes from an alkaline, hypersaline lake and an estuary.
abstract: We examined the genetic and physiological characteristics of chitin degrading enzymes expressed by fosmids cloned from two strains of chitinolytic gammaproteobacteria isolated from alkaline, hypersaline Mono Lake, California; and from a metagenomic library derived from an estuarine bacterial community (Dean Creek, Sapelo Island, GA, USA). The Mono Lake chitinolytic enzymes presented unique adaptations in terms of halo- and alkalitolerance. The sequence from one of the Mono Lake isolates (strain 12A) was a conventional family 18 glycosyl hydrolase; however, the expressed protein had a novel secondary activity peak at pH 10. We obtained a novel family 20 glycosyl hydrolase sequence from Mono Lake strain AI21. The activity of the expressed protein had a pH optimum of 10, several pH units higher than any other enzyme currently assigned to this family, and the enzyme retained 80% of its activity at pH 11. The enzyme was also halotolerant, retaining activity in salt solutions of up to 225 g l(-1). Sequence analysis indicated a molecular weight of approximately 90 kDa for the protein, and that it contained two active sites. Culture supernatant contained two chitinolytic proteins, 45 and 31 kDa, suggesting possible post-expression modification of the gene product. In contrast, the sequence found in the estuarine metagenomic library and the functional characteristics of the protein expressed from it were those of a conventional family 18 glycosyl hydrolase.
authors.affiliation: Department of Marine Sciences, University of Georgia, Athens, GA, USA.
authors.forename: Gary R
authors.initials: GR
authors.lastname: LeCleir
authors.forename: Alison
authors.initials: A
authors.lastname: Buchan
authors.forename: John
authors.initials: J
authors.lastname: Maurer
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Moran
authors.forename: James T
authors.initials: JT
authors.lastname: Hollibaugh
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics*; D002140:California; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005618:Fresh Water / Q000382:microbiology*; D015723:Gene Library; D019015:Geologic Sediments / Q000382:microbiology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D012623:Seawater / Q000382:microbiology*; D016415:Sequence Alignment; D012965:Sodium Chloride
chemical_list: D012965:Sodium Chloride; D002688:Chitinases