Comparison of chitinolytic enzymes from an alkaline, hypersaline lake and an estuary.
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.
LeCleir
Gary R
GR
Department of Marine Sciences, University of Georgia, Athens, GA, USA.
Buchan
Alison
A
Maurer
John
J
Moran
Mary Ann
MA
Hollibaugh
James T
JT
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
D012965:Sodium Chloride; D002688:Chitinases