Chitin in the marine environment: Genetic studies of a chito-oligomer hydrolyzing enzyme from Vibrio vulnificus, and the evolution of chitinolytic enzymes

Chitin, a homopolymer of {dollar}beta{dollar}-1,4 linked N-acetyl-D-glucosamine (NAG), is extremely abundant in nature. Each year billions of tons of chitin are produced by fungi, insects and crustaceans in terrestrial and aquatic habitats. Chitin is not only vital in the life cycle of these organisms, but it also represents an integral part of the food web, and plays an important role in the distribution and survival of bacteria in the environment. Methods for detecting chitinolytic bacteria were compared. Results indicated that the chitinolytic phenotype probably includes several heterologous genotypes, and that hydrolysis of chito-oligomer analogs can not be used to accurately predict hydrolysis of native chitin. Two novel methods are described for tracking these and other genes in environmental samples. The first method facilitates the isolation of chromosomal DNA, plasmid DNA, ribosomal RNAs, and messenger RNAs from aquatic samples without cell culture. The second method enhances the sensitivity afforded by small gene probes, thereby increasing their utility in the detection of rare molecules. Chitin hydrolysis in the marine bacterium Vibrio vulnificus involves a suite of enzymes which are inducible by chitin and chito-oligomers. The gene for one of these enzymes has been cloned from V. vulnificus; it is expressed consitutitively in Escherichia coli, and codes for the degradation of a wide range of chito-oligomers. The product of the cloned gene is 94 kDa in size, is predominantly hydrophilic, and probably remains cell associated in vivo. The activity of the cloned enzyme against both high and low molecular weight chito-oligomers suggests that it is best described as an N-acetyl-{dollar}beta{dollar}-hexosaminidase. The sequence of the hexosaminidase gene was determined, and the primary structure of the enzyme was inferred from the nucleotide sequence. The amino acid sequence indicates that the hexosaminidase of V. vulnificus is closely related to the chitobiase of V. harveyi, and that both are more closely related to human hexosaminidase than to plant chitinases. Chitin chemistry and commercial applications are reviewed. The numerous industrial, medical, and agricultural applications for chitin and chitin derivatives, indicate the scope and importance of chitin-related research.