Chitinases from Uncultured Marine Microorganisms

Abstract

Our understanding of the degradation of organic matter will benefit from a greater appreciation for the genes encoding enzymes involved in the hydrolysis of biopolymers such as chitin, one of the most abundant polymers in nature. To isolate representative and abundant chitinase genes from uncultivated marine bacteria, we constructed libraries of genomic DNA isolated from coastal and estuarine waters. The libraries were screened for genes encoding proteins that hydrolyze a fluorogenic analogue of chitin, 4-methylumbelliferyl β-d-N,N′-diacetylchitobioside (MUF-diNAG). The abundance of clones capable of MUF-diNAG hydrolysis was higher in the library constructed with DNA from the estuary than in that constructed with DNA from coastal waters, although the abundance of positive clones was also dependent on the method used to screen the library. Plaque assays revealed nine MUF-diNAG-positive clones of 75,000 screened for the estuarine sample and two clones of 750,000 for the coastal sample. A microtiter plate assay revealed approximately 1 positive clone for every 500 clones screened in the coastal library. The number of clones detected with the plaque assay was consistent with estimates of the portion of culturable bacteria that degrade chitin. Our results suggest that culture-dependent methods do not greatly underestimate the portion of marine bacterial communities capable of chitin degradation.

Use of a chiA probe for detection of chitinase genes in bacteria from the Chesapeake Bay

“…Up to 30% of total culturable heterotrophic bacteria have previously been reported to be chitinoclastic on phenotypic examination [7]. In a recent study on chitinases from uncultured marine microorganisms, the estimates of the per-centages of chitin degraders for the estuarine and coastal communities were 5.5 and 0.12%, respectively [35]. Chitinase activity can be much higher on particles than in the water column [36].…”

Kinetic and crystallographic analyses of the catalytic domain of chitinase from Pyrococcus furiosus– the role of conserved residues in the active site

“…deviation of 0.10 Å , and they made a sharp turn at NAG3. Although NAG1, NAG2, NAG4 and NAG5 residues adopted standard 4 C 1 chair conformations, the central NAG3 residue was distorted into the 1,4 B boat conformation. In addition, the dihedral angles of the third glycosidic bond (NAG3-NAG4) were very different from those of the other glycosidic bonds (NAG1-NAG2, NAG2-NAG3 and NAG4-NAG5) ( Table 3).…”

Functional screening of a metagenomic library for genes involved in microbial degradation of aromatic compounds

“…One advantage of this approach is that it can, in theory, accelerate the identification of novel genes with industrial value. For example, efforts have been made to retrieve genes such as chitinase, dehydrogenase, oxidoreductase, amylase, esterase, endoglucanase and cyclodextrinase from metagenomic libraries by functional screening (10 4 -10 5 ; Cottrell et al, 1999;Henne et al, 2000;Knietsch et al, 2003;Yun et al, 2004;Ferrer et al, 2005).…”