Cloning and expression of a novel chitinase gene from Citrobacter sp. P isolated in the intestines of large yellow croakers and characterization of its protein product, an exo-chitinase with broad pH stability

The gut microbiota of large yellow croaker (Pseudosciaena crocea) juveniles in cages fed formulated feed or trash-fish were characterized by 454 pyrosequencing as Proteobacteria and Fusobacteria of the main bacterial phyla. Chitinase gene (chi-X) from Citrobacter sp. P, a strain belongs to Proteobacteria and isolated from the digesta of trash-fish-fed individuals was cloned, expressed in Escherichia coli, and its protein product characterized. Chitinase CHI-X contains 493 amino acid residues with a predicted glycoside hydrolase family-18 catalytic domain. Purified CHI-X is most active at pH 4.0 and 60°C and is stable below 60°C, retaining 24.5-39.8% of its activity between 30-40°C, ~90% between pH 3.0 and 11.0. Its Km and Vmax is 6.24 mg/mL and 2.49 µmol/min, respectively. Its activity was stimulated by Mn2+ , Li+, and K+, and inhibited strongly by Ag+. Meanwhile, proteases had no effects on CHI-X activity.That CHI-X hydrolyzes colloidal chitin into GlcNAc and GlcNAc2 indicated its exo-chitinase characterization. CHI-X also degrades efficiently shrimp-shell chitin with the specific activity of 137.65 U/mg (the same as colloidal chitin), and showed significantly synergetic effect (P < 0.05) with ChiB565 (our previously published chitinase). Its lyophilized powder is stable. These findings indicate that CHI-X may adapt well to the environment of marine fish gastrointestines and, thus, could be potentially used by the aqua feed industry of warm water marine finfish as the single enzyme or the composite supplementation.

Identifier
Source https://data.blue-cloud.org/search-details?step=~012A637FDF63CA99AEB13614BBFC378305E58D801B2
Metadata Access https://data.blue-cloud.org/api/collections/A637FDF63CA99AEB13614BBFC378305E58D801B2
Provenance
Instrument 454 GS FLX; LS454
Publisher Blue-Cloud Data Discovery & Access service; ELIXIR-ENA
Contributor CAAS
Publication Year 2024
OpenAccess true
Contact blue-cloud-support(at)maris.nl
Representation
Discipline Marine Science
Spatial Coverage (119.310W, 26.390S, 119.310E, 26.390N)
Temporal Point 2012-12-15T00:00:00Z