ena-STUDY-CAAS-12-10-2013-01:37:25:513-401
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.
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.