PMID:22322871

Citation	Peng, N, Xu, W, Wang, F, Hu, J, Ma, M, Hu, Y, Zhao, S, Liang, Y and Ge, X (2013) Mitsuaria chitosanase with unrevealed important amino acid residues: characterization and enhanced production in Pichia pastoris. Appl. Microbiol. Biotechnol. 97:171-9
Abstract	A chitosan plate assay was employed to screen for chitosanase-producing bacterial strains and isolate 141 was found to exhibit high activity. Characterization of this isolate revealed that it belonged to Mitsuaria (designated as Mitsuaria sp. 141). The encoded chitosanase (choA) gene was then cloned by PCR and the deduced amino acid sequence showed 98% identity to a formerly described Mitsuaria chitosanitabida 3001 ChoA (McChoA). Surprisingly, the ChoA encoded by Mitsuaria sp. 141 (MsChoA) appeared to have a much higher optimum temperature compared to McChoA. Site-directed mutagenesis was then employed to generate five MschoA mutant genes encoding MsChoA K204Q, R216K, T222N, R216K/T222N, or K204Q/R216K/T222N. All the ChoA mutants exhibited a much lower specific activity and a lower optimum temperature. The results confirmed that the substitution of three non-conserved amino acids accounts for the major reduction of the enzyme activity in MsChoA. Furthermore, the MschoA gene was cloned for over-expression in Pichia pastoris after coding sequence optimization. One of the P. pastoris transformants with Mut(S) phenotype was found to produce 1,480.2 ± 340.9 U ChoA mL(-1) of cell culture by high-cell-density fermentation. This represents the highest yield of recombinant ChoA production that has ever been reported thus far. The recombinant P. pastoris strain should therefore be well suited for industrial-scale production of chitosanase.
Links	PubMed Online version:10.1007/s00253-012-3901-y
Keywords	Amino Acids/genetics; Betaproteobacteria/enzymology; Betaproteobacteria/genetics; Chitosan/metabolism; Cloning, Molecular; DNA, Bacterial/chemistry; DNA, Bacterial/genetics; Enzyme Stability; Glycoside Hydrolases/chemistry; Glycoside Hydrolases/genetics; Glycoside Hydrolases/metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutant Proteins/genetics; Mutant Proteins/metabolism; Pichia/genetics; Polymerase Chain Reaction; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Temperature
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
9BURK:G4WUC8		GO:0016977: chitosanase activity	ECO:0000314:		F		Fig. 5	complete CACAO 8934

References

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