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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 phenotype was found to produce 1,480.2 ± 340.9 U ChoA mL 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. [ABSTRACT FROM AUTHOR]