Beauveria bassiana chitinase (Bbchitl) is an important cuticle degrading enzyme involved in pathogenesis of fungi against insect. To obtain enough active chitinase for performing in vitro functional analysis, Bbehitl gene was expressed in Escherichia coli and Pichiapastoris, respectively. The high-level production of recombinant Bbchitl was detected in E coli expression system, however mainly located in inclusion bodies. Refolding of solubilized inclusion body proteins was achieved by dialysis. In P. pastoris expression system, Bbchitl was secreted into the culture medium under the induction of methanol. Active Bbchitl was purified to near 90% purity from culture medium by desalting chromatography and anion exchange chromatography. The yield of Bbchit I produced by P. pastoris was estimated at 153 mg/L, significantly higher than that of the refolded Bbchitl from E coli inclusion bodies (50 mg/L). Additionally, the specific activity of Bbchit] from P. pastoris was also higher than that from E. coli (3.9 U/mg versus 2.8 U/mg). These results indicated P. pastoris was a convenient expression system for the efficient production of Bbehit]. (c) 2007 Elsevier Inc. All rights. reserved.