Abstract: The xylose metabolic fusants were prepared by fusion of inactivated parental protoplasts with Pichia stipitis 1960(Ps1960) and Saccharomyces cerevisiae AADY(ScAADY) as parental strains. And the preparation conditions were optimized. The results showed that the Ps1960 protoplast was prepared by enzymolysis with 2% snailase and 1% cellulase for 45 min at 28℃, and then inactivation with 20 W ultroviolet lamp 10 cm away from the culture for 3 min. While ScAADY protoplast was prepared by enzymolysis with 1.5% snailase and 1% cellulase for 50 min at 28℃, and inactivation in water bath for 50 min at 55℃. And 0.6 mol/L sorbitol was used as osmotic stabilizer. And then, 22 fusants were obtained. The biomasses of the fusants under different culture medium conditions were determined to evaluate the ability of metabolism of xylose and ethanol tolerance. Thus, from 22 fusants, the fusant D2, which could ferment ethanol from xylose, with good hereditary feature was selected for ethanol fermentation. Furtherly, fusant D2 could produce 40.58 g/L ethanol under the conditions of 8% mixed sugar, mass ratio of xylose and glucose 6:1, inoculum size of 5% and cultured for 72 h with shaking 160 r/min at 30℃.

Key words: Saccharomyces cerevisiae, Pichia stipitis, protoplast fusion, inactivation, bioethanol