The long lag time in basal salts medium (BSM) and an occurrence of proteolysis are major problems for recombinant protein production in Pichia pastoris KM71. In this study, optimal conditions were explored for fed-batch cultivation of recombinant fungal endoglucanase in P. pastoris KM71. It was found that lag and process times were much reduced when the synthetic FM22 medium was used for the inoculum compared with enriched buffered glycerol complex (BMGY) medium. The highest endoglucanase activity was obtained at 30 degrees C which was more than 10 fold higher than that produced from shake flask. At 30 degrees C, the specific endoglucanase activity was dependent on culture pH and a higher specific activity was observed at pH 5.0 than at pH 6.0. The higher activity was likely due to lower rate of proteolysis, since a truncated protein species was apparent at pH 6.0, but not pH 5.0. Thus, production of endoglucanase at 30 degrees C and pH 5.0 is the optimal condition suitable for economical production in large scale. The combination of using synthetic FM22 medium for inoculum and proteolysis control by growth at lower pH could be applied for production of other recombinant proteins in P. pastoris. (c) 2013, The Society for Biotechnology, Japan. All rights reserved.