BACKGROUND: This work investigated the monokaryogenesis of dikaryon strains of Trametes hirsuta by protoplasts regeneration for extracellular ligninolytic enzyme production. Saccharification of corn straw was enhanced by alkali pretreatment combining crude ligninolytic enzymes. RESULTS: Effectiveness of alkali pretreatment of corn straw on delignification was evaluated under different concentrations. About 45% lignin loss was achieved at the concentration of 1.5% NaOH. In addition, 79.0% sugar yield was obtained after combined pretreatment with NaOH and crude ligninolytic enzyme produced from monokaryotic strains of Trametes hirsuta. Scanning electron microscopy (SEM) images showed that the porosity and surface area increased significantly after combined pretreatment. The FTIR spectra indicated that great intensity changes occurred at the 890900 cm-1, 15091513 cm-1 and 1595 cm-1 bands. CONCLUSION: The proposed combined pretreatment removes lignin and enhances saccharification of corn straw effectively. Copyright (c) 2012 Society of Chemical Industry