The mannan endo-1,4-beta-mannosidase gene man26A from Aspergillus niger CBS 513.88 was optimized according to the codon usage bias in Pichia pastoris and synthesized by splicing overlap extension PCR. It was successfully expressed in P. pastoris using constitutive expression vector pGAPz alpha A. The recombinant endo-beta-1,4-mannanase could work in an extremely board temperature range and over 30% relative activity were retained in the temperature range of 5-60 degrees C. The optimal pH value and temperature for activity were 5.0 and 45 degrees C, respectively. It was highly thermotolerant with a half-life time of 15 min at 90 degrees C. A novel fed-batch strategy was developed successfully for high cell-density fermentation and mannanase activity reached 5069 U/mL after cultivation for 56 h in 50 L fermenter. The broad working temperature range, high thermotolerance and efficient expression made this enzyme possible to be applied in food, animal feed and the production of biofuels. (C) 2011 Elsevier Ltd. All rights reserved.