A process for efficient production of an alkaline beta-mannanases from Bacillus sp. N16-5 was established by heterologous expression using Pichia pastoris. A high producing strain was generated by removing the native beta-mannanases signal peptide and increasing the copy number of the mature beta-mannanases gene. High cell density fermentation of this strain in 1-L bioreactor led to a production level of 4164 U/mL after 9611 of induction. Sorbitol co-feeding and temperature-lowering strategies both increased the beta-mannanase production levels. Combined usage of these two strategies achieved the most effective result the enzyme level reached 6336 U/mL within 84 h, which to our best knowledge is the highest production level reported for the expression of extreme beta-mannanase thus far. The strategy described in this work can also be adapted to express other important industrial enzymes with extreme properties. (C) 2011 Elsevier Inc. All rights reserved.