A Thermotolerant and Cold-active Mannan Endo-1,4-β-mannosidase from Aspergillus Niger CBS 513.88: Constitutive Overexpression and High-density Fermentation in Pichia Pastoris

The mannan endo-1,4-β-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 pGAPzαA. 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°C. The optimal pH value and temperature for activity were 5.0 and 45°C, respectively. It was highly thermotolerant with a half-life time of 15min at 90°C. A novel fed-batch strategy was developed successfully for high cell-density fermentation and mannanase activity reached 5069U/mL after cultivation for 56h in 50L 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.