Fusion expression of β-mannanase Man5A and xylanase Tlxyn11B in Pichia pastoris

Abstract

Mannanase and xylanase, the main hemicellulolytic enzymes, are widely used in food, feed, textile and papermaking industries, and usually they are used in combination. Mannanase Man5A from Talaromyces leycettanus JCM12802 consist of the carbohydrate binding module (CBM), linker region and catalytic domain. The CBM coding region of Man5A was removed and fused to C-terminal of the xylanase gene Tlxyn11B. The fusion gene Tlxyn11B-linker-man5A was successfully expressed in Pichia pastoris and the fusion protein Tlxyn11B-Man5A was purified and characterized. The theoretical molecular weights of Tlxyn11B, Man5A without CBM region, and Tlxyn11B-Man5A are 21.6 kDa, 41.0 kDa, and 62.6 kDa, respectively. The fusion protein had high xylanase and mannanase activities. The optimal temperature of the fused xylanase is 70 °C, which is 5 °C higher than Tlxyn11B-w (xylanase before fusion). The fused mannanase exhibited maximal activity at 90 °C, which is similar to Man5A-w (mannanase before fusion). More than 48% of xylanase activity of Tlxyn11B-Man5A was residual after the condition of 60 °C with 1 h, which is significantly higher than Tlxyn11B-w (only 20% of activity was left at 60 °C for 20 minutes). The optimal pHs of Tlxyn11B-Man5A for xylanase and mannanase activity are 4.0 and 5.0, respectively, which are 0.5 and 1.0 units higher than those of Tlxyn11B-w and Man5A-w. The pH range of fused enzymes got wider and the pH stability is improved. The specific activities of xylanase and mannanase of Tlxyn11B-Man5A are 1 784.3 U/mg and 1 639.6 U/mg, respectively, which is lower than those of Tlxyn11B and Man5A (8 300.0 U/mg and 1 979.0 U/mg). It may be due to of the high molecular weight of fusion enzyme. The Km and Vmax of the fused xylanase and mannanase are 1.2 mg/mL and 1.7 mg/mL, 2 000.0 μmol/(min·mg) and 2 831.6 μmol/(min·mg), respectively. Tlxyn11B and Man5A were successfully fusion expressed in P. pastoris, and the good properties of fusion of xylanase and mannanase make it has great application potential in animal feed, food and other industrial production, and it provided new ideas for the improvement of enzyme performance.