Removal of bacterial dextran in sugarcane juice by Talaromyces minioluteus dextranase expressed constitutively in Pichia pastoris

A gene construct encoding the mature region of Talaromyces minioluteus dextranase (EC 3.2.1.11) fused to the Saccharomyces cerevisiae SUC2 signal sequence was expressed in Pichia pastoris under the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP). The increase of the transgene dosage from one to two and four copies enhanced proportionally the extracellular yield of the recombinant enzyme (r-TmDEX) without inhibiting cell growth. The volumetric productivity of the four-copy clone in fed batch fermentation (51 h) using molasses as carbon source was 1706 U/L/h. The secreted N-glycosylated r-TmDEX was optimally active at pH 4.5-5.5 and temperature 50-60 degrees C. The addition of sucrose (600 g/L) as a stabilizer retained intact the r-TmDEX activity after 1-h incubation at 50-60 degrees C and pH 5.5. Bacterial dextran in deteriorated sugarcane juice was completely removed by applying a crude preparation of secreted r-TmDEX. The high yield of r-TmDEX in methanol-free cultures and the low cost of the fed batch fermentation make the P. pastoris pGAP-based expression system appropriate for the large scale production of dextranase and its use for dextran removal at sugar mills.

Automated summary

The gene construct encoding Talaromyces minioluteus dextranase (r-TmDEX) was expressed in Pichia pastoris with an increase in transgene dosage resulting in higher extracellular yield of the enzyme. The optimal activity of the N-glycosylated r-TmDEX was observed at pH 4.5-5.5 and temperature 50-60 degrees C, while the addition of sucrose as a stabilizer maintained enzyme activity under harsh conditions. The use of the P. pastoris pGAP-based expression system proved to be suitable for large-scale production of dextranase and its application in dextran removal.