High-level expression of a novel α-amylase from Thermomyces dupontii in Pichia pastoris and its application in maltose syrup production

Abstract

A novel α-amylase gene (TdAmyA) with an open reading frame of 1431 bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314 U/mL was obtained with protein content of 28.7 mg/mL after 168 h high-cell density fermentation. Molecular mass of purified TdAmyA was 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2 kDa by gel filtration. TdAmyA was a glycoprotein with 5.3% (w/w) of carbohydrate. TdAmyA exhibited maximal activity at 60 °C and pH 6.5, and was thermostable up to 55 °C within pH 4.5-10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. TdAmyA produced the highest maltose content of 51.8% after 8 h hydrolysis. Thus, TdAmyA might be a candidate α-amylase for maltose syrup production.

Keywords: Alpha-amylase; High-cell density fermentation; Maltose syrup; Thermomyces dupontii.