Enhanced soluble expression of a thermostble β-glucosidase from Thermotoga maritima in Escherichia coli and its applicaton in immobilization

Abstract

The β-glucosidase Tm-bglA gene from hyperthermophile Thermotoga maritima was cloned into expression vectors pET-28a, producing two proteins of 55 kDa and 52 kDa in cell, which could be referred to Tm-BglA with and without 23 amino acids. The results showed that fusion of 23 amino acids to Tm-BglA improved its soluble expression and product tolerance, resulting over 60% yield of recombinant Tm-BglA secreted into the growth medium in Escherichia coli JM109 (DE3). Tm-BglA with 23 amino acids had higher k cat and K M values for p-nitrophenyl-β-D-glycopyranoside and much stronger product inhibition than Tm-BglA without 23 amino acids. Subsequently, the Tm-BglA was immobilized on chitin efficiently by genetically fusing the chitin-binding domain of chitinase A1 from Thermoanaerobacterium thermosaccharolyticum DSM571. The immobilized Tm-BglA had higher optimal temperature (95°C) and was more thermostable at the range from 75 to 100°C than its free form. This immobilized protein exhibited high stability and the conversion yield exceeding 90%. The high-level soluble expression, combined simultaneous purification and immobilization of the enzyme on chitin offers a novel approach for the low cost production of β-glucosidase to produce lactose-free fresh dairy products.