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 con version 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.

Immobilization of β-Glucosidase from Thermatoga maritima on Chitin-functionalized Magnetic Nanoparticle via a Novel Thermostable Chitin-binding Domain

“…No significant differences were observed from both types of enzymes (free and immobilized β-Glu) at pH 5. This corresponds to previously published results for Tm-β-Glu-Tt-ChBD immobilized to a variety of supports chitin beads 7,13 . Increased pH stability for immobilized-enzyme systems could be related to diffusion constraints or secondary interactions between the enzyme molecules and supports 29 .…”

“…on Tt-ChBD and the expression plasmid Tt-ChBD has explained the sequences of the recombinant fusion proteins 7 . Tm-β-Glu-Tt-ChBD was constructed and successfully expressed in E. coli JM109 (DE3).…”

“…2B, (lanes1 Tm-β-Glu) shows the molecular weight and purity of the fusion proteins. In order to verify the binding abilities of Tt-ChBD fused Tm-β-Glu heparinases 7 , a study was started to Magnetic nanoparticles immobilization of tm-β-Glu-Tt-ChBD on Ch, Cs, and SA. The immobilization yield is considered a vital parameter in the immobilization process, which can indicate how much of the applied protein is bounded to the carrier.…”

“…T. maritima is a thermophilic bacterium that grows typically in extreme conditions (between 60-90 °C) with an ideal growth temperature of 85 °C and is of considerable interest due to the hyper-thermostability of these enzymes (TmBglA & TmBglB). The TmBglA has a broad substrate specificity and belongs to glycoside hydrolase family 1 β-glucosidases (GH1), which make it ideal to be applied in numerous industrial tenders 7 . As reported, β-glucosidases from T. maritima (Tm-β-Glu) present an efficient catalyst to break the glycosidic bond with a high conversion percentage in thermal conditions 8 .…”

“…Among the various available immobilization techniques, the approach of confining enzymes on chitin (Ch) by fusing target proteins with chitin-binding domain (ChBD) received considerable attention, mostly due to the desirable characteristics of the Ch supports for instance low-cost and accessibility, elevated stability to both temperature and pH, and microbial resistance 7,22,23 . Therefore, the application of applying Tm-β-Glu in the immobilized form with chitin-Fe 3 O 4 magnetic nanoparticles (Ch-MNPs) by a novel thermostable chitin-binding domain (Tt-ChBD) will be more commercially advantageous.…”