Characterization of cold-active trehalose synthase from Pseudarthrobacter sp. for trehalose bioproduction

Abstract

Trehalose is a functional sugar that has numerous applications in food, cosmetic, and pharmaceutical products. Production of trehalose from maltose via a single-step enzymatic catalysis using trehalose synthase (TreS) is a promising method compared with the conventional two-step process due to its simplicity with lower formation of byproducts. In this study, a cold-active trehalose synthase (PaTreS) from Pseudarthrobacter sp. TBRC 2005 was heterologously expressed and characterized. PaTreS showed the maximum activity at 20 °C and maintained 87% and 59% of its activity at 10 °C and 4 °C, respectively. The enzyme had remarkable stability over a board pH range of 7.0-9.0 with the highest activity at pH 7.0. The activity was enhanced by divalent metal ions (Mg²⁺, Mn²⁺ and Ca²⁺). Conversion of high-concentration maltose syrup (100-300 g/L) using PaTreS yielded 71.7-225.5 g/L trehalose, with 4.5-16.4 g/L glucose as a byproduct within 16 h. The work demonstrated the potential of PaTreS as a promising biocatalyst for the development of low-temperature trehalose production, with the advantages of reduced risk of microbial contamination with low generation of byproduct.

Keywords: Pseudarthrobacter sp; Cold active enzyme; Maltose; Trehalose; Trehalose synthase.

Fig. 1

Overall predicted three-dimensionals structure of PaTreS. A Cartoon representation of four distinct domains of ArTreS including domain A, B, C, and subdomain S7 represented in pink, yellow, green, and blue, respectively. B The superimposition of PaTreS and TtTS from Thermobaculum terrenum (PDB 5 × 7u) represents the proposed catalytic residues (H128/D225/E267/H334/D335) and Mg²⁺ ions

Fig. 2

SDS-PAGE analysis of recombinant PaTreS expression in E. coli BL21(DE3) A and PaTreS purification B. The recombinant strain was cultivated at 25 °C and induced with 0.1, 0.25, and 0.5 mM IPTG for 3 h. Lane M: standard protein marker

Fig. 3

Effects of pH on the activity and stability of PaTreS with maltose as substrate. The 50 mM sodium acetate (pH 3.0–5.0), 50 mM potassium phosphate (pH 6.0–8.0), and 50 mM glycine–NaOH (pH 9.0–11.0) were used to assay the enzyme activities at various pH values (pH 4.0–11.0). To examine pH stability, the enzymes were incubated at various pH values (pH 4.0–11.0) at 30 °C for 1 h. The residual activities were measured at pH 7.0 using 1% (w/v) maltose as a substrate

Fig. 4

Effects of temperature on the activity and stability of PaTreS with maltose as substrate. Enzyme activities at various temperatures (4–70 °C) were assayed. To examine the thermal stability, the enzymes were incubated at various temperatures (4–70 °C) for 6 h and then were immediately cooled. The residual activities were measured at 40 °C

Fig. 5

Effects of metal ions on PaTreS activity. The enzyme was incubated with 0.1, 1, 10, and 20 mM of different metal ions at 40 °C in 50 mM phosphate buffer pH 7

Fig. 6

Effects of enzyme loading on trehalose production by PaTreS. The reactions were carried out in 50 mM phosphate buffer (pH 7.0) at 20 °C for 24 h

Fig. 7

Effect of substrate concentration on the yield of trehalose by PaTreS. The reactions were carried out in 50 mM phosphate buffer (pH 7.0) at 4, 20, and 30 °C for 24 h. A Trehalose yield B Glucose yield

Fig. 8

Production of trehalose from maltose by PaTreS. The conversion was carried out in phosphate buffer (50 mM, pH 7) at 20 °C for 20 h. A Trehalose yield B Glucose yield