A Novel Trehalose Synthase for the Production of Trehalose and Trehalulose

Abstract

A novel putative trehalose synthase gene (treM) was identified from an extreme temperature thermal spring. The gene was expressed in Escherichia coli followed by purification of the protein (TreM). TreM exhibited the pH optima of 7.0 for trehalose and trehalulose production, although it was functional and stable in the pH range of 5.0 to 8.0. Temperature activity profiling revealed that TreM can catalyze trehalose biosynthesis in a wide range of temperatures, from 5°C to 80°C. The optimum activity for trehalose and trehalulose biosynthesis was observed at 45°C and 50°C, respectively. A catalytic reaction performed at the low temperature of 5°C yielded trehalose with significantly reduced by-product (glucose) production in the reaction. TreM displayed remarkable thermal stability at optimum temperatures, with only about 20% loss in the activity after heat (50°C) exposure for 24 h. The maximum bioconversion yield of 74% trehalose (at 5°C) and 90% trehalulose (at 50°C) was obtained from 100 mM maltose and 70 mM sucrose, respectively. TreM was demonstrated to catalyze trehalulose biosynthesis utilizing the low-cost feedstock jaggery, cane molasses, muscovado, and table sugar. IMPORTANCE Trehalose is a rare sugar of high importance in biological research, with its property to stabilize cell membrane and proteins and protect the organism from drought. It is instrumental in the cryopreservation of human cells, e.g., sperm and blood stem cells. It is also very useful in the food industry, especially in the preparation of frozen food products. Trehalose synthase is a glycosyl hydrolase 13 (GH13) family enzyme that has been reported from about 22 bacterial species so far. Of these enzymes, to date, only two have been demonstrated to catalyze the biosynthesis of both trehalose and trehalulose. We have investigated the metagenomic data of an extreme temperature thermal spring to discover a novel gene that encodes a trehalose synthase (TreM) with higher stability and dual transglycosylation activities of trehalose and trehalulose biosynthesis. This enzyme is capable of catalyzing the transformation of maltose to trehalose and sucrose to trehalulose in a wide pH and temperature range. The present investigation endorses the thermal aquatic habitat as a promising genetic resource for the biocatalysts with high potential in producing high-value rare sugars.

Keywords: low-cost sucrose feedstocks; metagenome; trehalose; trehalose synthase; trehalulose.

FIG 1

A dendrogram showing the phylogenetic relationship among the previously characterized trehalose synthases and TreM. The number mentioned at the points of branching indicate the bootstrap values (A). Superimposition of TreM (blue) over its template (red), i.e., trehalose synthase from Thermobaculum terrenum. The computed RMSD value was 0.075 Å (B). Homology model of TreM showing amino acid residues critical in substrate binding and catalytic action (C). SDS-PAGE showing protein bands (D); M, ladder; 1, cell-crude extract of vector-transformed E. coli; 2, cell-crude extract of TreM-expressed E. coli; 3, TreM eluted using 100 mM imidazole; 4 and 5, purified TreM eluted using 200 mM imidazole.

FIG 2

Effects of pH (A) and temperature (B) on the activity of TreM for the biosynthesis of trehalose and trehalulose. Residual activity of TreM exposed to 4.0 to 10.0 pH. The reaction performed using the enzyme fraction that was not pre-exposed to acidic or alkaline pH was considered control (C). Thermal stability of TreM at different temperatures (D).

FIG 3

Maltose-to-trehalose conversion by the TreM catalytic reaction during different time points at 45°C (A), 30°C (B), 20°C (C), and 5°C (D). Production of trehalulose from sucrose, table sugar, and muscovado by the TreM catalytic reaction during different time points at 50°C (E). Mean values not sharing common alphabets in the assessment time points are statistically different at a P value of <0.05.

FIG 4

HPLC and TLC chromatograms showing sugar profiling before and after yeast treatment. The purified fractions of trehalose (A) and trehalulose (B) have been shown.

FIG 5

Distribution of Glu, Asp, Arg, and Lys residues, possibly involved in the formation of a salt bridge. A salt bridge has been represented between two ion pairs using a black dashed line. Distribution of acidic residues (A), Glu and Asp (B), and nonpolar residues (C) in TreM.