Structural Characteristics and Function of a New Kind of Thermostable Trehalose Synthase from Thermobaculum terrenum

Abstract

Trehalose has important applications in the food industry and pharmaceutical manufacturing. The thermostable enzyme trehalose synthase from Thermobaculum terrenum (TtTS) catalyzes the reversible interconversion of maltose and trehalose. Here, we investigated the structural characteristics of TtTS in complex with the inhibitor TriS. TtTS exhibits the typical three domain glycoside hydrolase family 13 structure. The catalytic cleft consists of Asp202-Glu244-Asp310 and various conserved substrate-binding residues. However, among trehalose synthases, TtTS demonstrates obvious thermal stability. TtTS has more polar (charged) amino acids distributed on its protein structure surface and more aromatic amino acids buried within than other mesophilic trehalose synthases. Furthermore, TtTS structural analysis revealed four potential metal ion-binding sites rather than the two in a homologous structure. These factors may render TtTS relatively more thermostable among mesophilic trehalose synthases. The detailed thermophilic enzyme structure provided herein may provide guidance for further protein engineering in the design of stabilized enzymes.

A Novel Trehalose Synthase for the Production of Trehalose and Trehalulose

“…The amino acid sequence alignment with previously characterized trehalose synthases, prediction of secondary structure, and creation of a 3D homology model of the protein identified the conserved A, B, and C domains, conserved motifs, and the critical catalytic residues in TreM, which were in agreement with the previous studies ( 19 , 26 ). The SDS-PAGE analysis of recombinantly expressed and purified proteins determined the molecular mass of TreM (60 kDa) in the range of previously characterized trehalose synthases, e.g., Thermomonospora curvata (60 kDa) ( 23 ), Thermobifida fusca (66 kDa) ( 29 ), Thermobaculum terrenum (65 kDa) ( 19 ), Deinococcus geothermalis DSMZ 11300 (65 kDa) ( 27 ), Pimelobacter sp. R48 (62 kDa) ( 24 ), Deinococcus radiodurans R1 (64 kDa) ( 27 ), Picrophilus torridus (65 kDa) ( 30 ), Enterobacter hormaechei (65 kDa) ( 31 ), Deinococcus radiodurans ATCC 13939 (61 kDa) ( 27 ), and a saline-alkali soil-derived metagenomic enzyme (63 kDa) ( 32 ).…”

“…The temperature activity profile of TreM for trehalose synthesis was in accordance with the trehalose synthases characterized from T. terrenum ( 19 ), D. geothermalis ( 27 ), Meiothermus ruber ( 44 ), and P. torridus ( 30 ). A slight variation in the temperature optima of TreM for trehalose and trehalulose biosynthesis could be ascribed to the changes in the shape of the enzyme after binding with the different acceptor molecules ( 45 ).…”

“…The enzymatic process of trehalose synthesis is preferred due to its low-cost and more straightforward method. Primarily, trehalose biosynthesis is performed via three biocatalytic pathways, comprising different enzymes, as follows: (i) trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, (ii) maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase, and (iii) trehalose synthase ( 19 ). Trehalose synthase executes the reversible conversion reaction of maltose to trehalose.…”

Screening, Cloning, Expression and Characterization of New Alkaline Trehalose Synthase from Pseudomonas monteilii and Its Application for Trehalose Production

“…The performance of TreS enzymes from D. radiodurans [17], M. ruber [22], and P. torridus [16] was demonstrated in trehalose production at 30-45 o C and led to a trehalose yield of 56.0-61.0% with relatively higher glucose formation (7.8-9.2%) using a longer reaction time (24-72 h). The yield obtained in our study was only slightly lower than that from Thermus antranikianii [4] and Thermobaculum terrenum [40], which achieved more than 70% trehalose yield; however, their specificity as reflected by the formation of glucose byproduct was not reported for these enzymes. The alkalophilicity of PmTreS also potentially inhibits the growth of contaminated microorganisms in the production process [34].…”

In-Situ Biocatalytic Production of Trehalose with Autoinduction Expression of Trehalose Synthase

“…This reaction proceeds by an intramolecular rearrangement of the α-1,4 into an α-1,1 glycosidic bond, which represents a simple, fast, and low-cost method . To date, dozens of treS genes from different species have been reported in the literature and heterologously expressed in the Gram-negative bacterium Escherichia coli using lac -derived promoters, which are usually induced by the addition of isopropyl β- d -1-thiogalactopyranoside (IPTG). − However, the relatively high cost of IPTG and the inhibition of bacterial growth during strong and sudden induction with IPTG can have a huge impact on the high-throughput, economical production of recombinant TreS protein. Furthermore, since TreS is an intracellular enzyme, it is usually necessary to break the cells by ultrasonication and subsequently purify the enzymes from the crude extract before the enzymatic reaction.…”