Molecular cloning and characterization of maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase genes from hyperthermophilic microorganism sulfolobus hakonesis

Abstract

Trehalose (¥á-D-glucopyranosyl-[1,1]-¥á-D-glucopyranose), a nonreducing disaccharide of glucose, is found in various living organisms. In nature, trehalose serves not only as a carbohydrate reserve but also as a protectant against a variety of physical and chemical stresses. This compound has been expected to serve for various applications, for example as a sweetner, a stabilizer for dried or frozen foods, in cosmetics, and as a drug additives. Recently this property is practically applied to food and phamaceutical industries. Two genes encoding maltooligosyltrehalose synthase (ShMTSase) and maltooligosyltrehalose trehalohydrolase (ShMTHase) were isolated from a hyperthermophilic microorganism, Sulfolobus hakonesis (JCM 8857). ORFs of the ShMTSase and ShMTHase genes are 2,142 and 1,677 bp long and encode 713 and 558 amino acid residues, respectively. There are four domains for ShMTSase and three for ShMTHase in the deduced amino acid sequences, which are conserved in ¥áamylase family. Recombinant ShMTSase and ShMTHase were overexpressed in E. coli BL21(DE3)plysS and purified by Ni2+-NTA affinity chromatography. ShMTSase acts on the ¥á-1,4 linkage at the reducing end of maltodextrin, and transfers an oligomer segment of the maltodextrin to the C1-OH position of glucose to produce a ShMTHase hydrolyzes the ¥á-1,4-glucosidic linkage between the maltooligosyl group and trehalose unit. Thus trehalose is produced from maltodextrins by ShMTSase and ShMTHase in a sequential reaction. They were active on starch and produced trehalose from it, and the efficiency increased with the treatment of 4-¥á-glucanotransferase. The amount of trehalose from reaction with 4-¥áglucanotransferase was about 10 % more than that of trehalose from reaction without 4-¥á-glucanotransferase, after 24 hours incubation using maltopentaose as a substrate. Optimum temperature of each enzyme was 70 ¡É and optimum pH of each enzyme was 5.0. Each enzyme was stable at temperature below 85¡É. and Half life of ShMTSase/ShMTHase was 97.7 hours at the optimal condition.