Cloning And Expression Of Gene Encoding Trehalose Synthesis Relative Enzymes From Corynebacterium Glutamicum

Trehalose is a non-reducing disaccharide, consisting of twoα-1,1-linked glucose molecules. It is found in various organisms, including bacteria, fungi, yeasts, insects, and some plants. In nature, trehalose serves not only as a carbohydrate reserve but also as an agent that protects organisms against environmental stresses such as drought, heat, salinity. In addition, trehalose can be used as a sweetener, stabilizer for dried and frozen foods, moisture retainer in medicines and cosmetics, and drug preservative. Trehalose was initially produced by extraction from plants and baker's yeast grown on glucose, but both processes were extremely expensive. In order to lower the production costs, investigations have focused on searching for efficient synthetic processes and abundant raw sources for production of trehalose. The enzymes maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase) transforms starch into trehalose directly, the yield of trehalose up to 80%, together withα-amylase and isoamylase .This provides a potential route to trehalose industrial applications.The trehalose biosynthesis genes treY and treZ, encoding a MTSase and a MTHase, respectively, had been cloned from the Corynebacterium glutamicum by PCR technique. The ORF of MTSase was 2436bp long, and encoded 811 amino acid residues. MTHase was 1701bp long, and encoded 566 amino acid residues. The two genes were inserted into vector pET-30a respectively, and then were introduced into the host Escherichia coli BL21(DE3) pLysS. A high yield of the recombinant MTSase at a molecular weight of 90 kDa was obtained by IPTG induction. The soluble recombinant enzyme accounted for 45.3% of the supernatant protein. The recombinant MTHase had a molecular mass of 67kDa, but most of recombinant enzyme was found as aggregate in inlusion bodies. Purified recombinat MTSase showed its optimal activity at 35℃and pH8.0, enzyme activity up to 45.09U/ml fermentation medium and specific activity up to 1588U/mg protein .The recombinant enzyme was capable of decreasing the DE value of dextrin and producting a maltooligosyl trehalose mixture. Recombinant MTHase, together with recombinant MTSase, could catalyze dextrin into trehalose. The two genes were cloned into vector pPIC9K, respectively. And then, the two resultant recombinant plasmides were introduced into the host Pichia pastoris GS115 by PEG method, trying to express in the Pichia pastoris expression system.