Trehalose Production From Starch Catalyzed By MTSase And MTHase Expressed In Recombinant Escherichia Coli

Trehalose is a non-reducing disaccharide which is widely present in plants, algae, bacteria, fungi, yeasts and insects. Trehalose can be used to preserve the stabilization of biological membranes and proteins, and used as preservatives in pharmaceuticals or food, showing wide application prospect.In this work, the genes of maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose tetrahydrolase(MTHase) from Sulfolobus solfataricus ATCC 35092 were amplified using PCR. Two recombinant strains, E. coli BL21(DE3) (pTrc99a-MTSase) and E. coli BL21(DE3)(pTrc99a-MTHase), were constructed. The specific activity of MTSase and MTHase in E. coli BL21(DE3) at optimal fermentation conditions reached 31.3 Ug(wet cell)~-1 and 403 Ug(wet cell)~-1, respectively.To increase the expression level, the rare codons in the 5'- and 3'- end of the MTSase gene were substituted with E. coli preferred ones, the secondary structure of mRNA TIR regine was optimized, and the present termination codon, UAG was substituted with more efficient translational termination sequence, UAAU. By these modifications in the DNA sequence of MTSase gene, the specific activity of MTSase in E. coli cell was increased to 55.0 Ug(wet cell)~-1.The biotransformation of partially hydrolyzed starch to trehalose catalyzed by MTSase and MTHase was carried out at 75℃ and pH 5.0. The highest yield of trehalose (ca. 53.6%), was gained when the original starch concentration was 15%(w/v) and the DE value was 10. The yield was 78.7% when amylose was used as the substrate.