Heterologous Expression Of Sulfolobus Acidocaldarius MTSase And MTHase And Its Application

Trehalose is a safety, reliable and non-reducing disaccharide, and is widespread in nature.Trehalose is a particular stabilizer of organisms or biological macromolecules, these unique biological features and physical and chemical properties, make its has wide application in biopharmaceutical, food, agriculture and other industries.Maltooligosyl trehalose synthase(MTSase,EC5.4.99.15) and maltooligosyl trehalose trehalohydrolase(MTHase, EC3.2.1.141) are key enzymes in the synthesis of trehalose from starch. In the present study, Sulfolobus acidocaldarius(S. acidocaldarius)ATCC 33909 MTSase and MTHase was cloned and expressed in Escherichia coli(E. coli) BL21(DE3) and Brevibacillus brevis(B.brevis).Then optimized the fermentation conditions of the recombinant bacteria E.coli.The enzymatic properties of the recombinant enzymes were characterized and the optimum conditions for enzymatic conversion were investigated. The main results were listed as follows:(1) The genetic engineering bacteria E. coli BL21(DE3)/pET-24a(+)-treY and E. coli BL21(DE3)/pET-24a(+)-treZ were constructed and cultivated. Then we studied on how to improve the soluble expression of recombinant proteins, and we discovered that the enzyme activity of fusion enzymes Trx-MTSase and Trx-MTHase were improved when fusing Thioredoxin(Trx) with MTSase or MTHase together. the enzyme activity of fusion enzymes Trx-MTSase and Trx-MTHase were 41.0 U?mL-1 and 42.0 U?mL-1, 1.7 and 4.0 fold higher than that of non-fusion proteins(MTSase 24.2 U?m L-1,MTHase 10.4 U?m L-1). The enzyme activities of MTSase and MTHase were 1.26 and 1.31 fold higher than before after digesting the N-Trx,and 2.1 and 5.2 fold higher than that of non-fusion proteins.(2) After various purification steps to obtain the pure MTSase and MTHase.Then, the properties of the enzymes were studied. The optimum temperature of the recombinant enzymes were the same 75℃, the half-live period of MTSase were 12 h and 6 d at 60℃ and 75℃, and the half-live period of MTHase were 16 h and 8 d at 60℃ and 75℃. Respectivety, the optimum pH of MTSase and MTHase were 5.5 and 6.0, relatively stable in the range of pH 5.0-9.5.(3) Optimization of 3-L fermentor fermentation conditions of recombinant bacteria E. coli Origami(DE3)/pET-32a(+)-treY、E.coli Origami(DE3)/pET-32a(+)-treZ.The results show that the optimum culture conditions of E. coli Origami(DE3)/pET-32a(+)-treY was 30℃, and the lactose was added at flow speed of 0.4g·L-1·h-1 when OD600 reached 40, the maximum activity of MTSase was 229.0 U·m L-1 5.6 fold higher than production in shake flask after 24 h of induction.And the optimum culture conditions of E. coli Origami(DE3)/pET-32a(+)-treZ was 30℃, and the lactose was added at flow speed of 0.2g·L-1·h-1 when OD600 reached 40, the maximum activity of MTHase was 204.0 U·m L-1 4.9 fold higher than production in shake flask after 24 h of induction.(4) The MTSase and MTHase from S. acidocaldarius ATCC 33909 were expressed at B. brevis for the first time.The genetic engineering bacteria B. brevis/pSVN-treY and B. brevis/pSVN-tre Z were constructed and cultivated in the shake flask and 3 L fermentor,and the maximum activity of MTSase and MTHase were 66.0 U?mL-1 and 36.0 U?m L-14.0 and 4.6 fold higher than that of shake flask after 60 h cultivated in the 3 L fermentor at 30℃.(5) The optimal conditions for trehalose production were studied, and the optimal comdition as follows, potato starch concentration was 10%(w/v), the control temperature is 60℃,pH 5.5,the amount of enzymes were MTSase 80 U per gram of substrate,MTHase 20 U per gram of substrate,Pullulanase 5 U per gram of substrate,incubated for 48 h.Under the optimal conditions, the conversion rate of the trehalose was 78.8%. The conversion rate of the trehalose was 74.2%,and the trehalose production was 185.5g?L-1 at substrate concentration of 25%. Besides, the conversion rate of the trehalose was only 68.4%, but the trehalose production reaches the highest of 205.2 g?L-1 at substrate concentration of 30%. So, in consideration of industrial production, the best substrate concentration is 25%- 30%.