| Trehalose synthase(referred to as Tre S, EC 5.4.99.16), is an intramolecular transglycosylase, involves the direct conversion of maltose into trehalose by intramolecular transglucosylation of the α-1,4-linkage of maltose to α,α-1,1-linkage of trehalose. Trehalose has biological functions, it has very widely industrial application in fields such as pharmaceutical, cosmetic, agriculture and food industries and so on.In the present study, The Tre S from Thermus thermophilus ATCC 33923 was cloned and expressed, and the conversion rate of trehalose enzymatic preparation was improved through site directed mutagenesis. The Tre S mutant codon was optimized, and then constructed recombinant genetic engineering bacteria of the enzyme. The high level expression of trehalose synthase was realized through the optimization of high density fermentation of engineering bacteria. The main results were listed as follows:(1) The Tre S from Thermus thermophilus ATCC 33923 was successfully obtained by PCR, and then connected to the expression vector p ET-24a(+). The recombinant plasmids, p ET-24a(+)-Tre S, was constructed and transformed into E. coli BL21(DE3). The trehalose synthase activity was 6.4 U×m L-1 after the engineered cells cultivated 28 h in shake flasks. The factors of trehalose preparation by recombinant enzyme was studied, under the optimum conditions(concentration of maltose: 30 %, p H 7.5, an enzyme to substrate ratio of 15 U×g-1, temperature 40°C, 150 r×min-1), after 24 h the yield of trehalose was 49.0%.(2) In order to improve the yield of trehalose, site-directed mutagenesis of Phe 251 was performed to obtain P251 L, then P25 L was expressed in E. coli BL21(DE3). The trehalose synthase activity was 6.9 U×m L-1 after the engineered cells cultivated 28 h in shake flasks. Heat treatment, centrifugal filtering and DEAE anion exchange column was used to separate and purify the wild and mutant enzymes. The specific activity of them were 87.8 U ×mg-1 and 137.9 U×mg-1, the optimum temperature, optimum p H, temperature stability and p H stability of them had no significant difference. The factors of trehalose preparation by mutant were studied, under the optimum conditions(concentration of maltose: 30%, p H 7.5, an enzyme to substrate ratio of 20 U×g-1, temperature 50°C, 150 r×min-1, 24 h), the yield of trehalose was 62.0%, 13.0% higher than that of the wild-type enzyme.(3) The mutant gene(P251L) was optimized according to the codon preference of E. coli and subcloned into E. coli BL21(DE3). In shake flask, the enzyme activity was 34.6 U ×m L-1 after cultivation of 26 h. Different induction conditions were investigated in 3 L fermentor. The optimal conditions were as follows. The combinant E. coli was cultured under p H 7.0, 30% DO concentration, the temperature was 37°C in growth stage, when OD600 reached 50, the induction was performed with 0.2 g×L-1×h-1 lactose and the induction temperature was 32°C, the trehalose synthase enzyme activity reached 472.8 U×m L-1 after 35 h of cultivation. Then with the condition, the experiments were carried out in 30 L fermentor, the enzyme activity reached 356.2 U×m L-1 after 39 h of cultivation. Because of the good thermal stability, high temperature treatment was tried to broke the E. coli cells to release trehalose synthase. The results showed that treated it 30 min under 80°C the yield of trehalose synthase reached 72.3%. It has industrial application prospect. |
PDF Full Text Request