Recombinant Expression Of 4-α-glucanotransferase And Its Application In The Preparation Of Cycloamylose

The 4-α-glucanotransferase is a multifunctional enzyme, which can catalyze cyclization reaction, coupling reaction, disproportionation reaction, hydrolysis reaction with starch. It catalyzes intramolecular transglycosylation of α-1,4 glucans to produce cycloamyloses. cycloamylose is a common name for cyclic α-1,4-glucans with more than nine D-glucopyranose units, which has the larger hydrophobic cavity and higher water solubility to forming inclusion complex with some poor stability material. As a result, cycloamyloses have the potential to be widely used in the food, biotechnology, pharmaceutical and chemical industries.In this study, the genes of 4-α-glucanotransferase from Aquifex aeolicus and Thermus aquaticus ATCC 33923 were cloned and expressed in Escherichia coli BL21（DE3）. The process of cycloamyloses preparation from this two enzyme（AaAM and TaAM） was optimized, and the performance of TaAM was better by comparison. Then mutant TaAM-Y54 G was constructed by using site-directed mutagenesis. The double enzyme method with TaAM-Y54 G and pullulanase was used to improving the yield of cycloamyloses preparation. We also optimized the conditions for the production of TaAM-Y54 G by recombinant strain in the 3 L fermentor in this study, which realized the high level expression of 4-α-glucanotransferase. The main results are as follows:（1） Constructed the E. coli BL21（DE3）/pET-24a（+）-AaAM and E. coli BL21（DE3）/pET-24a（+）-TaAM. The activity of AaAM and TaAM were 4.6 U×mL-1 and 139.8 U×m L-1, respectively, in shake flask.（2） The conditions for cycloamyloses preparation with potato starch by this two enzyme were optimized as follows: The substrate concentration was 1%（w×v-1） debranched potato starch, the reaction pH was 7.0, the reaction temperature was 75°C, AaAM concentration was 100 U×g^-1 starch for AaAM reaction system, in which cycloamyloses yield reach to 24.8% for 10 hours. The reaction pH was 7.0, the reaction temperature was 70°C, TaAM concentration was 250 U×g^-1 starch for TaAM reaction system, in which cycloamyloses yield reach to 33.6% for 12 hours, so the TaAM was selected for the follow-up study.（3） The mutant Y54 G was constructed by site-directed mutagenesis to improving the yield of cycloamyloses preparation. Successfully constructed the recombinant E. coli BL21（DE3）/pET-24a（+）-TaAM-Y54 G, the activity of recombinant strain was 203.1 U×mL-1 in shake flask. The recombinant enzyme was purified by ammonium sulfate precipitation and DEAE-Sepharose FF cation exchange chromatography.The purified wide enzyme and mutant enzyme were exhibited a specific activity（cyclization activity） of 163.1 U×mg^-1 and 189.2 U×mg^-1, respectively. The optimum temperature of the purified wide enzyme and mutant enzyme were 75°C and 70°C, The optimum pH of the purified wide enzyme and mutant enzyme were 7.0 and 6.5. The wide enzyme and mutant enzyme showed 56.8% and 46.6% of its initial activity after incubation at 70°C for 40 hours. This two enzyme showed more than 90% of its initial activity after incubation at 4°C for 24 hours in pH 5-10 buffer.（4） The conditions for cycloamyloses preparation with potato starch by TaAM-Y54 G were optimized as follows: The substrate concentration was 1%（w×v-1） potato starch, the reaction pH was 5.5, the reaction temperature was 65°C, the TaAM-Y54 G concentration was 250 U×g^-1 starch, pullulanase concentration was 50 U×g^-1 starch, in which cycloamyloses yield reach to 43.2% for 12 hours. The 10%（w×v-1） potato starch was liquefied with 5.0 U×g^-1 TaAM-Y54 G, after which the reaction pH was 5.5, the reaction temperature was 65°C, TaAM-Y54 G concentration was 250 U×g^-1 starch, pullulanase concentration was 50 U×g^-1 starch, in which cycloamylose yield reach to 43.2% for 12 hours.（5） The optimization of TaAM-Y54 G production from engineered E. coli BL21（DE3） in a 3 L fermentor was investigated. Investigation of different induction strategies revealed that induction was optimal when the temperature was maintained at 30°C, the inducer（lactose） was fed at a rate of 0.8 g×L-1×h-1, and protein expression was induced when the cell density（OD600） reached 50. Under these conditions, the enzyme activity reach to 2525.9 U×m L-1, was 12.4 times higher than the shake flask fermentation.