Fermentation And Optimization Of γ-Cyclodextrin Glycosyltransferase In Recombinant E.coli

Cyclodextrin Glycosyltransferase (CGTase, EC2.4.1.19), which is capable of generating cyclodextrins(CDs) through cyclization utilizing starch, maltose and other polymers of glucose. As CDs has wider applications in many fields, such as food, pharmaceuticals, chemistry, agriculture and makeup industries, CGTase has become research hotspots.Previously, our laboratory achieved the extracellular production ofγ-CGTase in recombinant E.coli BL21 (DE3) with OmpA as signal peptide. To increase excellular production ofγ-CGTase, the expression optimization of recombinant strain E.coli BL21(DE3)/pET20b(+)-γcgt was studied in flasks and 3 L bioreactor. To improve the expression ofγ-CGTase,several recombinant strains were selected as research objects, the fermentation process were optimized in 3 L bioreactor. The main results are listed as follows.(1) The culture medium for E.coli BL21(DE3) /pET20b(+)-γcgt in flasks was investigated. According to the single-factor experiments and orthogonal experiment results, the best media composition was listed as following, 8 g/L glycerol, 10 g/L tryptone, 6 g/L yeast extract, 6 mmol/L Ca2+, 4 mmol/L Mg2+, 2 mmol/L Cu2+, 0.05 mol/L PO43-. Through experiments verification,γ-CGTase activity was 5818 U/mL the best fermentation condition was also listed as following, when strain was cultured in 30 mL/250 mL,220 rpm,pH 6.5-7,25℃,extracellularγ-CGTase activity reached 7214 U/mL.(2) The expression optimization for E.coli BL21(DE3) /pET20b(+)-γcgt in 3 L bioreactor was investigated. The glycine concentration, induction temperature and induction point were studied. When OD600=15, added with 0.75% glycine, when OD600=70, induced with lactose by 0.20 g/L/h in 25℃, extracellularγ-CGTase activity was 8145 U/mL, periplasmγ-CGTase activity was 2010 U/mL.(3) As T7 promoter is very strong, there are much inclusion body in cell, to improve this status, study reduced synthesis ofγ-CGTase. Study chose two vectors, pTrc99A and pKK223-3, three hosts, E.coli BL21(DE3), E.coli JM109 and E.coli W3110. Six recombinant strain was cultivated in shaking flasks, E.coli JM109/pTrc99A-γcgt and E.coli JM109/ pKK223-3-γcgt got higher expression protein. Extracellularγ-CGTase activity was 1210 U/mL and 1070 U/mL respectively, periplasmγ-CGTase activity was 1309 U/mL and 1214 U/mL respectively.(4) The fermentation optimization of E.coli JM109/pTrc99A-γcgt was performed in 3 L bioreactor, effects of lactose concentration, glycine, induction point, induction temperature, TritonX-100 in production ofγ-CGTase were studied. When OD600=15, added 0.75 % glycine into media, when OD600=40, induced with lactose by 0.30 g/L/h in 30℃, extracellularγ-CGTase activity was 465 U/mL, and the periplasmγ-CGTase activity was 23270 U/mL.