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Cyclodextrin glycosyltransferase (referred CGT enzyme, EC2.4.1.19) is a glycosyl hydrolase α-amylase family (family 13) an important member of it through the cyclization reaction starch into cyclodextrins ( abbreviated CD). CGT enzyme reaction according to the initial stage of the main types of cyclodextrin generated, the CGT enzyme into α-, β-and γ-CGT enzyme. With α-cyclodextrin in food, molecular recognition and nanomaterials such extensive application, a-CGT enzyme has become a hot research. In this thesis, where the laboratory building and saved containing secretory signal peptide OmpA E. coli E. coli BL21 (DE3) {OmpA-pET-20b () / α-cgt} as the starting strain, high yield, high secretion and high intensity of production as the goal, in shake flask and fermentor studies 3L fermentation medium and culture conditions on α-CGT enzyme exocytosis affected. The main contents are as follows: 1, recombinant E. coli in seed medium of the best activation time is 6-8h. In shake flask level, logarithmic cell growth in pre-added 0.75% (w / v) glycine can improve a-CGT extracellular enzyme production to 36h when the fermentation, the extracellular enzyme activity of 18.9 U / mL, which was earlier not add glycine 2.2 times. In shake flask level, through a series of single-factor experiment was optimized fermentation medium consisting of: glycerol 10 g / L, industrial grade peptone 18 g / L yeast extract industrial grade 20 g / L, K2HPO4 · 3H2O 16.43 g / L , KH2PO42.31 g / L, MgCl2 5mM, initial pH 7.0; fermentation conditions were: 30 ℃, 7% of the inoculum size, liquid volume of 20mL medium / 250mL flask. Conditions in shake flask extracellular production of recombinant E. coli enzyme α-CGT 60h, from the fermentation ability of 20.5 U / mL up to 48h of fermentation 56.0 U / mL, the intensity of production from 341.7 U / L / h up to 1166.7 U / L / h. 2, the use of optimized complex medium of dissolved oxygen 3L tank batch fermentation and partial - feeding inducer fermentation fermentation of recombinant E. coli enzyme α-CGT, the results show batch fermentation in the large intestine 30% DO bacilli extracellular obtained under the highest activity, to 17.8 U / mL, respectively, for the same period 20% DO and 40% DO levels of 1.7 and 1.2 times. Batch - batch fermentation of E. coli in the induction of enzyme production under conditions better than the non-induced conditions, which employs IPTG induction, the fermentation 30h extracellular enzyme activity of 49.7 U / mL, 1.6 under non-inducing conditions X; using lactose induction, the fermentation activity of extracellular 27h 44.0 U / mL, under non-inducing conditions was 1.4 times. 3, in the 3L of the tank under the uninduced E. coli synthetic media for high-density fermentation of recombinant α-CGT enzyme, the results show a synthetic medium suitable for high-density cell growth, cell OD600 up to 150.0, but bacterial cells do not produce the enzyme. To induce expression of heterologous recombinant E. coli proteins, experimental comparison of different inducer concentration and induction time for the induction of cell density fermentation enzyme α-CGT, the results showed that: 1) in 0.5 mM IPTG induction, cell 1h begin immediately after the induction of autolysis, no extracellular enzyme activity; at 8.0 g / L / h lactose induction, cells within 4h after induction continues to grow exponentially, up to 71.1 OD600 bacterial extracellular enzyme activity up to 10.0 U / mL. 2) cells in the 0.8 g / L / h induced extracellular lactose highest activity, extracellular 27h fermentation yield of 46.44 U / mL, respectively, 0.4 g / L / h and 8.0 g / L / h 1.8 fold and 4.6-fold. 3) recombinant E. coli obtained at OD600 = 50-induced extracellular enzyme activity were highest OD600 = 25 and 100 induced 5.5-fold and 66.3-fold. 4, in the 3L tank of liquid nitrogen source feeding and temperature-induced high density fermentation of recombinant Escherichia coli producing α-CGT enzymes. The results showed that: 1) cells containing a nitrogen source in the feeding solution obtained from the fermentation of extracellular activity in the highest, 105.1 U / mL, a control experiment 2.3 times; containing an inorganic nitrogen source obtained from the fermentation liquid feeding extracellular enzyme activity lowest, 8.8 U / mL, only 19.0% of control experiments. 2) induced by bacterial fermentation at 25 ℃ 35h extracellular enzyme activity obtained the highest, reaching 275.3 U / mL, is 20 ℃ and 30 ℃ induced 5.1-fold and 3.6-fold, the production is all reported extracellular production a-CGT The highest yield of the enzyme.
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