| Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is an extracellular enzyme that capable of converting starch or starch derivates into cyclodextrins through an intramolecular transglycosylation reaction, and can be classified asα-,β-, andγ- type CGTase. The CD has the unique sparse-water cyclo-round barrelmolecular structure, can carry on the package and the molecular recognition to each kind of organiccompound, thus change and protect the physics and chemistry nature of object compound. Therefore, the CDs has the widespread utilization in food, medicine, cosmetics domain and so on. The overproduction of CGTase is a prerequisite for the industrial of CGTase in various reactions. Therefore, to overcome the lower productivity of wild strains, the utilization of recombinant Escherichia coli has recently drawn much attention for the industrial production of CGTase.In this study, engineering strain E. coli FIA 08-07, constructed and conserved in our lab, has the ability to extracellularly express and secrete the recombinantα-CGTase, was selected forα-CGTase production. According to the principles of fermentation optimization including the optimization of culture conditions based on microbial reactions (outer factor), and the optimization based on metabolic flux analysis (quantitativeness of inner factor), a series of feasible approaches or strategies were carried out to achieve high product concentration, high yield and high productivity ofα-CGTase in the optimization of E. coli FIA 08-07 cultivation process. The main results of this thesis were described as following:(1) Seed medium significantly influenced the seed activation and growth and enzymatic productivity. By the implement of an orthogonal array experiment design, the importance order of factors on enzyme production was obtained, and the most suitable seed medium was determined to be consisted of yeast extract 15 g/L, peptone 5 g/L, glucose 10 g/L, NaCl 10 g/L. The seed growth was investigated, the results were: the logarithmic growth phase was from 4 to 12 h, and the seed age was determined at 8 h, which was beneficial toα-CGTase fermentation.(2) For genetic engineering bacteria, plasmid stability and cellular insoluble inclusion bodies have an important effect onα-CGTase yield during fermentation. The experiment results showed that plasmid stability was over 90% during the whole fermentation. But there were manyα-CGTases accumulated in the cytosol as biologically inactive inclusion bodies which should be investigated in later research.(3) The fermentation culture medium and condition for recombinantα-CGTase production in shaking flask were investigated. The optimum medium was composed as following: 12 g/L peptone, 24 g/L yeast extract, 8 g/L glucose, 0.5 g/L lactose, 16.5 g/L K2HPO4·3H2O, 2.3 g/L KH2PO4, and 2.5 mmol/L CaCl2. The optimal initial pH, culture temperature and medium volume in 500 mL shaking flasks for enzyme fermentation were 7.0, 25°C and 100 mL. The appropriate inoculum size was 4 %.(4) The influences of various agitation speeds and dissolved oxygen (DO) levels onα-CGTase production by E. coli FIA 08-07 were investigated in a 3 L fermentor. Between these two different control strategies, control the constant agitation speed was more efficient. When the air flow rate was controlled at 1.5 L/min, in the constant agitation speed of 300 r/min, theα-CGTase production rate and the cell growth were both highest.(5) The effect of temperature, varied from 20 oC to 37 oC, on cell growth andα-CGTase production the batch fermentation of cultivation was investigated. It was found that cell growth was hastened along with the increase of temperature (such as 37 oC ) but the plasmid stability declined obviously. The optimum temperature for extracellularly secrete production ofα-CGTase in E. coli FIA 08-07 was 25 oC, the extracellular activity ofα-CGTase achieved 15.2 U/mL. The extracellular secretion of the recombinant enzyme was suppressed at higher temperatures. The reason was that the nascent recombinant polypeptides aggregated as inclusion bodies in the inner side of inner membrane due to too high rate of enzyme synthesis at high temperature. Such inclusion bodies may subsequently block the protein secretion system. Lower temperature resulted in too low rate of enzyme synthesis, which limited the extracellular secretion of recombinant enzyme. Two-stage temperature control strategy was confirmed not able to enhance cell growth orα-CGTase production, so the optimum temperature ofα-CGTase fermentation in a 3 L fermentor was controlled at 25 oC constant.(6) The batch fermentation processes of E. coli FIA 08-07 forα-CGTase production under diverse initial glucose concentration were investigated. It was concluded that high concentration, high yield and high productivity of both cell andα-CGTase could not be consolidated by improving initial glucose concentration in a batch process. With the aim of enhancing theα-CGTase production by E. coli FIA 08-07, fed-batch culture should be investigated in detail. The effects of different cultivation modes such as batch feeding and constant-rate feeding onα-CGTase production were both studied. As a whole, the resrults shown that it was efficient to realize high cell and highα-CGTase concentration by the two kinds of feeding strateges. However, constant feeding fermentation was the most suitable approach to attain not only high concentrations but also high yields and high productivities of cell andα-CGTase in the cultivation of E. coli FIA 08-07. The OD600 andα-CGTase activity were 35.3 and 26.8 U/mL after 48 h constant feeding batch cultivation, which were 1.7-fold and 76.3 % higher than the corresponding value of batch fermentation, and also the activity ofα-CGTase was about 52-fold higher than that of the parent strain, P. macerans JFB05-01.
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