The Study On Immobilization Of E.coli Expressing P450 BM-3 For Indigo Production By Intra-hollow Ca-alginate Capsule

Intra-hollow Ca-alginate capsule comes of the Ca-alginate bead. It not only has the advantages of broad source, inexpensive cost, gentle immobilization condition, easy operation, good biocompatibility, and less resistance to the substrates, but also can overcome the disadvantages of the difficulty that the substrates and oxygen can hardly transfer into the bead core and the limited space that the cells can only grow on the bead surface and in the gel holes. Therefore, the intra-hollow Ca-alginate capsule is a promising immobilization carrier.P450BM-3 (CYP102) isolated from Bacillus megaterium is a self-sufficient fatty acid monooxygenase, it catalyzes the ω-2 hydroxylation of long chain saturated fatty acids (C12-C20) and the hydroxylation and epoxidation of unsaturated fatty acids. Cytochrome P450 monooxygenases can catalyse many kinds of substrates. Recent studies show some kinds of P450BM-3 rebuilded by genetic engineering are capable to transform indole into indigo carmine. Indigo carmine is a kind of common dye and mainly synthesized by chemical methods. There are many advantages by using biological methods: the simplified process, the decrease of cost, the reduce of the hazard of cancinogenic substance in product and the lower pollution to the environment. At the same time, through modern stain screening, new ways of dyes synthesis by microbes will come true.In this thesis, we studied the effects of operation conditions (the concentrations of SA / CaCl₂/CMC, dropping velocity and height, the injection velocity and aperture and gelation) on the inner-diameter, membrane thickness, diameter and compression intensity of capsules. Conditions in preparing microcapsule were determined. Also, addition of a certain concentration of CaCl₂ in the culture was proved to be effective in improving the strength of the capsules, which was related to the long-time cultivation.Then, based on the effect of three components (SA, CMC, CaCl₂) on the external diameter, membrane thickness and inner-diameter of the Intra-hollow Ca-alginate capsule, in order to find the correlation between the concentration changes and the properties of the capsules, regression analysis was used to analyze the experimental data. Point estimation and interval estimation of new experiment were estimatedunder 95% confidence level. As a result, all the experimental results were in the estimation interval, the error of diameter between the experimental data and model estimation was 0.2% and the thickness 1.3% and the inner-diameter 0.5%. So the model was effective in estimation of the concentration changes and the size of the capsules. The research showed that, under the same operation conditions, inner-diameter of the Intra-hollow Ca-alginate capsule only related to the concentration of CMC andAfter that, E. coli, which was used to produce P450BM-3, was immobilized in capsules. Effects of biological compatibility of E. coli and the capsules, growth curvature, thermal stability, pH stability, numbers of capsules, FeC^ concentration on the production of the enzyme were investigated. At last, indole was catalyzed by the capsules and factors such as substrate concentration, temperature, pH and glucose amount were studied. The largest production of Indigo carmine was about 30mg/mL culture, conversion rate about 45.56%, and it was nearly 3 folds larger than other reported P450 synthesis systems (lOmg/L culture).