| Inulin is a special kind of carbohydrate of fructose polymer with the linkage beta-2,1 glycoside bond, inulo-oligosaccharides have been regarded as a kind of functional food ingredients. Using endo-inulinase from microorganism to produce inulo-oligosaccharides has become the focus of these studies, but most endo-inulinase from the natural microorganism is exo-inulinse or the mixtures of endo-inulinase and exo-inulinase. Only few microorganism produce endo-inulinase, however, the disadvantages of which stand out, that is , lower quantity and difficulty of being isolated and purificated, to sum up, it is urgent to solve these problems to benefit the industrial product. So, the majority of studies of endo-inulinase aim at the enzymatic properties, gene cloning and expression, recombinant strain construction, production and application of microorganism's endo-inulinase, The object of the present study is to optimize the transformant growth in shake flasks and high-density cultivation to utilize the root of chicory for producing inulo-oligosaccharidies. 1 Expression of endo-inulinase.1.1 Production of endo-inulinase in flask cultivation. The results showed: (l)the tranformant utilized carbon sources glucose better than the others; (2) the best concentration of the trace elements is 100ml/L; (3)the optimum range of pH is fiom 6.0 to 7.0, (4)the optimum methanol concentration as sole is 0.1%; (5)fermentation period through methanol inducement is about 96 hours. When the cell optical density is up to 100, the enzyme activity can reach to 35U/mL, which is better than INVITROGEN system; orthogonal experiments showed that the optimum conditions for yeast growthing was glucose concentration 4%, (NH4)2SO4 concentration 11.5g/L.1.2 Production of endo-inulinase in high -cell density cultivation. Using three different culture medium F1, F2 and F3, the lag phase of cell growth was 1.36h, 2.29h and 2.48h. The relationship between cell fresh weight and culture time fit the exponential model, the average specific growth rate of the fed-batch culture is 0.4846h-1, 0.2856h-1 and 0.2813h-1. The FW of the cells in the batch cultivation was about 280g/L, the inulinase activity was reach to 112U/mL, the U/g,FWwas 0.36 unit.2 Properties of enzyme activity. The optimal conditions for enzyme reaction were pH5.5 and 55 C, the enzyme activity at pH4.5-6.5 and below 60C was stable. The various metal ions and chemicals ragents were added to the enzyme solution at the final concentration of 1mmol/L, After preincubation at 55C for 1h, the results showed that Hg2+, Pb2+ , Ba2+ abviously inhibitate the enzyme activity; Ca2+ can increased the enzyme activity; Mg2+, Fe2+, Cu2+, Zn2+ have no evident effection on the enzyme activity; EDTA has partially inhibition action. The enzyme has no effection on raffinose, but could hydrolyze inulin and partially hydrolyzed sucrose.3 Extraction of inulin. Using of dry chicory powder extract inulin, in conditions of liquid/material ratiol:15, 80C, 2 hours, the ratio of extraction reach to 98%. To determination of the total dissolved suga from chicory on the conditions of 0.2mol/l HC1, 100 C, 40min, the accuracy was best.4 Enzyme reaction. To determine the optimum enzyme dosage, enzyme reactions were carried out for 30h with several enzyme concentration reaching to 200g inulin per liter water , which was the most acceptable concentration within the solubility limit and under the reaction temperature, the maximum yield of total inulo-oligosaccharides of each reaction mixture was 62.0% without any significant effect of enzyme dosage, in contrast with 12U/g substrate, a reaction time of 20-30h was needed to reach maximal yield;Maximum yield in total inulo-oligosaccharides was abtained when 40g inulin/L was used; The products of the enzyme reaction in the end of time were analyzed by TLC and the ion chromatogram (IC2500), the results showed that the range of inulo-oligosaccharides from GF2 to GF10. The relation between the constitution of the product and the time of the enzyme reaction is not clo...
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