| Objects:To identify a strain producing high activity konjac glucomannanase, investigate theregularity of bacterial growth and fermentation, verify the conditions of producingglucomannanase, probe into the purification methods and the characteristics of glucomannanase, found a process producing konjac oligosaccharide.Methods:The strain was identified with the characteristics of morph and physiological biochemistry. According to Bergey's Manual of Determinative Bacteriology and Determinative Manual of Common Bacteria, the strain was grouped into genus or even species. Based on shake flasks the optimum culture and fermentation time were studied and then the fermentation conditions were verified on 5 L fermentator with the orthogonal experiment. Konjac glucomannanase was separated and purified through ammonium sulphate precipitation, gel filtration chromatography(Sepharose 6B) and ion exchange chromatography(DEAE-52), then the protein purify was determinated by PAGE and the molecular weight by SDS-PAGE. Furthermore, the enzyme characteristics were investigated about the optimum pH and temperature and so on to improve enzyme activity through adjusting the reaction environments. Based on the above results, the small scale of KOS production is studied including the agglutinator and the crude enzyme's reaction conditions. Meanwhile the parameters of KOS specimen, such as the sugar contents and components, transformation efficiency and so on, are determined.Results:1. According to morph characteristics and physiological biochemistry analysis, this strain is identified as Bacillus Cohn. 6ue to its deviation with other species of Bacillus Colin, this strain maybe a new species, for the time being it was named Bacillus thermodurans.2. On the shake flasks, the optimum culture is peptone 1%, beef extract 0.3%, konjac glucomannan 2%, NaCl 0.5%, K2HPO4 0.2%, the optimum temperature 40℃, pH 7.2, rotating velocity 120 rpm. The crude enzyme specific activity is average to 3859 U/mg.3. Through the orthogonal experiment, the optimum magnification conditions on 5 L fermentator are temperature 50℃, pH 6, at prophase stirring velocity 200 rpm, aeration rate 40 L/h, then after 6 h stirring velocity 100 rpm, aeration rate 20 L/h. The average specific activityis 4403 U/mg. when monitored on line, the growth of bacteria entered logarithmic phase after 6 h and stationary phase after 26 h. The enzyme specific activity decreased after 24 h. In this course, DO changed largely while pH was stable. In view of the relation between the bacteria growth and the production, the fermentation was coupling kinetics.4. Purification of Konjac glucomannanase includes three steps: ammonium sulphate precipitation, gel filtration chromatography and ion exchange chromatography.Glucomannanase precipitation concentration is at the range of 40~80%0 Sepharose 6B and DEAE-52 have better resolving power. After the three-step treatment, purification fold is 6.7 with 67.14% enzyme activity recovery and single band shows by PAGE determination. When substrate is konjac glucomannan, enzyme has Fmax of 6mg/mL-min and Km of lOmg/mL.According to the SDS-PAGE result, the purified enzyme has three subunits and it will be studied deeply whether the enzyme has single protein or it is another isoenzyme. This glucomannanase shows the maximal activity at temperature 50癈 and pH 5.6~5.8. When temperature is below 40癈, It remained 90% activity for 20 hours. At the range of pH 5'~7 enzyme activity can remain for 10 hours or so. Zn2+ and Fe2+ have great inhibition on enzyme activity while other trial ion and organic compound have little effect on activity.5. After fermented mash was pretreated by the mixture of 0.05% CaCl2 and 0.1% Na2HPO4, The bacteria and solid particles were separated and then salted out at 40%~80% under 0℃. the crude enzyme was dialyzed and mixed konjac glucomannan at the scale of 1:300 in water, then acted under 50℃ for 10 hours. At last, the hydrolyzate was spray-dried to the konjac oligosaccharide powder. In this cour...
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