Studies On Fermentation Optimization And Enzyme Characteristics Of Pullulanase From Bacillus Subtilis FB14

Initial pullulanase production of Bacillus subtilis FB14 being original strain was 110.34U/mL.Through UV mutagenesis,a mutan FB14-35 whose production reached 166.85U/mL was obtained.Then a mutan FB14-35-llwas obtained through plasma mutagenesis.The pullulanase production of FB14-35-11 reached 216.53U/mL,96.24%higher than that of the original strain.The fermentation optimization of FB14-35-1 was studied.The single factor screening test of culture medium and fermentation conditions and the uniform design of nitrogen source were conducted.The result showed that the optimum culture mediums were malt syrup 10%,urea 0.275%,(NH4)2SO4 0.1,corn 0.8,KH2PO4 0.5%,K2HPO40.25%.The optimum fermentation conditions were inoculum concentration 1%,shaker speed 220r/min,initial pH7.0,temperature 34℃.After aboved fermentation optimization the pullulanase fermentation level reached 259.62U/mL,19.90%higher than that of before optimization.The metabolic process of FB14-35-11 producing pullulanase fermentation was studied.the results showed the coupling relationship between the cell growth and enzyme production.The kinetics model of cell growth,pullulanase formation,sugar consumption were established,and the parameters of model were got,they were μmax 0.1378h-1,α59.06,p-4.59,Yx/s 0.418 g.g-1,Ms 0.00886 g-g-1-h-1.The characteristics of the pullulanase were studied.The results indicated that the optimum reaction conditions were temperature 60 ℃ and pH 5.5.The enzyme showed better stability in low temperature(<55℃)and neutral pH conditions(7.0-7.5).The enzyme kinetics of pullulanase was studied on the substrates of pullulan.The enzymatic reactions were accord with the Michaelis-Menten equation,the reaction activation energy(Ea)was 17.08kJ/mol,the Michaelis constant(km)was 1.38 g/L,and the Vm was 10.62μg/mL-min.The different effectors on pullulanase were studied.The results showed that the monovalent metal ions and the divalent ion Zn2+ has a lirttle influence on enzyme activity,Mg2+ and Ca2+ has a certain role in promoting enzyme acivity,Cu2+ and the trivalent metal ions had strong inhibitory effect.In addition to acetone and low concentration of ethanol,other organic solvent had different degrees of inhibition and only low concentrations of ethanol had a certain role in promoting.The most of modifier of pullulanase had different degrees of inhibition,initially concluded the amino-acid residues including lysine,methionine,serine were active groups of pullulanase.The studies of pullulanase immobilization showed when Fe3O4 magnetic nanoparticles modified by anhydrous ethanol-SMNP was used for enzyme carrier,the highest adsorption rate(50%)was got.And the immobilization conditions optimization results showed the ratio of enzyme to carrier 0.6mg:20mg,pH 5.0,immobilized time 10h.In the end the fixation rate reached 55.16%.The studies on enzymatic properties of immobilized enzyme showed that the optimum reaction conditions of the immobilized enzyme were 60℃ and pH4.5.Compared to free enzyme the immobilized enzyme showed more stablity of heat and acid resistance.The immobilized enzyme still had a high residual activity after using 14 times.