Research On The Breeding Of Dextranase Producing Strain And The Enzymology Properties

Dextranase?Dextranase;EC 3.2.1.11?specificity catalytic cracking the a-1,6 glucoside keys in Dextran?Dextran?molecules,for reduce viscosity of sugar cane juice in sugar industry,preparation of low molecular weight medicinal dextran in medicine medicinal,remove the glycosidic deposition in the gums in dentist industrythe,and other fields,Industrial application is extensive and the market demand is huge.At present,the level of enzyme production by dextranase production strains is low,make the scale of industrial fermentation was limited,the production efficiency is not high enough.This research to carry out the Penicillium cyclopium dextranase production strains of mutation breeding work to improve the dextranase production to adapt to the needs of practical application.Carried out several rounds of mutation processing by the Atmospheric and Room Temperature Plasma?ARTP?and Ultraviolet irradiation?UV-ray?technology for dextranase producing strain Penicillium cyclopium?P.c yclopium 4022?.Research the pertinence about transparent circle diameter of blue dextran culture dish and shake flask enzyme activity,establish the method to screen the mutant by transparent circle diameter of culture dish for obtain the mutant strains can product high yield dextranase.On this basis,research on the medium nutrition conditions and enzyme production culture conditions of strain Penicillium 12p27,in order to enhance the dextranase production ability by Penicillium cyclopium.And Separate and purify the dextranase producted by P.cyclopium 12p27 to research the stability of temperature,pH and enzymology properties.The main results are as follow:1.Mutagenize the P.cyclopium 4022 by ARTP and UV-ray technology.Get a stability genetic performance of dextranase high yield mutant strains P.cyclopium 12p27 through transparent circle diameter of culture dish and shake flask,the enzyme activity of this strain respectively increased 49.64%?ARTP?and 128.18%?UV-ray?by the starting strain.Build the mothod to screen the mutant strains combined with transparent circle diameter of Blue-Dextran 2000 culture dish and shake flask screening:Show a correlation between these two mothods.2.Through the single factor experiment research of the influence of carbon source,nitrogen source and surface active substance on enzyme activity,and then combined with the mathematical statistics method research of the fermentation condition about product dextranase by Penicillium cyclopum.Through the response surface experiment design optimization,the optimal fermentation conditions of Penicillium cyclopium 12p27 is:49.5g/L of D70:Sucrose,4.1g/L of beef extract,19.70mmol/L of sorbitol,0.5 g/L of MgS04.7H20,0.5g/L of KCl,0.01g/L of FeSO4·4H20,1.0g/L K2HPO4,pH value of 6.5.The dextranase activity reached 10.95 U/mL under this condition,increased 173.07%than 4.01 U/mL before of the optimization.3.Optimize the different factors in the culture conditions,further improve the enzyme activity that producted by the high yield strains 12p27.Including the cultivation in the process of fermentation temperature,shaking table speed,medium initial pH,bottled liquid volume and inoculation amount of per flask for meritocratic selection.Access to the most beneficial to fermentation strain culture conditions for enzyme production is:below 30?,control table speed of 230r/min,40mL bottled liquid volume,initial pH of 7.0,0.5mL inoculation amount,dextranase activity reached 14.48U/mL,increased 1.32 times than 10.95U/mL of the optimization before.4.Using 75%saturation of ammonium sulfate precipitation and DEAE Cellulose DE-52 ion exchange chromatography,separate and purify the dextranase of P.cyclopium 12p27.The dextranase specific activity increased from 124.53U/mg to 1052.71U/mg,purified by 8.45 times,dextranase's ultimate recovery was 10.98%.By SDS-PAGE electrophoresis appraisal,dextranase with 65kDa molecular weight purified has reached the electrophoresis purity pure.5.Research on enzymology properties about the P.cyclopium 12p27 dextranase shown the good thermal stabilitythe when the temperature is 30?,the optimum catalytic temperature is 40?,the optimum pH is 6.0.The preservation effect that toward to Alkaline conditions is better than those toward to Acidic conditions.Dextranase showed good stability when under the condition of pH 4?7.Fe2+ and K+ on the enzyme activity has a promoting effect,but the Pb2+,Al3+,Ag+,Mn2+,Co2+ and Zn2+,has certain inhibitory effect of dextranase.This enzyme present a substrate specificity of different substrates,hydrolysis ability for macromolecule dextran is better than small molecule dextran.The hydrolysis ability for molecular weight is greater than D70,is higher than small molecule dextran substrates such as D3 D40.Take the form of conversion mie equation,make Lineweaver-Burk double bottom diagram,get the michaelis constant Km value is 4.4827mmol/L,Vmax value is 1.461 ?mol mL^-1·min^-1.