Screening Of Thermostable Pullulanase Strains,Fermentation Optimization And Mutation Breeding

Pullulanase is a kind of debranching enzyme that can specifically cut the α-1,6 glycosidic bond in the branching branch of amylopectin so as to cut the entire collateral to form amylose.Pullulan can maximize the use of starch raw materials,so it has important applications in starch processing industry.At present,pullulan is mainly used in high concentration maltose,resistant starch,high glucose syrup,beer,feed and alcohol production,and shows great application value.Due to the high temperature of the starch hydrolysis process,it is urgent to screen for strains that produce high temperature resistant pullulanase.This paper aims to screen out new heat-resistant pullulanase-producing bacteria from soil,,and to characterize them,morphological and physiological and biochemical characteristics and molecular biology.In addition,the method of isolation and purification of pullulanase was established,and the enzymatic properties of pure enzyme were studied.By optimizing the fermentation medium and fermentation conditions and mutagenesis to improve the ability of heat-resistant pullulanase-producing bacteria.At the same time,the mutant strain with the highest activity of stable heredity and pullulanase was cultured in 10L fermentor.The detailed results are described as follows:1.screening and identification of strains producing thermostable pullulanase.A themostable pullulanase strain T-10 of 1.22U/mL enzyme activity was Isolated from soil samples in a crater.By applying morphological observation,physiological and biochemical test of characteristics and 16S rDNA sequence homology analysis to determine the strain of Bacillus amyloliquefacienss.2.Separation,purification and characterization of pullulanase.The crude pullulan was eluted by ammonium sulfate precipitation,DEAE-Sepharose ion exchange chromatography and Sephadex G-75 gel chromatography to obtain electrophoretic pure pullulan with molecular weight of about 80 kD.The specific activity of the purified pullulan was 16.06 U/mg,the purification factor was 45.71 times,and the recovery rate was 4.33%The optimum temperature for the pullulanase activity was at 60℃and the pullulanase maintained more than 60%of its maximum activityafter incubation for 4h at 70℃;The optimum pH value for the pullulanase activity was at 6.0,and the pullulanase maintained about 70%of its maximum activityafter in the pH range of 3.0-8.0 after incubation for 4h.It has good thermal stability and a wide range of pH stability.It is a I type pullulanase,It is the most suitable substrate of Pullulan.The higher the concentration of Al3+、Fe3+、Mn2+、Ni2+、Zn2+、Fe2+ and Pb2+,the more obvious inhibition of pullulanase.Ca2+、Co2+、Ba2+、Mg2+ and NH4+ have a certain activation effect on pullulanase at low concentration,and they have negative effects on pullulanase at high concentration.EDTA、Cu2+ and Hg2+ at low concentrations will have a strong inhibitory effect on pullulanase;Na+ and K+ have a certain effect on pullulanase,and their concentration has little effect on pullulanase.The kinetic parameters of luminolase were 0.023mmol/L and the maximum reaction rate was 32.23μmol/min..3.Optimization of medium and fermentation conditions of producing thermostable pullulanase strain T-10 fermentation.The medium composition and fermentation conditions of the heat-resistant pullulanase strain T-10 were optimized by single factor test,Plackett-Burman test and Box-Behnken response surface test The optimal fermentation medium and fermentation conditions were as follows:Soluble starch content 21.7g/L,soybean peptone content 16.0g/L,potassium nitrate content 7.0g/L,dipotassium hydrogen phosphate content 1.5g/L,magnesium sulfate heptahydrate content 0.3g/L,culture medium initial pH7.0,Inoculation amount 2%,fermentation temperature 37℃,shaking speed 180rpm,fermentation time 16h.Under this condition,the enzyme activity of pullulan was 5.04U/ml,and the predicted value was 4.82U/ml,which was 4.33%,which indicated that the regression model could predict the activity of pullulase.By optimizing the fermentation medium and fermentation conditions,the final enzyme activity was increased by 313%compared with the initial enzyme activity.The growth and production curve of strain T-10 were studied.It was found that Biosynthesis of pullulanase is synchronous mode synthesis.4.Mutation breeding of thermostable pullulanase strain T-10.The strain T-10 was mutagenized by UV mutagenesis,NTG mutagenesis and ARTP mutagenesis,and the stable mutant strain with high activity of heat-resistant pullulanase was selected.By calculating the lethality rate,the UV treatment time was 30s,the NTG concentration was 0.2 mg/mL and the ARTP treatment time was 10s,when the other conditions were determined.At last,2 strains with high yield were obtained.After 10 subculture,The mutant strain N1 with the highest increase in enzyme activity was obtained,and its activity was 6.74U/mL,which was 32.26%higher than that of the original strain.The mutant strain N1 was fermented in a 10L fermentor according to the optimized fermentation medium and fermentation conditions.When the fermentation time was 18h,the enzyme activity reached the maximum value of 11.84U/ml,which was 1.76 times higher than that in shake flask fermentation.