Optimization Of Fermentation And Transglycosidation Conditions Of α-glucosidase-producing Strains,Research On Enzymatic Properties And Mutation Breeding

Isomaltooligosaccharide（IMO）,as a cost-effective functional oligosaccharide,is widely used as a prebiotic in food,medicine and medicine because of its excellent physiological and biochemical properties and the effect of promoting the proliferation of intestinal probiotics.Feed and other fields.α-glucosidase（α-glucosidase,AGase,EC 3.2.1.20）is a key enzyme preparation in the IMO production process,and its enzyme activity is directly related to the quality of the product.Aspergillus niger GXZ-239 is an alpha-glucosidase-producing strain preserved in the laboratory.In order to further explore its industrial production potential,the strain GXZ-239 was subjected to a 5 L fermentation tank scale-up process study.Use fermentation medium（8%corn starch,8%corn steep liquor dry powder,0.06%Zn SO₄,0.4%KH₂PO₄,p H 6.5）to carry out a single factor optimization study of aeration ratio,rotation speed and inoculation amount,and obtain the optimal condition:the aeration ratio is 1.34 vvm,speed of 400 rpm,inoculation volume of 3%（v/v）spore suspension.Under these conditions,the maximum enzyme activity of strain GXZ-239 is 279.8±4.8 U/m L,and the maximum biomass is 135.0±3.6 g/L Compared with the initial conditions of the5 L fermentor,the enzyme activity increased by 247.3%,and the maximum biomass increased by 90.2%.Then,the optimization study of transglycosidation conditions of strain GXZ-239 was carried out at the shake flask level.First,single-factor optimization is carried out,and then orthogonal experimental design is carried out on the basis of single-factor experiment.Choose the substrate concentration,the amount of bacteria to be added,the initial p H value,the speed and the temperature to optimize the results,in 25%high maltose syrup,5%of the amount of bacteria added,initial p H 4.0,shaker temperature The IMO content was 65.3±1.7%,and the effective trisaccharide e IMO（Effective Isomaltooligosaccharide,e IMO）content was 32.4±1.3%when tested at 37°C and a shaker speed of 160 rpm.Discuss the enzyme stability test of Aspergillus niger GXZ-239 mycelium.It is found that the mycelium still has high enzyme activity after repeated use to the 15th time.The IMO content in the product is maintained at more than 50%,and the enzyme activity gradually decreases when it is used..At the 23rd time,the enzyme activity is reduced by half.When it is reused to the 30th time,the IMO content is 20.8±0.1%,and the e IMO content is10.6±0.6%.Carry out the separation and purification ofα-glucosidase and study its enzymatic properties.Theα-glucosidase was obtained through purification steps such as ultrasonic cell destruction,ammonium sulfate precipitation,T-Butyl hydrophobic chromatography and DEAE-Sepharose FF anion exchange chromatography.A single band was detected by SDS-PAGE gel electrophoresis,indicating that a higher purity enzyme was obtained.According to the electrophoresis results,the molecular weight of thisα-glucosidase was about 40KDa.After the above experimental steps,the final recovery rate of the enzyme was 26.2%.Using maltose as a substrate for the study of enzymatic properties:the optimal reaction temperature is p H 6.0,the optimal reaction temperature is40℃,Km and Vmax are 512.7 mmol/L and 70.9μmol/（min·mg）,respectively.When the temperature is below 60℃,the enzyme has good stability,and the relative enzyme activity remains 73.7%when incubated at 60℃for 1 h.In a weak acid environment of p H 6.0-7.0,the enzyme has good stability.After incubating at a constant temperature of p H 7.0 for 1 h,the relative stability of the enzyme is good,and it can still maintain 79.4%relative enzyme activity.Enzyme activity is lost in alkaline conditions.Ba²⁺,Cr²⁺can improve enzyme activity,while EDTA,β-mercaptoethanol,K⁺,Mn²⁺,Na⁺show inhibitory effect on enzyme activity.In order to further improve the enzyme activity of strain GXZ-239,Aspergillus niger strain GXZ-239 was subjected to ultraviolet mutation breeding.The ultraviolet mutagenesis of the strain was carried out at a vertical height of30 cm from the ultraviolet lamp（18 W,253.7 nm）and irradiation for 120seconds.Through the primary screening and re-screening,4 strains with a greater increase in enzyme activity were obtained.After 5 times of subculture,the enzyme activity of Aspergillus niger YB-4 was stable at 339.6±5.73 U/m L,which was 25.6%higher than the initial strain GXZ-239.