Screening Of Aspergillus Oryzae Strains With A High Of Fructosyltransferase Activity And Its Optimization For Production

As a kind of functional oligosaccharides, Fructooligosaccharide can regulate theintestinal flora balance, runchang purge, cut down the hematic fat and cholesterol andpromote the absorption of minerals and vitamin synthesis function.Because of these functions,Fructooligosaccharide has got great attention and been widely used in the life of modernpeople. In modern industry, we use the sucrose as substrate and aspergillus oryzae which cancompose fructosyltransferase as the manufacturer to produce fructooligosaccharide.However,fructosyltransferase activity of aspergillus oryzae is very low, about200U/g.So selectingaspergillus strains with high fructosyltransferase activity and researching on the productionprocess to increase the activity of fructosyltransferase have great significance on industrialproduction.Aspergillus oryzae SBB201was mutated through the uv-lithium chloride compoundmutagenesis. The positive mutation strains were chose and enzymolysised to prepareprotoplast which were used to be start strains for protoplast fusion. Based on the theory ofdouble inactivated markers and lethal damage complementary, genome shuffling technologywas used to breed target strains and we gained a stable aspergillus strains with highfructosyltransferase activity.The research on the fructosyltransferase fermentation condition was conducted throughflask-shaking test and the influence of different culture medium and culture conditions onproducing fructosyltransferase was studied. The most important factors that influence thefructosyltransferase synthesis were selected by PB experiment and the ascent path wasemployed to determine the response central point. The flask-shaking optimization tofructosyltransferase fermentation condition was carried by response surface method.And thenwe explore the influence of agitation speed, aeration and inoculation amount onfructosyltransferase synthesis further in a2L fermentater. At last, the pilot test in10L,500Lfermentater were used to verify the results. The results showed as follows:The mutant Râ…¢-7was induced by two times of uv-lithium chloride compoundmutagenesis and the enzyme activity of Râ…¢-7reached to291.01U/g which was increased21.8%comparing with the original strain whose enzyme activity is233.69U/g. Through three rounds of genome recombination, sieving fusion mutants and measuringthe fructosyltransferase activity of the fusants, fushion mutant Râ…¢-7was obtained with itsenzyme activity reached to498.24U/g. The enzyme activity of Râ…¢-7was71.21%higher thanthe starting strain whose enzyme activity is291.01U/g.and20%higher than the original strainwhose enzyme activity is233.69U/g.The research on the fushion mutant showed a goodgenetic stability.Through the single factor experiment to confirm the optimum medium component whichis: carbon sources:5%sucrose;nitrogen sources:6%defatted Soy-bean; cornmeal:0.3%;inorganic salts:NaCl0.1%and the optimum culture conditions which is:shakingspeed:150rpm;temperature:30℃; pH:5.0.The most important factors that influence the fructosyltransferase production was carbonsources concentration, nitrogen sources concentration and shaking speed.These factors wasdetermind by PÎ' experiment.And then through the response surface experiment, we confirmthe optimum conditions, that is: carbon sources concentration:5.94%;nitrogen sourcesconcentration:1.60%and shaking speed:165rpm.In the optimum conditions,the enzymeactivity of Râ…¢-7can reach to895.687U/g.The fermentation process research in2L fermenter shows: when the speed set at200rpm,the aeration set at1.0L/min, the inoculation amount at the1.25%,the growth is easy tomonitor and its enzyme activity is518.08.U/g. When at the condition of the inoculationamount at the1.25%and the200rpm shaking speed, compared the enzyme activity effectedby the different aeration(1.0L/min，2.0L/min，3.0L/min), we found that at1.0L/minaeration,the enzyme activity is higher. When at the condition of the inoculation amount at the1.25%and aeration at1.0L/min, compared the enzyme activity effected by differentspeed(100rpm，200rpm，300rpm，500rpm),we found that the speed at200rpm, the enzymeactivity is higher.When total sugar remains unchanged the study of the effect on enzyme activity causedby the feeding fermentation process is made. In the fermentation experiment of2L fermenter,the mycelium enzymes activity reached to731.45U/g while using the process ofsemi-medium combined with feeding carbohydrate and the mycelium enzymes activity was518.08U/g using the process of the full medium. Compared with the process of full medium, the enzyme activity improved41.11%While using the process of semi-medium combinedwith feeding carbohydrate. In the fermentation experiment of10L fermenter, the myceliumenzymes activity reached to804.04U/g while using the process of semi-medium combinedwith feeding carbohydrate and the mycelium enzymes activity was566.26U/g using theprocess of the full medium. Compared with the process of full medium, the enzyme activityimproved41.90%. In the fermentation experiment of500L fermenter, the mycelium enzymesactivity reached to640.00U/g while using the process of semi-medium combined withfeeding carbohydrate and the mycelium enzymes activity was426.00U/g using the process ofthe full medium. Compared with the process of full medium, the enzyme activity improved50.23%.