Study On The Fermentation Of Aminopeptidase From Bacillus Subtilis Zj016

Exo-type aminopeptidase in protein hydrolysis products of the preparation of compound combinations can be used as starting off with a bitter enzyme,such as the role to improve protein utilization,as well as tools for N terminal sequencing enzymes and enzyme used in medical diagnostic applications.Optimizations of the fermentation condition for aminopeptidase of Bacillus subtilis Zj016 was as follows:soybean dregs 12g/L,corn flour 30g/L,CoCl20.2mmol per liter,K2HPO4 3 g/L,MgSO(V7H2O 0.6 g/L, the initiative pH8.0,inoculum size 4%,rotate speed 210r/min, temperature 38℃,fermentation time 26h.The aminopeptidase activity was about 5500U/mL.The fermentation conditions of the 15L fermentor are determined as follows:fermented Fermenter(B. Braun Biotech international)are installed with liquid volume 8L,fermentation temperature 38℃,inoculation 4%(V/V), agitation speed for the 300-500r/min, aeration rate of 1:0.7,dissolved oxygen control about 50%,oxygen and agitation speed are not coupled, Pretreatment of raw materials, the activity was higher when the initial sugar in the fermentation medium 8-10g/L.By the middle make sugar fermentation enzyme activity 4000U/mL.Fermentation by fed-batch,respectively for 7 hours,14 hours of feeding,the activity has greatly improved.The activity of fermentation broth was 4000U/mLEstablishment of a liquid extraction process aminopeptidase:fermentation broth by centrifugation,clarification,ultrafiltration,desalination,and alcohol precipitation to obtain the liquid after the dissolution of aminopeptidase.Clarification,the yield of 90% of the activity, ultrafiltration yield of 91% of the activity,the recovery rate of 78% of desalination,the yield of 85% by alcohol precipitation, the total recovery was 55%.liquid enzyme in the pH 6,a months later, the activity of preservation rate of 85%.Bacillus subtilis Zj016 as the original strain by low energy nitrogen ion implantation, I screened a stable genetic mutants with high enzyme production N80-25,its capacity for enzyme production,compared with the original strain Zj016,increased by 1.2 times.Aminopeptidase collaboration with the neutral protease hydrolyzed soy protein isolate, substrate concentration of 6%,the amount of neutral protease E/S=5000U/g,the amount of aminopeptidase E/S=9.75×105U/g,pH8.5,temperature 50℃,4h,the hydrolysis of total free amino acid content of 13.6mg/mL,which Leu was relatively the highest,14.9% of total free amino acids,followed by Ala 14.5%,Arg 11.5%,Glu 9.6%,Val8.03%,Ile7.93%.Double-enzyme ydrolysis of free amino acid content is ProteAXG enzyme hydrolysis of free amino acid content of 1.95 times.Hydrolysis of peptide molecular weight of 1100D below,which accounts for about 600D peptide of about 16% dipeptide tripeptide about 75%,The degree of hydrolysis of formaldehyde titration is up to 50.8%.After optimizations of increasing aminopeptidase,the degree of hydrolysis is up to 62%.