Optimization Of Fermentation Conditions And Characterzation Of A Raw Starch-digesting Glucoamylase From Athelia Rolfsii

Raw starch amyla is a kind of amylase with the ability to hydrolyze raw starch,its application could save energy for the traditional sugar making process.However,e recent reported microorganism producing this enzyme was mostly cultivated with peptone as main nitrogen source,and liquefied enzyme is in the majority while hydrolyzed starch glucoamylase is rare.In this paper,the maximum yield of active raw starch-digesting glucoamylase was obtained and the properties of enzymes were represented,with corn yellow pulp as the source of nitrogen,and based on optimization of Athelia rolfsii after bacteria fermentation conditions,to optimize the traditional sugar making process and save energy.In this study,the inoculation amount was fixed to 5%?volume fraction?,using raw starch-digesting glucoamylase yield as indicators,fermentation temperature???,fermentation time?h?and shaking speed?r/min?as experimental factors,the optimal fermentation conditions of raw starch-digesting glucoamylase from Athelia rolfsii growing in culture medium containing corn starch and corn soak solution were determined by single factor tests and response surface methodology.The optimal fermentation conditions were,fermentation temperature 38 ?,fermentation time 81 h,shaking speed 215 r/min.The primary enzyme production was 26.2386U/mL.The protein?enzyme?in the study was identified by Nanotechnology-High Performance Liquid Chromatography and multi-stage series ion trap MS(NanoLC-ESI-MS/MS.The analysis results of NanoLC-ESI-MS/MS were used to search against the non-redundant protein database?NR database,NCBI?to show that the protein?enzyme?from Athelia rolfsii were glucoamylase,the molecular weight was 61966.69 u,the number of peptide was 51,the relative abundance was 97.9%.The crude enzyme of raw starch-digesting glucoamylase from Athelia rolfsii was purified by ammonium sulfate precipitation,ultrafiltration membrane package removing salt,freeze concentration,Sephadex G75 chromatography to reach electrophoresis grade purity.Then the enzyme was determined as amylase by Native-PAGE and the molecular weight was determined in the scope of 53000-66000 u by SDS-PAGE.The characterization results of enzymatic properties showed that the raw starch-digesting glucoamylase from Athelia rolfsii enzyme was used for hydrolysis of raw corn,wheat,cassava,sweet potato,pea starch granules at 30?;the optimum reaction temperature was 60?,the thermal stability is good,the optimum reaction pH is 5.5,acid resistance is good;Mg²⁺,Ca²⁺and Mn²⁺could promote the enzymatic activity of the raw starch-digesting glucoamylase,Cu²⁺ and Ni²⁺ had significant inhibitory effect on the enzyme activity,the effects of different concentrations of Fe²⁺ on the activity of the raw starch-digesting glucoamylase were promoted and inhibited;Km was determined as 2.5mg/mL and Vmax was determined as 1.2mg/mL·min according to the kinetic study of the raw starch-digesting glucoamylase;the results of hydrolysis degree determination of raw starch-digesting glucoamylase showed that the enzyme had the best hydrolysis effect on raw corn starch and the worst hydrolysis effect on raw sweet potato starch;the hydrolysis effects of raw starch-digesting glucoamylase on corn starch and soluble starch were better than commericial ?-amylase.