Purification And Physical-Chemical Characteristic Of A Type Of Antifreeze Protein From Wheat Bran (wbAFP)

Antifreeze proteins(AFP)are a family of protein which has three basic characteristics,thermal hysteresis activity,inhibit recrystallization of ice and modify the growth of ice.Binding on the surface of ice,AFP can lower the freezing temperature non-colligatively without affecting the melting temperature(usually to be 0?).The gap between the freezing and melting temperature is known as Thermal Hysteresis Activity(THA).AFP could be divided to four categories,antifreeze protein in fish,plant,insects,bacteria and fungi.Antifreeze protein has been successfully used food industry,medicine industry and agriculture industry.In this paper,wheat bran was used as the research object,wheat bran antifreeze protein(wbAFP)with THA was extracted,the crude protein was purified,and the secondary structure was studied.The ratio of solvent to material,time of extraction,PBS pH,PBS concentration and saturated ammonium sulfate concentration were studied to find the relationship with protein extraction rate and THA.In addition to the extraction time,other four factors have something with protein extraction rate and THA.And on this basis,the central composite experiments and response surface analysis were used to optimize these factors.The experiments results showed that 41.46% protein extraction ratio and THA 0.0195 ? were obtain under the optimized condition of ratio of solvent to material 7.5:1,PBS pH 7.8,PBS concentration 29mmol/L and saturated ammonium sulfate concentration 100%.The actual protein extraction ratio was 41.98%,and THA 0.019 ?,which showed that the optimized conditions were accurate.This AFP was purified by ammonium sulfate precipitation,anion-exchange chromatography on a DEAE-cellulose-26 column,and size-exclusion chromatography on a Sephadex G75 column and two fractions were got.The result of SDS-PAGE confirmed that these two fractions were the same protein and the molecular weight is 11.2 kDa.The THA of this AFP is 0.169? at a concentration of 5mg/mL.As a result,AFP was purified 449.99 fold,yielding a productivity of 4.17%.The amino acid composition of AFP was determined.The relative molecular mass of these amino acid residues was 11502 Da,which is similar with the result of SDS-PAGE(11.2 kDa).This AFP is glycine-rich(52.08%).It was compared with several common Antifreeze Glycoproteins(AFGPs)and was inferred that this AFP is related with cold-inducible protein.Thermal gravimetric analysis was used to determine the hydrophilicity,and it was found that this AFP had a relatively high hydrophilicity compared with BSA,which was in accordance with the result of amino acid composition.The Secondary structural compositions were determined by Circular Dichroism(CD),FT-IR spectra and Raman spectra.It was found that the results of FT-IR spectra and Raman spectra were dovetailed,but the result of CD was relatively differed with another two results.The results were summarized as ?-helix of 25-32%,?-sheet of 15-24%,?-turn of 33-38% and random coil of 13-18%.