| The main target of this study was to evaluate protein extraction technics from Chimonanthus praecox L.flowers,which were traditionally regarded as highly edible and officinal materials.Fristly, the native protein was extracted from the Chimonanthus praecox L.flower in buffer.Then,albumen was precipitated from the extracted solution by adding acetone at frozen temperature,combined with salting out and dialysis.By this way,the native protein concentrate was prepared.Further studies were conducted to investigate the anti-oxidation ability,amylase and proteinase activities of the native protein concentrate.Then,α-amylase was purified and its activity in different temperatures and pH were investigated.Therefore,theoretical foundation has been established for further exploitation of the Chimonanthus praecox L.flower resource.The experimental results of this study are as follows:(1)Concentrating techniques of native protein were studied.The extraction rates of native protein by acetone washing and direct extracting with buffer were compared.Experimental results showed that the extraction rate of protein by directly extracting with buffer would be better than that by acetone washing.Further study was also undertaken to investigate the effect of ionic strength,pH, temperature,sample-solution ratio and time on the extraction rate of native protein.Purifications of crude protein concentrate by acetone washing,ammonium sulfate salting out and dialysis were also studied.It was found that the optimal conditions for the extraction of native proteins would be:0.2 mol/L NaCl,pH 7.2,25℃,solid/liquid 1:6,4h and under these conditions,31%protein was extracted.The purified product contains 66%protein.(2)Anti-oxidation ability as well as amylase and proteinase activities of the native protein concentrate were investigated.The aim of this study was to evaluate the biology functional properties of the native protein concentrate.Experimental results showed that anti-oxidation ability changed a little bit when proteins in the native protein concentrate were precipitated by TCA and separated.This result may tell that the prepared native protein concentrate is not a very good anti-oxidant.However,more studies would be necessary for finding single protein,which owns high anti-oxidation ability.Studies on amylase and proteinase activities of the native protein concentrate revealed that they were purified 3.62 and 1.49 times,respectively.Their activities were 262.94U·mg-1and 12.32 U·mg-1,respectively.(3)The amylase activity of the native protein concentrate from Chimonanthus praecox L. flower is 262.94U·mg-1.Based on this finding,α-amylase was further purified and the best temperature and pH conditions for the purification were studied.When further purification of the native protein concentrate with acetone washing was carried out followed by sectional salting out, dialysis and the purified the native protein concentrate was dried by polyethylene glycol 20000 followed by chromatographic isolation on a DEAE-52cellulose column,theα-amylase was purified 7.4 times,and its recovery rate is 10.4%.Experimental results showed that the best pH forα-amylase absorption by DEAE-52 cellulose was 7.5 and the best solvent for washing outα-amylase from the DEAE-52 cellulose column was 0.5mol/L NaCl.Theα-amylase obtained is most active at 40℃and pH 4.It is quite active at 20～70℃,pH3～8.By this study,theoretical foundation has been established for further exploitation of Chimonanthus praecox L.flower resource,especially for the preparation of the native protein concentrate.Experimental results obviously showed that theα-amylase adapts to wide range of temperature and pH,and this implies that the native protein concentrate possesses a bright future of application to industry.
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