Separation,purification And Structural Characterization Of Selenoproteins From Selenium-enriched Agaricus Blazei Murrill

Selenium was an essential micronutrient for many organisms,and the bioavailability of organic selenium was higher than that of inorganic selenium.Exogenous supplementation played an important role in increasing the organic selenium content of organisms.As a high selenium-enriched plant,Agaricus blazei Murrill(ABM)can effectively biodemine selenium and convert inactive and highly toxic inorganic selenium into safe and effective organic selenium.Therefore,in this study,selenium-enriched ABM was used as raw material to analyze its protein content,amino acid composition,selenium form and selenium content in different selenium-containing proteins.On this basis,the selenoprotein was isolated,purified and structurally characterized,in order to provide technical parameters for the preparation of high quality selenoprotein and related product development.The main findings were as follows:(1)The protein content of Se-enriched ABM was 42.88%,which was rich in essential amino acids,and the glutamic acid was the highest.The first limiting amino acid was tryptophan.The total selenium content of selenium-enriched ABM was 9.150μg/g,of which organic selenium accounts for 85%,the main form was selenocystine,and inorganic selenium only contained selenite.(2)Selenium protein was extracted by fractional extraction of selenoprotein(water-soluble selenoprotein,salt-soluble selenoprotein,alcohol-soluble selenoprotein,alkali-soluble selenoprotein)and subcritical water extraction of selenoprotein.The selenium content,infrared structure and free radical scavenging activity of five selenoproteins were compared.Based on the single factor experiment,the extraction process of subcritical water extraction with selenoprotein was optimized by BBD response surface analysis.The conditions for extracting the selenoprotein of the ABM were obtained: extraction p H = 7.50,extraction time was 15 min,the extraction temperature is 167℃,and the ratio of material to liquid was30:1.The yield of selenoprotein of ABM reached a maximum of 42.738%.The order of Se content of the four selenoproteins was: water-soluble selenoprotein > salt-soluble selenoprotein > alkali-soluble selenoprotein > alcohol-soluble selenoprotein.The order of protein yield was: alkali soluble selenoprotein > water soluble selenoprotein > salt soluble selenoprotein > alcohol soluble selenoprotein.The yield of selenoprotein extracted by subcritical water was 1.5 times that of water-soluble selenoprotein,but the selenium content of subcritical water extract protein was only 60% of the water-soluble selenoprotein.(3)Fourier transform infrared spectroscopy results show that water-soluble selenoprotein,salt-soluble selenoprotein,alcohol-soluble selenoprotein and alkali-soluble selenoprotein were a variety of complex proteins,which had obvious characteristics in the characteristic absorption band.Further analysising the secondary structure of the five selenoproteins in the amide I band and the amide III band,it can be seen that the amino acid composition and content of the five selenoproteins were different.Infrared results indicated that the amino acid composition and content of the five selenoproteins were different.The high-low trend of the content of crude selenoprotein selenium had a good consistency with the anti-oxidation activity,and the effect of crude selenoprotein amino acid composition on its antioxidant activity was also obvious.Therefore,the water-soluble selenoprotein in selenium-enriched ABM was selected as a further research object.(4)The water-soluble selenoprotein was sequentially purified using a Q Sepharose Fast Flow strong anion exchange column and a Sephadex G-100 gel chromatography column.After SDS-PAGE electrophoresis purity testing,there were three bands named PR-Se-1,PR-Se-2 and PR-Se-3.After LC-MS/MS detection,the database was combined with de novo sequencing analysis to identify three selenoproteins.PR-Se-1 identified an unknown protein with a molecular weight of 114024.PR-Se-2 was identified as dihydrolipoyl dehydrogenase,and it was considered that sulfur was replaced by selenium into a selenium-containing enzyme;PR-Se-3 was a fusion of transaldolase.