Study On Removal Of Protein From Chitin And Chitosan

The residual protein in chitin and chitosan limits its use in biomedical applications,the purpose of this paper is to remove residual proteins in chitin and chitosan by different methods.Screened the most efficient enzymes from several different proteases and optimized its conditions;the effects of different pretreatment combined with protease on protein removal were studied;attempted to remove residual proteins with low concentrations of sodium hydroxide and various denaturants,surfactants and other chemical reagents;and compared the effects of different removal methods on the properties of chitin and chitosan;the molecular weight range of the protein in chitosan was determined.The main results were as follows:(1)The effects of proteases such as acid protease?alkaline protease?papain? pepsin?neutral protease?flavor protease and trypsin on the removal of protein from chitin were compared,found that alkaline protease is most effective on deproteinization among all proteases tested,on this basis,the conditions of protease were further optimized,the results were as follows: temperature 45 ?,reaction pH 8.5,solid-liquid ratio 1:15,reaction time 4h,enzyme addition 0.4%(3500 u/g),under such conditions,the residual protein in chitin decreased from 5.91% to 3.21%,the removal rate was 45.68%.(2)The effects of low concentration NaOH solution?denaturants?surfactants and other chemical reagents on the removal of protein in chitin was studied.Orthogonal test results showed that 2% NaOH solution could effectively remove the protein in chitin,reduced the residual protein from 5.91% to 0.37%,and the removal rate was 93.7%;the effects of different reagents on the removal of protein from chitin were compared,found that sodium sulphite is the most effective on deproteinization,reduced the residual protein in chitin from 5.91% to 3.99%,the removal rate was 32.49%;Subcritical water and ultrasound can effectively promote the protease catalysis,the protein removal rate by protease in chitin treated by subcritical water was 46.6%,in chitin treated by ultrasound was 45.67%,the protein removal rate is higher than only use protease.(3)The degree of deacetylation,crystallinity and protein secondary structure of chitin treated by different methods were analyzed.From the chitin infrared spectrum we can see the degree of deacetylation of chitin treated by different methods were increased,and chitin treated by subcritical water had the highest degree of deacetylation,followed by protease treatment of chitin;from the X-ray diffraction results we can see that the crystallinity and grain size of chitin treated by subcritical water and sodium hydroxide solution were significantly reduced,and subcritical water treatment of chitin had a smaller degree of crystallinity,while the grain size of chitin treated by sodium hydroxide solution was smaller.(4)The accuracy of bis-quinoline carboxylic acid method(BCA)?coomassie brilliant blue method and NaOH solution extraction protein on the determination of protein in chitosan were studied,the results showed that: the alkaline color reagent made chitosan precipitation,and the results was not accurate;for coomassie brilliant blue method,chitosan would interfered with the results,affected the accuracy of determination;finally,the residual amount of protein was determined to be 0.414% by the lye extraction protein method.(5)The effects of proteases such as acidic protease?alkaline protease?papain? flavor protease and complex protease on the removal of protein from chitin were compared,found that alkaline protease was most effective on deproteinization among all proteases tested,on this basis,the conditions of protease were further optimized,the results were as follows: temperature 50 ?,reaction pH 9.0,solid-liquid ratio 1:25,reaction time 4 h,enzyme addition 0.4%(3500 u/g),under such conditions,the residual protein in chitosan decreased from 0.414% to 0.322%.;the effects of low concentration sodium hydroxide solution(1%?2%? 3%)on the removal of protein from chitosan were studied,derived from the orthogonal test results,1% sodium hydroxide solution could effectively removed the protein in chitosan to 0.22%;compared the effects of different reagents on the removal of protein from chitosan,found that urea was most effective on deproteinization,reduced the residual protein in chitosan to 0.338%;the result of removal of protein from chitosan by ultrasound treatment combined with protease showed that the amount of protein residue in chitosan could be reduced to 0.304%,the protein removal rate is higher than only use protease.(6)The degree of deacetylation and viscosity of chitosan treated by different methods were analyzed,the results showed that the degree of deacetylation of untreated chitosan was 87.64%,and it would increased treated by protease and urea,wherein the degree of deacetylation of the urea-treated chitosan increased to 91.01%,followed by protease-treated chitosan,the degree of deacetylation increased to 89.38%;the viscosity of untreated chitosan was 14.17 mPa·s,the viscosity of chitosan treated by protease and sodium hydroxide would reduced to14.00 mPa · s and 13.17 mPa · s respectively,while the viscosity of urea-treated chitosan increased to 17 mPa · s.(7)C hitosan was degraded by sodium nitrite and hydrochloric acid,the molecular weight range of the residual protein was determined to be 1700~2200 Da by tricine SDS-PAGE,chemical analysis gave an average molecular weight of 554 Da.