Study On The Degradation Mechanism Of Silver Carp Myofibrillar Protein By Alkali Protease From Bacillus Licheniformis

Generally,high nutrient content and high moisture content of aquatic would contribute to microorganisms growth,therefore,microorganisms would be one of major factors of aquatic spoilage.The degradation mechanism of alkali protease of bacillus licheniformis was investigated in this study,and the platform of two-dimensional electrophoresis(2-DE)of sliver carp myofibrillar proteins was established.Using proteomics to research the degradation pathway of myofibrillar proteins of sliver carp which degradated by alkali protease of bacillus licheniformis,and revealed the initial spoilage mechanism induced by spoilage organisms.The main results were summarised as follows:1.The enzyme characterization of alkali protease of bacillus licheniformis was determined by conventional methods,including optimal temperature,optimal pH,temperature stability,pH stability and effect of inhibitors on enzyme activity.The results showed that:the optimal temperature of alkali protease was about 50?,the optimal pH was about 10.And the temperature stability showed that after treated in 80? for 0.5 h,the enzyme activity decreased to 1.9%.The enzyme activity was relatively stable at pH 8?11.The inhibitor of PMSF had a good suppression effect on alkali protease,after treated by 50 mmol/L PMSF,the enzyme activity decreased to 0.34%.2.The changes of structure and property of myofibrillar proteins of sliver carp treated by alkali protease were determined by some indexes as content of sulfhydryl group and surface hydrophobicity,and the propertis of gel formed by myofibrillar proteins treated by alkali protease were determined by methods of texture profile analysis and gel strength.The results showed that surface hydrophobicity increased then decreased,which touched the maximum at 2 h for 25? and 2.5 h for 4?,respectively;the activity of Ca2+-ATPase also increased then decreased,and obtained the max value at 0.5 h for both temperature;the content of sulfhydryl group increased then decreased as changes of surface hydrophobicity.The water holding capacity and gel strength decreased;the peak ratio of T21 and T22 increased then decreased,and the peak ratio of T23 decreased,the peak ratio of T24 increased;the whiteness of gel decreased then increased;the microstructure showed that network formed by myofibrillar proteins was ruptured by alkali protease gradually.3.The platform of 2-DE for sliver carp myofibrillar proteins was estabilished.The key process of isoelectric focusing(IEF)and the pH gradient of immobilized pH gradients(IPG)strip etc were selected as the main causes of 2-DE.The gels were stained with sliver nitrate,scanned and analyzed using PDQuest software,a high resolution map of myofibrillar proteins of sliver carp was obtained finally,and this research would be useful for qualitative and quantitative investigation of myofibrillar proteins of sliver carp.4.Using the platform of 2-DE to study degradation of myofibrillar proteins of sliver carp treated by alkali protease at 4? and 25?,respectively.With treatment time increasing,number of spots increased then decreased.Finally,75 spots were matched by software PDQuest,and using matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS)to identify 24 spots,evidencing actin,myosin heavy chain(MHC)etc.Bioinformatics for these 24 spots were analyzed by GO functional annotation.Combined cellular component,molecular function,biological process with biochemical indexes above,pathway of degradation of sliver carp myofibrillar proteins treated by alkali protease was speculated:firstly skeleton structure of myofibrillar protein was destroied,actomyosin disintegrated to actin and myosin,then actin and myosin were degraded into derivatives such as MHC etc.