Purification,Properties And Application Of Protease From Monopterus Albus Zuiew

Monopterus albus Zuiew is typical flesh-eating fish, and as a source of meat, it is very delicious and nutritious. Consumers are so like to eat it that the supply of it is inadequate . the protease is one of the most important digestive enzyme .To find a solution to the contradiction,the study of the protease from Monopterus albus is important,because it not noly makes us know assimilation character of the Monopterus albus,but also afford some informations that conduce to man-made breeding of it.So this experiment have great significance in enhancing output, economizing cost and resolving outstanding contradiction of Monopterus albus.Protease is one of important industry enzymatic preparations, which is abroad applied in food, chemical industry and medicine etc..At present, because people are more and more interest in natural and healthy consumption, and detergents that contain phosphorus and food additives are forbided using step by step, natural enzymatic preparation will be used more abroad.But the natural source of protease is shortage, especially animal protease,in the light of which ,it is only applied in medicine.Taken together, it is urgent to explore new source of protease..As offals,the guts of Monopterus albus weren't be taken full advantage all the time,while the outputs of Monopterus albus constantly increase, and there are 0.3kg bowels from 1kg Monopterus albus,That is,there are a million tons of guts in our country every year.If this huge resource isn't made good use, it will not only be wasted, but contaminate environment.Therefore,the study of protease from the guts of Monopterus albus can effectively solve deficiency of protease,and give a new way to integratively explore Monopterus albus as well.This article studied on purification, properties and some applications of protease. the results of study as follows: 1.Purification of Protease from Monopterus albus ZuiewAfter mash.deposition of (NH)4SO2, DEAE-Sepharose chromatography and Sephacry S-200 gel chromatography, the homogeneous protease from Monopterus albus was acquired. There is only one strip after SDS-PAGE and IEF,which further proved the protease is homogeneous. The molecular weight is 26 KDa and 25.5 KDa by SDS-PAGE and Sephacryl S-200 gel chromatography and the isoelectric point of enzyme is PH 6.2.The recovery of purified protease is 9.8% and the purified fold is 245.2. Characterization researchThe protease showed optimum activity at 55 °C and pH10.5 and it is stable at 20°C-40°C and pH 7- pH12 respectively. Using the casein as substrate,the Km is 0.55%(W7V).3. Chemical Modification of enzymeThe protease of Monopterus albus is a kind of alkaline protease. In order to investigate the relationship of the structure and function of it, the effects of protein modification reagents , EDTA and metal ions on the protease activity have been studied. The enzyme activity was significantly decreased after modified by NBS, I-AC, TNBS, BrAcandPMSF, and PCMB and 2-ME didn't obviously effect enzyme activity. Cu2 \ Ba2*can activate enzyme activity while Pb2+, Mn2+ and EDTA can strongly inhibit it. MgJ* > Li *and K+ only slightly inhibit enzyme activity, and Ca +and Na+have no effects on enzyme activity.4. Application Properties of the ProteaseZymography: Zymography analysis of the extract from the the compound of the guts of Monopterus albus suggested that three distinct proteases were present.one strip of those is identical with the strip of the purified enzyme from bowels by SDS-PAGE.So we can forecast that the identical protease can gain from the compound of the guts.Effects of store temperature and time on activity of protease: The activity of the crude enzyme rised frist ,and then decreased after a week at 4'Cand 25°C respectively.The dry enzyme that was purified is stable at 4'C and 25 "C, while it is unstable in the water at 4°C and 25°C. Effects of four surfactants and some chemistry reagents on protease:The alkaline protease is a important ingredient of detergent.To study the cooperation of enzyme with surfactant, four surfactants including LAS, SDS, CHAPS. Triton X-100 were used in this study.The result suggested that the enzyme is stable in surfactant solution and four surfactants didn't inhibit the activity of it. The common thickening agents ,such as Soil temperature80, Agar, Poly vinyl alcohol,...