nyenaceyg
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 Wysłany: Pią 22:18, 14 Sty 2011 Temat postu: Proteolytic enzyme activity _2123 new method for d |
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Determination of proteolytic enzyme activity of new methods
L) 37 ℃ preheating 10rain, adding a certain amount of the enzyme solution preheated 5min. Reaction after a certain time, directly into the 5mL solution of Coomassie brilliant blue. In the 0.1-1h, the determination of 595mm light absorption. To the same solution without enzyme as control reaction; to without enzyme and without the same reaction of casein solution as blank. Ordinate for the As. s spider reduce the value of the abscissa and the amount of enzyme or reaction time. Coomassie brilliant blue solution preparation: lOOmg Coomassie brilliant blue G250 dissolved in 50mL95 ethanol,[link widoczny dla zalogowanych], adding lOOmL85 phosphoric acid, and finally with distilled water to lOOmL. Results and discussion was determined by the above-mentioned five proteolytic enzymes on casein hydrolysis and enzyme concentration. Such as Fig. ① Received t1993 1-5 by a O6-25, Revised draft date 19,981,021,630 Gen said, the more sensitive method, can be detected 0.05t ~ g of protein enzymes. If prolonging the reaction time to the lh,[link widoczny dla zalogowanych], the sensitivity can be improved. {Ee (g) F. 1Eazym ~ 0.15mol/LNBCLTemp: 37 ℃ 2 Division 00.20.40.60.8Enzyme (g) F.2Enzyme: ChymotrypsiFReaet [onperiod: lOminBuffer, 50retool card .6 Trls-HC1.Temp: 37 ℃; l Secretary-yme (g) .4 E ~ yra ~ {SuBtili ~ iaFReactionperio [I10minBuffer: 50 ~ ol/LpH7.6Tria-HCI.Temp: 37 ℃ at the optimum pH and trypsin 37 ~ C the reaction lOrain,[link widoczny dla zalogowanych], the amount of trypsin is proportional to the reduction in the amount of 595nm absorbance (A595). linear the range of 0.05 a O.4, ug. elastase, and subtilisin in the 0.1-1.0 and 0.1-1.6g Fig.6 show a good linear relationship is subtilisin CH time course of protein. This method measured the results with traditional methods have a good correspondence between 0. measured by the method of Bacillus subtilis and papain activities were 376.OZU/mg protein and 184.70U/mg protein ( 1 unit is defined as 1 mg per hour out of casein hydrolysis). measured using traditional methods these two enzyme activities were 3.69U/rag protein and 1.84U/rag protein. the stability of this method time for a 60min 2O . the same sample parallel to the Shu-five tubes, the reproducibility of the A values were 0.6840.680,[link widoczny dla zalogowanych],0,679,0.679,0.681. 631l I Time (rain) F · 5Enzyme ~ papainIRe ~ 'tionp0d {20minlF.6EnzFrae: Subm | Buffer {50mmol/LpH7 · 6Tri ~ HC1.2mmol/LCys.Buffer {50mmcl/LpH7.6TnHCI.O · lmmol/LEDTA.Temp.I37 \
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