Kinetic determination of fruit bromelain

, the purpose
is a biocatalyst produced by
cells
proteins and proteins as the main component. Enzymes catalyz various chemical reactions in organisms are carried out in an orderly and efficient manner. Enzyme reaction dynamics studies the effects of various factors on reaction speed, including substrate concentration, enzyme concentration, type and concentration of inhibitors, pH and temperature. The most basic dynamic relationship is the effect of substrate concentration on the speed of enzymatic reaction. In the simplest case of no inhibitors, the Miyman equation correctly describes this dynamic relationship:reaction velocity v and substrate concentration ( S) have a one-to-one correspondence, in order to deeply understand an enzyme, the key is to determine its maximum reaction speed V
max
and the Meter constant K
m
. By deforming the Mi-Man equation, 4 methods can be found to find the V
max
and K
m
. Among the more commonly used is the Lineweaver-Burk graphing method (double countdown graph method), which is based on the deformation equation:




V
max
. This experiment takes fruit pineapple
protease
crude products as the experimental material, first purifys the enzyme with ion exchange column layer analysis, and then through the time process curve, enzyme activity, protein content determination, to find the K
m
value, to deepen the understanding of enzyme activity, purification than live protein, master the general principles and methods of enzyme dynamics determination.
2, principle
fruit pineapple protease is a plant -based protease, a single peptide chain, containing 4% of the sugar components, 4 degrees C isoelectrine point pI is 9.4, molecular mass of about 28,100. In order to shorten purification time and prevent or reduce self-degradation, DEAE one cellulose ion exchange layer can be used to purify it as the first exculation peak to be purified.
Fruit pineapple protease can hydrolysate the substrate casein into a small peptide that can be dissolved in the solution of triclosan, after a certain period of time with triclosan to terminate the enzyme reaction, with the small peptide generated in the filter to make the added value of the absorbent to indicate the speed of the enzymatic reaction. Based on this, the time process curve of the enzymatic reaction can be made to determine the range of the
V
the reaction. The protein content of crude and purified enzyme fluids can be determined by the Coomas Bright Blue G-250 method. Based on this, the return yield of
can be calculated.