Good day,

Pectinase is complex sets of enzymes

Polygalacturonase is a hydrolase comes under pectinase, it will cleaves you water soluble polygalactouronic acid into galactouronic acid, similarly pectic lyase which uses different mechanism releases galactouronic acid.

The aim is to measure the galactouronic acid, which is a reducing sugar, you can test using DNA assay compare with known standard of galactouronic acid. You you will get amount of galactouronic acid. The following assay is for polygalactouronase.

the reaction mixture consisted of 0.5 ml of 1% soluble pectin dissolved in buffer (find a optimum buffer), and 0.5 ml of crude enzyme extract (without biomass). After 15 min of incubation at 50oC, the liberated reducing sugars (galacturonic acid equivalents) can be estimated by the dinitrosalicylic acid (DNS) method. One unit (IU) of pectinase was defined as the amount of enzyme releasing one μmolgalacturonic acid equivalent per minute under the assay conditions.

Specific Activity is Activity/ mg of protein present in the crude extract

you can find the conc. of protein by Biuret/ bradford method

-Good Luck

Specific activity is the measurement of the purity of enzyme.

In crude extract enzyme activity/ mg of protein is less, because the enzyme is not pure and has many other proteins. when you purify the enzyme, specific activity of the enzyme (enzyme activity/ mg protein) will increase. It is generally used to reprent the purity of the enzyme.  

Dear Bijesh,

I am agree with Dr. Mozhi Arasu and Rai. I suggest you  to  check the total soluble protein content by lowry method using bacillus culture that you have used then perform activity of pectinase with varying enzyme concentration, substrate concentration, different pH range, temparature and incubation time to find out the optimum condition of pectinase in bacillus culture. Maximum activity can be measured based on the maximum D-Galacturonic acid release at optimum level of above parameters. 

The specific activity of enzyme was expressed as the unit of enzyme activity per mg of protein per minute under the standard assay conditions”

                           Units per ml or gram of bacillus culture

   U/mg    =_______________________________________________

                       mg of protein per ml or gram of bacillus culture

Thank you all for your valuable suggestions