This website is written by experts in Pharmaceutical Industry Manufacturing Research and Development and Regulatory Affairs Since 2008 and in 2011 Awarded as one of top 10 websites across world in Pharmaceutical Manufacturing Regulatory
USFDA WHO GMP Guidelines Pharmaceutical Regulations Information about Pharma Quality Managment System Validations Process Validation Cleaning Validation Equipment Qualification Equipment Process Validation Computer System Validation Sterile Product Manufacturing Guidelines Media fill guideline Aseptic Techniques in Sterile dosage form Clean Room Clean Room Classification Guidelines on Area Qualification HVAC AHU HEPA Filter HEPA Filter integrity testing and leak test

Sunday, November 22, 2009

How to determine enzyme activity rate contant Michaela men ten constant KM and maximum velocity of reaction, or maximum enzyme activity V max of a enzyme example B- Amylase

Determination of km and V max of B- Amylase

To determination of km and V max of B-Amylase.

Theory: - B-amylase form higher plants catalyses the hydrolysis of the and 1- 4 glycoside linkage in the polysaccharides to give maltase. It is an example exoenzyme and therefore splits off maltose unit from free non reducing end of the chain.
If the activity of an enzyme is determined over a range of substrate concentration and graph is plotted. A curve similar to the rectangular hyperbola is obtained.

An equation relating activity [s] was first derived by Michaels Men ten.



Michaela men ten constant, km is a characteristic of each enzyme. It is independent of either enzyme or substrate concentration and many be obtained as the substrate concentration at high maximum velocity of reaction. Activity of B-amylase is determined by estimating the product (reducing sugar maltose) obtained. One of the regents used for estimating reducing sugar is 3, 5 DNA. In alkaline solution.



Product obtained after reduction is 3 – amino – 5 – nitro salicylic acid which is orange red in colour. The colour thus produced depends upon the concentration of reducing sugar and is measured calorimetrically at 540 nm.


Requirements: - 1) 5% stench solution. 2) DNS reagent.
3) 0.5% sodium chloride. 4) Buffer at PH 4.8
5) Amylase extract.


Procedure: - Proper two sets of test tubes (one set for sample and other for blank) as follows.
To one set of test tubes add 1ml of enzyme extract and incubate at 370C for 30 mins. After incubation add 1ml of DNS reagent, 1ml NAOH (bench reagent) and 2ml of water. To the second set add 1ml of DNS reagent, 1ml NaCH and then 1ml of enzyme extract. Add 2ml of distilled water to all the tubes. Heat both sets of test tubes in the boiling water bath for 10 mins. With a marble on top prevent loss of water by evaporation, cool the solution and dilute 1ml to 10ml and determine absorbance using respective blanks. Plot the graph of absorbance against [s] and determine km and Vmax.





===================================================================
This website is a guide for pharmaceutical manufacturing and for pharmacy students and pharmaceutical regulatory affairs proffesionals and for every one associated with pharmaceutical industry , we recommend you to subscribe to this website by entering your email ID in the box provided so that you will receive our articles as and when our website is udated.

This website is most favorite Amongst proffesionals in almost all pharmaceutical companies in USA , Pharmaceutical companies in UK , Pharmaceutical companies in Japan , Canada , UAE , Pharmaceutical companies in India 
Following are some of articles which;will be useful for you in further understanding of aspects of sterile dosage form manufacturing

Pharmaceutical Validation

Cleen Room Classification

Pharmaceutical Industry

Pharmaceutical Manufacturing

Clinical Trials

Media fill

Regulatory Affairs in Pharmaceutical Industry

No comments:

Pharma Guidelines Most Read

.

Readers Visit Number

This website is Authentic Source of Pharmaceutical Guidelines

This website is Authentic Source of Pharmaceutical Guidelines

Pharma Validations Quality Management System USFDA Guidelines WHO GMP Guidelines New Drug Update





This website consist of detail Information about Pharma Guidelines Pharma Regulatory Affairs USFDA Guidelines WHO Guidelines Pharmaceutical Quality Management System Pharma Validations Process Validation Cleaning Validation Equipment Qualification Equipment Process Validation Computer System Validation Sterile Product Manufacturing Guidelines Media fill guideline Aseptic Techniques in Sterile dosage form Clean Room Clean Room Classification Guidelines on Area Qualification HVAC AHU HEPA Filter HEPA Filter integrity testing and leak test