Preparation of penicillinase enzyme

1. PREPARATION AND USE
OF PENICILLINASE
ENZYME

2. ENZYME PRODUCTION
USING MICROBES
 Enzymes can be produced commercially making use of various
microbes, which are the most significant and convenient sources.
Rich quantities of enzymes can be produced under suitable growth
conditions using the microorganisms. They can be produced using
inexpensive media within a short time period. It is also easy to
manipulate the microorganisms in genetic engineering techniques so
that the production of desired enzymes can be increased.
EXAMPLE
Enzyme Source Application
Penicillinase Bacillus subtillis Penicillin removal.
Lipases Candida lipolytica
Aspergillus niger
Production of cheese, flavour
production.
Protease Aspergillus niger Digestive aid.

3. WHAT IS PENICILLINASE
ENZYME?
 Penicillinase is a bacterial enzyme that inactivates most of the penicillins. It is
an extracellular enzyme produced by the members of the Coliform group of
bacteria, by the Bacillus species, and some of the strains of Staphylococcus.
 Penicillinases are also known as B-Lactamase I, B-
LactamaseII,Cephalosporinase ,Penicillin amido-B-lactam hydrolase. Normally,
this is a group of enzymes with varying specificity for hydrolysis of B-lactam
complexes.
 The enzyme hydrolyses the penicillin into penicilloic acid and breaks beta
lactam ring and provide resistance to bacteria towards Beta lactam antibiotics
like penicillin, cephalosporins And carbapenems.
 CHEMICAL REACTION

4. PRODUCTION OF PENICILLINASE
ENZYME
 Steps in production
1. SELECTION OF MICROORGANISM - Bacillus cereus isolated from the
soil sample can be used for the production of Penicillinases.
2. FORMULATION OF MEDIUM– For the growth of microorganisms
culture medium adjusted at ph 9 with 10% Sodium bicarbonate
(NaHCO3). Content of medium is given below-
Chemical Quantity(gm/L)
Glycerol 2
Polypeptone 5
Yeast extract 5
K2HPo4 1
MgSO4 , 4H2O 0.2

5. 3)PRODUCTION PROCESS
1. 19 hours old inoculum culture used for fermentation.
2. Glycose or glycerol 0.2% added for enhancement of enzyme activity.
3. Keeping at PH-9 and temp. 30°c for 23 hours on rotatary shaker.
4. Benzyl penicillin used as an inducer.
5. Time – 4-5 days.
4)RECOVERY AND PURIFICATION OF ENZYME
1. 5 days culture is centrifuged and the cell debris is discarded to separate
the filtrate.
2. 1 liter quantity of filtrate at PH 6.5 was stirred in the cold for 30min with
50g of hyflo supercell and the adsorbing agent collected by filtration.
3. supercell was suspended in 500 ml of ammonia water with mechanical
stirring and carried out in the cold (4 degree centrigrade )for 30 min.
4. Add ammonium sulphate to precipitate out the enzyme as tiny floculs
which removed by filtration.

6.  Dry it using freeze dryer (lypophilization ).
 The final product obtained is grayish brown in color and of a light
,flaky or leathery consistency.

7. USES
 MEDICAL USES: penicillinase used in treatment of Penicillin
induced allergies like serum sickness, Acute arterial and non
arterial etc.
 ANALYTICAL USES : For measuring human prolactin in plasma.
 Sterility testing of bulk antibiotics.
 For microbiological cleanliness of antibiotics.

8. REFERENC
E
 Biotechnoligy by u .satyanarayan
 Pharmaceutical biotechnology fundamentals and application by Ashutosh kar and
k .sambamur .
 P. V. PHARMACEUTICAL BIOTECHNOLOGY.
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