Biotech M pharma 2nd send' dr harisingh gour sagar university

1. Bacterial Enzymes,
Industrial Enzymes
And Production Of
Enzymes

2. BACTERIAL ENZYMES, INDUSTRIAL ENZYMES AND
PRODUCTION OF ENZYMES
Assignment of
PHARMACEUTICAL BIOTECHNOLOGY
PHS CC 1203
Session 2023-2024
Department of Pharmaceutical Sciences
Dr. HarisinghGour Vishwavidyalaya,Sagar, (M.P.)
(A Central University)
Supervisors:
PROF.UMESHK. PATIL
DR. UDITA AGRAWAL
DR.PRIYANKA JAIN
MR.SATYAM
SHYAMVISHWAKARMA
Submitted by:
ADARSH SHARMA
Y23254001

3. ACKNOWLEGEMENT
I sincerely appreciate the assistance and support I received from my guide and other faculty
members during my assignment PROF. UMESH K. PATIL , DR. UDITA AGRAWAL
,DR. PRIYANKA JAIN and MR. SATYAM SHYAMVISHWAKARMA.

4. CONTENT:
1. INTRODUCTION
2. STEPS INVOLVED IN PRODUCTION OF ENZYMES
3. BACTERIAL ENZYMES
4. INDUSTRIAL ENZYMES
5. REFERENCES

5. • ENZYMES:
Enzymes are colloidal, organic, polymer, proteinaceous substance that acts as biocatalyst and alters the speed of
any reaction.
• Enzymes have an active site .
• This active site is the place where the substrate binds with the enzymes and holds the substrate.
• Active site has specific shape due to tertiary structure of proteins.
1. INTRODUCTION

6. • STEPS:
2. PRODUCTION OF ENZYMES
(1.) Selection of microorganism
(2.) Isolation of microorganism
(3.) Strain Improvement
(4.) Formulation of Medium
(5.)Production Process
(6.) Recovery and purification of enzymes

7. (2.1) Selection of microorganism
• Microorganism should not be pathogenic.
• Raw material should be cheap.
• Fermentation time taken should be less.
• Organism should be able to produce maximum
quantities of enzymes in short time.

8. (2.2) Isolation of microorganism
• We always get microorganism in mixture of different strains. So, isolation becomes essential from the
mixture to obtain PURE CULTURE.
Importance of pure culture:
 Once purified, isolated species can be cultivated with the knowledge that only desired microorganism is being grown.
 Pure culture are correctly identified.
 Experiments with pure culture ensures same result regardless of how many times experiment is performed.
METHODS OF ISOLATION
 STREAK PLATE METHOD
 POUR PLATE METHOD
 SPREAD PLATE METHOD
 SERIAL DILUTION METHOD

9.  STREAK PLATE METHOD
• Widely used technique used to isolate a pure strain from
single species especially bacteria.
• Streak literally means “a long, thin line”: and the streak
plate method is a microbiological culture technique
where a sample is spread in a petri dish in the form of a
long, thin line over the surface of solid media.
• The sample is picked by using different tools, mostly
using a sterile inoculating loop or swab.
• The sample is placed over a surface of sterile solid media
at one edge of the petri dish and a smear is prepared.
• Using the tool, the smear is successively streaked over
the agar medium on different patterns..
• As the streaking proceeds, the inoculum is gradually
diluted to the point where bacterial cells are separated as
individual cells or as a colony-forming unit (CFU) at a
gap of a few millimeters.
• When these inoculated plates are incubated, the isolated
bacterium or a CFU will give rise to a well-isolated
colony. This will allow us to get a pure culture as well as
describe the colony morphology of the organism

10.  POUR PLATE METHOD
• The sample is either added to the Petri plate and then the
molten agar medium is poured over it, or the sample is
mixed with the molten agar medium prior to pouring.
• After pouring in the Petri plate, the plate must be swirled
quickly to properly mix the sample with the medium.
• The mixed medium is allowed to solidify and is
incubated under the suitable condition to grow the
microorganisms present in the sample.
• Following the incubation, the numbers of isolated
colonies are counted.

11.  SPREAD PLATE METHOD
• Spread Plate Method is one of the widely
used culture techniques in microbiology
laboratories due to its ease and simplicity.
• The spread plate method is a microbiological
laboratory technique for isolating and
counting the viable microorganisms present
in a liquid sample by spreading a certain
volume of the sample over an appropriate
solidified culture media.
• The sample in the spread plate method must
be liquid or in suspension. Before plating,
the samples are serially diluted.

12.  SERIAL DILUTION METHOD
• This method is implied for pure culture which generally doesn’t grow on solid media and grow only in liquid media.
• A microorganism that pre-dominates in mixed culture can be isolated in pure form by series of dilutions.
• The inoculum is subjected to serial dilutions in sterile liquid medium and a large number of tubes of sterile liquid medium are
inoculated with aliquots of serial dilutions.

13. (2.3) Strain improvement:
• Once the microorganism is selected and isolated,
strain improvement for optimizing enzyme
production can be done.
 It is done to provide desired qualities to
microorganism.
 To increase production of enzymes.
• It is performed by:
Physical Methods-
X-Rays
UV Methods
Chemical Methods

14. (2.4) Formulation of Medium:
• Culture media should contain all nutrients to support adequate growth of microorganisms
that results in adequate quantity production of enzymes.
• Ingredients of media:
 Readily available
 Low cost
 Nutritionally safe
 Growth of microorganism should be proper
• Composition of media:
a) Macronutrients- Magnesium (Mg), Sulphur(S),Potassium(K) etc.
b) Micronutrients- Iron(Fe), Zinc(Zn), Manganese(Mn) etc.
c) Carbohydrates- Sugars, Starch, Cellulose
d) Vitamins- Thiamine, Adenine
e) Amino acids
f) Hormones
g) Antibiotics
* Gelling Agent added if solid culture media is made.

15. Preparation of 1L of Culture Media:
(1.) 700 ml of tissue grade level water was taken.
(2.) A portion of above was taken and 100ml of
macronutrients.
(3.) Micronutrients was added above. Then it was sterilised
using autoclaving.
(4.) Adjust desired pH according to the growth of
microorganism.

16. (2.5) Production Process:
• It is carried out by:
(a) Submerged Culture
(b) Solid Substrate Culture
• In submerged solid media, the yield is more, and chances of infection is less.
• The solid substrate culture is historically important and used for fungal enzymes such as amylase, cellulase.
The fermentation is started by inoculating the medium.
The growth conditions (pH, temperature, O2 supply) are maintained at optimal levels.
The bioreactor system must be maintained sterile throughout the fermentation process.
Duration of fermentation is 2-7 days. Besides desired enzymes, several other metabolites are
produced. So, enzymes have to be recovered and purified.

17. (2.6) Recovery and Purification of Enzymes:
• The desired enzyme produced may be excreted into culture or may be present within the cells.
• Depending upon requirement, the enzyme is purified.
For release of intracellular enzymes:
 Sonication
 High pressure
 Osmotic shock
 Done for microbial cell disruption
Removal of Cell Debris:
 Filtration or Centrifugation can be used
Removal of Nucleic acids:
 They are precipitated and removed by adding Poly-Cation such as Polyamines.
Enzyme Precipitation:
Using ammonium sulphate salts and organic solvents (isopropanol, ethanol, acetone).
More enzyme Purification by:
 Ion-exchange chromatography
 Size-exclusion chromatography
 Affinity chromatography
Enzymes is dried and stored using freeze dryers.

18. OUTLINE OF PRODUCTION OF ENZYMES:

19. BACTERIAL ENZYMES:
LIST OF ENZYMES OBTAINED
FROM BACTERIA:
 AMYLASE
 PROTEASES
 LIPASE
 ESTERASE
 CELLULASE
 GLUCANASE
 XYLANASE
 GLUCOSE ISOMERASE
 ᵦ - LACTAM AMYLASE
APPLICATION
OF
BACTERIAL
ENZYMES:
STARCH
INDUSTRY
DETERGENT
INDUSTRY
FOOD
INDUSRTRY
TEXTILE
INDUSTRY
FINE
CHEMICALS
BREWING
AND
JUICES
PAPER
AND PULP

20. BACTERIAL ENZYMES:
SOURCES
OF
BACTERIA:
BACILLUS SUBTILIS
BACILLUS
SUBTHERMOPHILUS
BACILLUS
LICHENIFORMIS
BACILLUS
AMYLOLIQUEFACIENS
HALOBACILLUS
SPECIES

21. PROPERTIES BACTERIAL ENZYMES:
 AMYLASE  Starch hydrolysing activity
 Thermotolerant
 Thermostable
 Alkali resistant
 Cyclodextrin producing enzyme
 PROTEASES  Proteolytic activity
 Acidic
 Thermophilic
 Active in presence of inhibitory
compounds
 LIPASE  Lipolytic enzyme
 Fat splitting
 Stereoselectivity
 Racemic-resolution activity
 CELLULASE  Cellulolytic complex enzyme
 Saccharification of crystalline and
amorphous cellulose
 Thermostable

22. INDUSTRIAL ENZYMES:
1. TEXTILE INDUSTRY-
AMYLASE  For desizing of fabric like cotton
 Hydrolyses starch into soluble dextrin and oligo saccharide
CELLULASE  Biopolishing of cellulosic fabrics under acidic conditions
 Partially digests excess yarns, loosening them from fabric
PECTINASE  Bio-scouring of cellulosic fabrics under alkaline conditions
 Hydrolyses pectin and associated hemicellulose matter from fabrics thus assisting eco-friendly
removal of waxes from fabrics
CATALASE  Breaks hydrogen peroxide into nascent oxygen and water
 Used for bleach cleanup.

23. 2. DETERGENT INDUSTRY-
ALKALINE
PROTEASE
 Decomposes protein-based stains like blood, mucus etc.
ALKALINE
AMYLASE
 Automatic dish-washing liquid detergent formulations to decompose starch-based stains like
potato, food, carbohydrates etc
ALKALINE LIPASE  Decomposes fatty based stains like fats, butter, salad etc.
ALKALINE
CELLULASE
 Degradation of cellulose and Modifying structure of cellulose fibre to increase colour brightness
3. PULP AND PAPER INDUSTRY-
CELLULASE  Pulp cleanliness
 Improves drainage
LIPASE  Allows secondary fibre to loosen up, releasing embedded ink with reduced usage of detergents
LIGNINASE  Removes lignin to soften paper
LACCASE  Bleach to improve brightness

24. 4. LEATHER INDUSTRY-
ALKALINE AND
ACID PROTEASE
 Removes unwanted proteins, materials like elastin, albumin, mucoids, globulins without damaging
collagen.
ALKALINE AND
ACID LIPASE
 Hydrolyses insoluble fat and oil matter into soluble fatty acids and glycerol giving high degreasing
performance.
5. STARCH AND SUGAR INDUSTRY -
AMYLASE  Hydrolyses alpha-1,4-glucosidic bonds to reduce viscosity of gelatinized starch, producing soluble
dextrin.
GLUCOAMYLASE  To saccharify liquified starch from various sources such as corn, wheat .
 Resultant are glucose rich syrups.
DEXTRANASE FOR
SUGAR INDUSTRY
 Dextran are undesirable compounds in sugar production which reduces viscosity and reduces
industrial recovery.
GLUCOSE
ISOMERASE
 Catalyses isomerization of glucose to fructose.

25. 6. DAIRY INDUSTRY-
CHYMOSIN,
LYSOZYME
 Cheese manufacturing
LIPASE  Enhances ripening of blue Mold cheese
7. BAKING INDUSTRY -
AMYLASE  For starch modification
XYLANASE  To break down Xylan.
8. ANIMAL FEED INDUSTRY -
PHYTASE  For breakdown of phytic acid
 Increases digestibility of feeds
BETA-GLUCANASE  To break down beta-glucans present in Animal Feed

26. REFERENCES:
1. Vyas S.P, Dixit V.P, “Pharmaceutical biotechnology”, CBS
publishers and distributors, 1st edition, 1998, Pg no.288-296
2. Gad S.C, “Handbook of pharmaceutical biotechnology”,
Wiley publications, 2007, Pg.no.691-698
3. Smith J.E, “Biotechnology”, Cambridge publications, 5th
edition, 1995, Pg.no 73-88
4. Sharma A.K, Beniwal V, “Industrial Enzymes”, Nova
publications, 2014, Pg.no 15-49
5. Quax w, “Bacterial enzymes”, Prokaryotes(2006), 1;777-796