This document provides an overview of enzymes, including their types, production methods, and industrial applications. It discusses the main types of enzymes like amylases, lipases, and pectinases. Amylases break down starch into sugars and have applications in food processing. Lipases split fats into fatty acids and are used in soap production. Pectinases break down pectin in plant cell walls. The document also covers enzyme production methods like submerged and semisolid fermentation and the microorganisms used to produce different industrial enzymes.
Polysaccharides produced by microorganism during their growth and especially at the stationary phase of growth when there is excess of carbon source in the medium.
High molecular weight carbohydrate polymers mainly produced by bacteria and fungi.
Microbial polysaccharides are of two types:
Storage polysaccharides like glycogen, inulin etc.
Exopolysaccarides like xanthans, dextrans, levans which are secreted by the cells.
Polysaccharides produced by microorganism during their growth and especially at the stationary phase of growth when there is excess of carbon source in the medium.
High molecular weight carbohydrate polymers mainly produced by bacteria and fungi.
Microbial polysaccharides are of two types:
Storage polysaccharides like glycogen, inulin etc.
Exopolysaccarides like xanthans, dextrans, levans which are secreted by the cells.
Glutamic acid, a key amino acid with diverse industrial applications, is primarily produced via microbial fermentation, particularly using strains of *Corynebacterium glutamicum* and *Bacillus subtilis*. These microorganisms are adept at synthesizing glutamic acid through various metabolic pathways, with glucose or other carbon sources serving as substrates. The fermentation process involves optimizing growth conditions such as pH, temperature, and nutrient availability to maximize yield. Glutamic acid production can be further enhanced through genetic engineering to improve strain productivity and tolerance to environmental stresses. The versatile nature of glutamic acid makes it a valuable component in food additives, pharmaceuticals, and biodegradable polymers, emphasizing the importance of efficient and sustainable production methods.
The presentation talks about the basics of bioprocess. Describes what is fermentation? Also lists the different modes of fermentation and the basis for selection of type of reactor. General requirements for a fermentation process. Components of a reactor
In this context, there is a need to use “biodetergent or biocleaners”, which offer a better option to the synthetic detergents with respect to their biodegradability, low toxicity, non-corrosiveness environmental-friendliness, enhanced cleaning properties and their increased efficiency and stability in different formulations.
To counter these limitations, enzyme-based detergents are fast emerging as an alternative to synthetic detergents owing to their
biodegradability,
low toxicity,
non- corrosiveness,
environmental friendliness,
enhanced cleaning properties,
increased efficiency and stability in different formulations.
They are therefore also being referred to as “green chemicals”
Presently, proteases, amylases, lipases and cellulases make up the major portion of the market for industrial enzymes in cleaning applications.
Protease enzymes were first hydrolases introduced into detergent formulations specifically for the degradation of protein-based stains. Proteases have been classified according to the nucleophile or reactive component found at their catalytic sites
AMYLASES AND PROTEASES ARE THE ENZYMES USED A LOT IN FOOD INDUSTRIES FOR THE PRODUCTION OF FOODS. THESE ARE SUPPOSED TO PRODUCE AT A LARGER QUANTITIES IN ORDER TO FULFILL THE DEMANDS FROM THESE INDUSTRIES, THE LARGE SCALE PRODUCTION OF THESE ENZYMES MUST BE CARRIED OUT. THIS METHOD OF LARGER PRODUCTION OF THESE ENZYMES ARE EXPLAINED IN THIS PRESENTATION.
UNIT-5 Protein Engineering: Brief introduction to protein engineering,Use of ...Shyam Bass
UNIT-5 6th Sem B.PHARMA PHARMACEUTICAL BIOTECHNOLOGY)
Protein Engineering: Brief introduction to protein engineering, Use of microbes in industry, Production of enzymes-general considerations, Amylase, Catalase, peroxidase, Lipase Basic principles of genetic engineering
BY- SHYAM BASS
Glutamic acid, a key amino acid with diverse industrial applications, is primarily produced via microbial fermentation, particularly using strains of *Corynebacterium glutamicum* and *Bacillus subtilis*. These microorganisms are adept at synthesizing glutamic acid through various metabolic pathways, with glucose or other carbon sources serving as substrates. The fermentation process involves optimizing growth conditions such as pH, temperature, and nutrient availability to maximize yield. Glutamic acid production can be further enhanced through genetic engineering to improve strain productivity and tolerance to environmental stresses. The versatile nature of glutamic acid makes it a valuable component in food additives, pharmaceuticals, and biodegradable polymers, emphasizing the importance of efficient and sustainable production methods.
The presentation talks about the basics of bioprocess. Describes what is fermentation? Also lists the different modes of fermentation and the basis for selection of type of reactor. General requirements for a fermentation process. Components of a reactor
In this context, there is a need to use “biodetergent or biocleaners”, which offer a better option to the synthetic detergents with respect to their biodegradability, low toxicity, non-corrosiveness environmental-friendliness, enhanced cleaning properties and their increased efficiency and stability in different formulations.
To counter these limitations, enzyme-based detergents are fast emerging as an alternative to synthetic detergents owing to their
biodegradability,
low toxicity,
non- corrosiveness,
environmental friendliness,
enhanced cleaning properties,
increased efficiency and stability in different formulations.
They are therefore also being referred to as “green chemicals”
Presently, proteases, amylases, lipases and cellulases make up the major portion of the market for industrial enzymes in cleaning applications.
Protease enzymes were first hydrolases introduced into detergent formulations specifically for the degradation of protein-based stains. Proteases have been classified according to the nucleophile or reactive component found at their catalytic sites
AMYLASES AND PROTEASES ARE THE ENZYMES USED A LOT IN FOOD INDUSTRIES FOR THE PRODUCTION OF FOODS. THESE ARE SUPPOSED TO PRODUCE AT A LARGER QUANTITIES IN ORDER TO FULFILL THE DEMANDS FROM THESE INDUSTRIES, THE LARGE SCALE PRODUCTION OF THESE ENZYMES MUST BE CARRIED OUT. THIS METHOD OF LARGER PRODUCTION OF THESE ENZYMES ARE EXPLAINED IN THIS PRESENTATION.
UNIT-5 Protein Engineering: Brief introduction to protein engineering,Use of ...Shyam Bass
UNIT-5 6th Sem B.PHARMA PHARMACEUTICAL BIOTECHNOLOGY)
Protein Engineering: Brief introduction to protein engineering, Use of microbes in industry, Production of enzymes-general considerations, Amylase, Catalase, peroxidase, Lipase Basic principles of genetic engineering
BY- SHYAM BASS
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Where can I sell my pi coins at a high rate.
Pi is not launched yet on any exchange. But one can easily sell his or her pi coins to investors who want to hold pi till mainnet launch.
This means crypto whales want to hold pi. And you can get a good rate for selling pi to them. I will leave the telegram contact of my personal pi vendor below.
A vendor is someone who buys from a miner and resell it to a holder or crypto whale.
Here is the telegram contact of my vendor:
@Pi_vendor_247
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How can I sell my pi coins?
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Tele-gram.
@Pi_vendor_247
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If you are looking for a pi coin investor. Then look no further because I have the right one he is a pi vendor (he buy and resell to whales in China). I met him on a crypto conference and ever since I and my friends have sold more than 10k pi coins to him And he bought all and still want more. I will drop his telegram handle below just send him a message.
@Pi_vendor_247
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@Pi_vendor_247
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Below is the contact information for my personal pi vendor.
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2. 2
Introduction
• Enzymes are macromolecular biological catalysts.
• Enzymes accelerate, or catalyze, chemical
reactions.
• The molecules at the beginning of the process are
called substrates and the enzyme converts these
into different molecules, called products.
• Microbial enzymes are the biological catalysts for
the biochemical reactions leading to microbial
growth and respiration, as well as to the
formation of fermentation products.
4. Types of Enzymes
ADAPTIVE
• Produced only when the need arises
Eg. When a cell is deficient of a particular
nutrient.
Constitutive
• Produced always irrespective the amount of
substrate.
4
5. 5
History
• The first enzyme produced industrially was the
fungal amylase Takadiastase which was employed
as a pharmaceutical agent for digestive disorders.
• By 1969, 80% of all laundry detergents contained
enzymes, chiefly Proteases.
• Due to the occurrence of allergies among the
production workers and consumers, the sale of
such enzyme utilizing detergents decreased
drastically.
6. 6
• Special techniques like micro-encapsulation of
these enzymes were developed which could
provide dustless protease preparation. It was
thus made risk free for production workers
and consumers.
• Microbial rennin is also one of the most
significant enzymes. It has been used instead
of Calf’s rennin in cheese production.
7. 7
Location of Enzymes
• Enzymes which are produced within the cell or
at the cytoplasmic membrane are called as
Endocellular enzymes.
• Enzymes which are liberated in the
fermentation medium which can attack large
polymeric substances are termed as
Exocellular enzymes. Eg: Amylases &
Proteases
8. 8
Improved Prospects of Enzyme
Application
• Microbial Genetics – High yields can be obtained
by Genetic manipulation.
Example – Hansenula polymorpha has been
genetically modified so that 35% of it’s total
protein consists of the enzyme alcohol oxidase.
• Optimization of fermentation conditions (Use of
low cost nutrients, optimal utilization of
components in nutrient solution, temperature
and pH)
9. 9
• New cell breaking methods like Homogenizer,
Bead mill, Sonication etc
• Modern purification processes like Counter
current distribution, Ion-exchange
chromatography, Molecular-sieve
chromatography, Affinity chromatography and
precipitation by using alcohol, acetone.
• Immobilization of enzymes
• Continuous enzyme production in special
reactors.
11. Semisolid Culture
The enzyme producing culture is grown on the
surface of a suitable semi-solid substrate
(Moistened Wheat or Rice Bran with nutrients)
Preparation of Production Medium – Bran is
mixed with solution containing nutrient salts.
pH is maintained at a neutral level. Medium is
steam sterilized in an autoclave while stirring.
11
12. The sterilized medium is
spread on metal trays upto a
depth of 1-10 centimeters.
Culture is inoculated either in
the autoclave after cooling or
in trays.
High enzyme concentration in
a crude fermented material.
12
14. 14
Enzymes produced by Semi-solid
culture
Enzyme Micro-organisms
α- Amylase Aspergillus oryzae
Glucoamylase Rhizopus spp.
Lactase A. oryzae
Pectinase A. niger
Protease A. Niger & A. oryzae
Rennet Mucor pusillus
15. Advantages of Semi-solid culture
It involves comparatively low investment
Allows the use of substrate with high dry matter
content. Hence it yields a high enzyme concentration in
the crude fermented material.
To cultivate those moulds which cannot grow in
the fermenters due to wall growth.
Allows the moulds to develop into their natural
state.
15
16. Disadvantages of Semi-solid culture
Requires more space and more labour
Involves greater risk of infection
Difficult to introduce automation in such
systems
16
17. 17
Submerged Culture
• Fermentation equipment used is the same as
in the manufacture of antibiotics.
• It’s a cylindrical tank of stainless steel and it is
equipped with an agitator, an aerating device,
a cooling system and various ancillary
equipment (Foam control, pH monitoring
device, temperature, oxygen tension etc)
• Good growth is not enough to obtain a higher
enzyme yield.
18. 18
• Presence of inhibitors or inducers should also be
checked in the medium.
Example – Presence of Lactose induces the
production of β- galactosidase.
• As the inducers are expensive, constitutive
mutants are used which do not require an
inducer.
• Glucose represses the formation of some
enzymes (α-amylases). Thus the glucose
concentration is kept low.
• Either the glucose can be supplied in an
incremental manner or a slow metabolizable
sugar (Lactose or metabolized starch)
19. 19
• Certain surfactants in the production medium
increases the yield of certain enzymes.
• Non- ionic detergents (eg. Tween 80, Triton)
are frequently used.
20. Advantages of Submerged culture
Requires less labor and space
Low risk of infection
Automation is easier
20
22. 22
After fermentation
• Once fermentation is finished, the fermented liquor is
subjected to rapid cooling to about 5o C in order to
reduce deterioration.
• Separation of micro-organisms is accomplished either
by filtration or by centrifugation of the refrigerated
broth with adjusted pH.
• To obtain a higher purity of the enzyme, it is
precipitated with acetone, alcohols or inorganic salts
(ammonium or sodium sulfate).
• In case of large scale operations, salts are preferred to
solvents because of explosion hazards.
24. 24
Introduction
• Amylase is an enzyme that catalyses the
hydrolysis of starch into sugars.
• Present in the saliva of humans
• Hydrolysis of Starch with amylase will first result
in the formation of a short polymer Dextrin and
then the disaccharide Maltose and finally
glucose.
• Glucose is not as sweet as Fructose. Thus the next
step would be the conversion of Glucose to
Fructose by the enzyme Glucose isomerase.
26. 26
α- Amylase
• Also called as 1,4-α-D-glucan glucanohydrolase.
• Calcium metalloenzymes which cannot function
in absence of calcium ions.
• Breaks down long carbohydrate chains of
Amylose and Amylopectin.
• Amylose is broken down to yield maltotriose and
Maltose molecules.
• Amylopectin is broken down to yield Limit dextrin
and glucose molecules.
27. 27
• Found in saliva and pancreas.
• Found in plants, fungi (ascomycetes and
basidiomycetes) and bacteria (Bacillus)
• Because it can act anywhere on the substrate,
α-amylase tends to be faster-acting than β-
amylase.
• In animals, it is a major digestive enzyme, and
its optimum pH is 6.7–7.0
28. 28
ß- Amylase
• Also called as 1,4-α-D-glucan maltohydrolase.
• Synthesized by bacteria, fungi, and plants.
• Working from the non-reducing end, β-amylase
catalyzes the hydrolysis of the second α-1,4
glycosidic bond, cleaving off two glucose units
(maltose) at a time.
• During the ripening of fruit, β-amylase breaks
starch into maltose, resulting in the sweet flavor
of ripe fruit.
• The optimum pH for β-amylase is 4.0–5.0
29. 29
γ- Amylase
• Also termed as Glucan 1,4-α-glucosidase.
• Cleaves α(1–6) glycosidic linkages, as well as
the last α(1–4) glycosidic linkages at the
nonreducing end of amylose and amylopectin,
yielding glucose.
• The γ-amylase has most acidic optimum pH of
all amylases because it is most active around
pH 3.
31. 31
Producing strains
• Bacteria – B. cereus, B.subtilis, B.
amyloliquefaciens, B. polymyxa, B.
licheniformis etc
• Fungi – Aspergillus oryzae, Aspergillus niger,
Penicillum, Cephalosporin, Mucor, Candida
eetc.
32. 32
Applications
• Production of sweeteners for the food industry.
• Removal of starch sizing from woven cloth
• Liquefaction of starch pastes which are formed
during the heating steps in the manufacture of
corn and chocolate syrups.
• Production of bread and removal of food spots in
the dry cleaning industry where amylase works in
conjunction with protease enzymes
34. 34
Introduction
• Lipases are also called as Glycerol ester
hydrolases
• They are a subclass of esterases
• It splits fats into mono or di- glycerides and
fatty acids.
• They are extracellular enzymes
• Mainly produced by Fungi
Eg: Aspergillus, Mucor, Rhizopus, Peniciilum
etc
35. 35
• Bacteria producing lipases include species of
Pseudomonas, Achromobacter and
Staphylococcus.
• Yeasts like Torulopsis and Candida are also
commercially used.
37. 37
• Enzyme production must be induced by
adding oils and fats.
• But in some cases the fats have effect on the
lipase production.
• Glycerol, a product of lipases action, inhibits
lipase formation.
• Lipases are generally bound to the cells and
hence inhibit an overproduction but addition
of a cation such as magnesium ion liberates
the lipase and leads to a higher enzyme titer
in the production medium.
38. 38
Applications
• Primarily marketed for therapeutic purposes
as digestive enzymes to supplement
pancreatic lipases.
• Since free fatty acids affect the odor and taste
of cheese, and the cheese ripening process is
affected by lipases, microbial affects during
the aging process can be due to lipase action.
• In the soap industry, lipases from Candida
cylindraceae is used to hydrolyze oils.
40. 40
Introduction
• Pectinase is an enzyme that breaks down
pectin, a polysaccharide found in plant cell
walls.
• Pectic enzymes include Pectolyase, Pectozyme
and Polygalacturonase.
• Pectin is the jelly-like matrix which helps
cement plant cells together and in which
other cell wall components, such as cellulose
fibrils, are embedded.
41. 41
• Basic structure of a pectin consists of α-1,4
linked Galactouronic acid with upto 95% of it’s
carboxyl groups esterified with methanol.
• Pectinase might typically be activated at 45 to
55 °C and work well at a pH of 3.0 to 6.5.
43. 43
Production Strains
• Aspergillus niger, A. wentii, Rhizopus etc
• Fermentation with Aspergillus Niger runs for
60-80 hours in fed batch cultures at pH 3-4
and 37o C using 2% sucrose and 2% pectin.
44. 44
Applications
• Pectinase enzymes are commonly used in
processes involving the degradation of plant
materials, such as speeding up the extraction of
fruit juice from fruit, including apples.
• Pectinases have also been used in wine
production since the 1960s
• Helps to clarify fruit juices and grape must, for
the maceration of vegetables and fruits and for
the extraction of olive oil.
• By treatment with pectinase, the yield of fruit
juice during pressing is considerably increased.
46. 46
Introduction
• Protease (Mixture of Peptidases and
Proteinases) are enzymes that perform the
hydrolysis of Peptide bonds.
• Peptide bonds links the amino acids to give
the final structure of a protein.
• Proteinases are extracellular and Peptidases
are endocellular.
• Second most important enzyme produced on
a large scale after Amylase
50. Classification Based upon the pH in
which the Proteases are Active
Alkaline serine Proteases
Acid Proteases
Neutral Proteases
50
51. 51
Alkaline Serine Proteases
• pH of the production medium is kept at 7.0 for
satisfactory results.
• Have serine at the active site
• Optimum temperature maintained is 30o to
40o C.
• Important producers are B. licheniformis, B.
amyloliquefaciens, B. firmus, B. megaterium,
Streptomyces griseus, S. fradiae, S. rectus and
fungi like A. niger, A. oryzae, A.flavus.
52. 52
• Enzymes used in detergents are chiefly
proteases from bacillus strains
(Bacillopeptidases)
• Best known proteases are Subtilisin Carlsberg
from B. licheniformis and Subtilisin BPN and
Subtilisin Novo from B. amyloliquefaciens.
• These enzymes are not inhibited by EDTA
(Ethylene diamine tetraacetic acid) but are
inhibited by DFP (Di isopropyl
fluorophosphate)
53. 53
Proteases for the Use in Detergent
industries
• Stability at high temperature
• Stability in alkaline range (pH- 9 to 11)
• Stability in association with chelating agents
and perborates
• But shelf life is affected in presence of surface
active agents.
54. 54
Screening
• Because the enzymes should be stable in
alkaline conditions, screening for better
producers is done by using highly alkaline
media.
• It was found that B. licheniformis and B.
subtilis showed growth is the range of pH 6-7
by new strains were found to grow even in pH
10-11.
• Genetic Manipulation can also be carried out.
55. Fermentation Process
Cultures are stored in the lyophilized state or
under Liquid nitrogen.
Initial cultures are carried out in shaken flasks
and small fermenters (40-100 m3) at 30-37o C
Fed-Batch culture is generally used to keep
down the concentration of ammonium ions and
amino acids as they may repress protease
production
55
56. High oxygen partial pressure is
generally necessary for
optimal protease titers
Time span for fermentation is
48-72 hours depending upon
the organism
Proteases must be converted
in a particulate form before
they are added to detergents..
56
57. 57
• To prepare a suitable encapsulated product, a
wet paste of enzyme is melted at 50-70o C
with a hydrophobic substance such as
polyethylene glycol and then converted into
tiny particles.
58. 58
Neutral Proteases
• They are relatively unstable and calcium,
sodium and chloride must be added for
maximal stability.
• Not stable at higher temperatures
• Producing organisms are B. subtilis, B.
megaterium etc
• They are quickly inactivated by alkaline
proteases.
59. 59
Acid Proteases
• Similar to Mammalian pepsin
• It consists of Rennin like proteases from fungi
which are chiefly used in cheese production
• They are used in medicine, in the digestion of
soy protein for soya sauce production and to
break down wheat gluten in the baking
industry
60. 60
Applications
• Textile industry to remove proteinaceous
sizing.
• Silk industry to liberate silk fibers from
naturally occurring proteinaceous material in
which they are embedded.
• Tenderizing of Meat
• Used in detergent and food industries.