Use of microbes in industry

Use of Microbes in Industry
28 June 2021 Abhijit Debnath BP605T and Biotech Unit-1 1
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida
Abhijit Debnath
Asst. Professor
NIET, Pharmacy Institute

Use of Microbes
in Industry
 Introduction
 Production of Enzymes
 General Consideration of Enzymes Production
 Amylase
 Catalase
 Peroxidase
 Lipase
 Protease
 Penicillinase
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida

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28 June 2021 Abhijit Debnath BP605T and Biotech Unit-1
INTRODUCTION (CO1.1)

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Some Examples Of Industrial Uses Of Enzymes:
 Rennin for coagulation of milk to make cheese
 Invertase from yeast and lactase in the food industry
 Cellulase and amylase to remove waxes, oils, and starch coatings on fabrics and to improve the look of the final product
 Amylase and protease for baking
 Lipases in fruit juices to break down cell walls for increased yield
 Proteases, lipases, amylases, oxidases, peroxidases, and cellulases in detergents to help break down stains and chemical bonds
 Carbohydrase to convert starch into corn syrup
 Zymase to convert carbohydrates into ethanol in alcoholic beverages
 Cellulases are used to convert cellulose into glucose to improve biofuel yield
 Lipase and phospholipase are used in the production of biodiesel by converting free fatty acids to fatty acid methyl esters
 Phytases to improve agricultural feed efficiency

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 Since the early 1960s, microbial enzymes have gradually and progressively replaced those from other sources.
 In a conservative estimate, they might now represent almost 90% of the total market.
 Microorganisms are excellent cell systems for enzyme production:
 They are metabolically vigorous,
 They are quite versatile and easy to propagate on a large scale by submerged or solid-state fermentation,
 They are simple to manipulate both environmentally and genetically, their nutritional requirements are
simple and
 Their supply is not conditioned by seasonal fluctuations

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Enzyme Functions Source Applications
 Amylase To hydrolyze the glycosidic bonds in starch molecules,
converting complex carbohydrates to simple sugars
Aeromonas Hydrophila
Alteromonas
haloplanktins
Starch Industry, Paper, Food
Industry, Pharmaceutical Industry
 Catalase It break down hydrogen peroxide (H2O2) to water and
oxygen molecules, which protects cells from oxidative
damage by reactive oxygen species
Aspergillus niger
 Peroxidase It reak down hydrogen peroxide (H2O2), which is one of
the toxins produced as a byproduct of using oxygen for
respiration
Bacillus sphaericus,
Bacillus subtilis,
Pseudomonas sp
Decolorization of synthetic dyes.
They help in the degradation of
pesticides, polycyclic aromatic
hydrocarbons (PAHs)
 Lipase Required for all aspects of fat metabolism. Lipases mediate
the digestion of dietary fats, the uptake of fats into various
tissues, and the mobilization of fats inside cells.
Pseudomonas Food, Detergent, Pharmaceutical,
leather, textile, cosmetic and paper
industries.
 Protease Catalyzes proteolysis, the breakdown of proteins into
smaller polypeptides or single amino acids.
Zanthomonas
Candida humicola
Pseudomonas
aeruginosa
Food, Detergent, Pharmaceutical,
leather, textile, cosmetic and paper
industries.
 Penicillinase

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PRODUCTION OF ENZYMES (CO1.1)
 Isolated enzymes were first used in detergents in 1914, although
their protein nature was not proven until 1926,
 The large - scale microbial production started in the 1960s.
 Most of the enzymes are commercially produced by
microorganisms through submerged fermentation though some
are produced by solid - state fermentation.
 The major industrial enzymes are produced by GRAS ( generally
recognized as safe ) - status microorganisms in large biological
reactors called fermenters. Steps involved in enzyme production

 Selection of a Suitable Enzyme
 Criteria used in the selection of an industrial enzyme include specificity, reaction rate, pH and temperature
optima and stability, effect of inhibitors, and affinity to substrates
 Enzymes used in industrial applications must usually be tolerant against various heavy metals and have no need
for cofactors.

 Selection of a Suitable Production Strain
 Microorganisms are the preferred because of their fast multiplication rate and ease of culture.
 Extracellular enzyme producers are preferred to intracellular producers because the recovery and purification
processes are much simpler;
 the production host should have GRAS - status, which means that it is “ generally recognized as safe. ”
 The organism should be able to produce large amounts of the desired enzyme in a reasonable time – frame.
 Most of the cases, the genetically modified organism possess a greater range of enzymes with varying
properties, such as improved activities or specificity, safe to handle and reduced content of foreign proteins.

 Production Methodology
 Once the organisms has been selected, the production process has to be developed.
 Submerged fermentation has been extensively used for the industrial production of enzymes to date,
 But solid - state fermentation is rapidly gaining interest worldwide for the production of primary and secondary
metabolites
 The optimization of a fermentation process includes choice of media composition, cultivation type, and process
conditions irrespective of the type of bioprocess and considerable effort and time needs to be expended to
accomplish these tasks

 Submerged fermentation  The large - volume industrial enzymes are produced in 50 – 500
m3 fermenters.
 The medium in submerged fermentation is liquid which remains
in contact with the microorganism.
 A supply of oxygen is essential in submerged fermentation.
 There are four main ways of growing microorganisms in
submerged fermenters: batch culture, fed - batch culture,
perfusion batch culture, and continuous culture.
 In perfusion batch culture, the addition of the culture and
withdrawal of an equal volume of used cell - free medium is
performed.

 Submerged fermentation

 Solid - state fermentation
 The high cost of enzyme production by submerged fermentation makes it uneconomical to use many enzymes in
several industrial processes. To reduce the cost of production, solid - state fermentation is an attractive alternative.
 Solid - state fermentation appears to possess several biotechnological advantages, such as
- higher fermentation productivity,
- higher end - concentration of products,
- higher product stability,
- lower catabolic repression,
- cultivation of microorganisms specialized for water - insoluble substrates or mixed cultivation of various fungi,
and
- lower demand on sterility due to low water activity

 Solid - state fermentation (SSF)  Solid State Fermentation is defined as fermentation involving
solids in the absence of free water, although the substrate
must possess sufficient moisture to support microbial growth
and metabolism.
 The process of SSF is performed on a solid substrate with a
low moisture content, with the advantages of a high product
concentration but only a relatively low energy being required.
 The required water content in SSF is absorbed by the
substrate in a solid matrix and offers more advantages for the
growth of microorganism for the transfer of oxygen .

 Solid - state fermentation (SSF)

 Methods for Enhancing Production
 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.
 Toobtain 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.
 Scale - up, purification of end-products, and biomass estimation are the major challenges that have led the researchers to search for
solutions.
 Scale- up in solid- state fermentation has long been a limiting factor, but recently with the advent of biochemical engineering a
number of bioreactors have been designed that overcome the problems of scale - up and, to an extent, also the on - line monitoring of
several parameters, as well as heat and mass transfer.
 Employing natural supports, their utilization supposes a reduction in production costs and usually much higher activities are obtained

 Downstream Processing
 The cost for the purification and conditioning of enzymes
for final use – that is the downstream processing of
enzymes – accounts for more than 50% of the total enzyme
production cost.
 The cost of downstream processing depends on the degree
of purity required, hence on the end use of the enzyme.
 The downstream processing cost for a therapeutic enzyme
will clearly be higher than that for a technical enzyme
because of the purity required and in order to reduce the
production cost downstream processing must be improved.

 Formulation of a Stable Product
 10% of the enzyme could be lost during each purification step, leading to low enzyme recovery.
 To reduce the cost of enzyme, increase in the enzyme recovery and thereby reduction in the number of purification steps
was very important.
 In recent years process engineering of chromatography has improved considerably,
 Including the development of continuous chromatographic process such as simulated moving bed and continuous
separation, which have been recently introduced in the downstream processing of proteins.
 Thus it is possible to purify the enzymes now with a high recovery rate and limited number of steps, which helps in
reducing the cost and produce stable enzyme.

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 Amylase is an enzyme that catalysesthe 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.
AMYLASE (CO1.1)
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.

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AMYLASE (CO1.1)

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 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.
 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-actingthanβ-
amylase.
 In animals, it is a major digestive enzyme, and its optimum pH is 6.7–7.0
α- AMYLASE (CO1.1)
Alpha Amylase (PDB ID: 1BAG)
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etails?id=com.molsoft.imolview&hl=e
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 Also called as 1,4-α-D-glucan maltohydrolase.
 Synthesized by bacteria, fungi, and plants.
 Working from the non-reducing end,β-amylase catalyzesthe
hydrolysisof the secondα-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.
 Theoptimum pHfor β-amylase is 4.0–5.0
ß- AMYLASE (CO1.1)
ß- AMYLASE (CO1.1)
Beta Amylase (PDB ID: 1BAG)
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etails?id=com.molsoft.imolview&hl=e
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 Also termed as Glucan 1,4-α-glucosidase.
 Cleaves α(1–6)glycosidic linkages, as well as the lastα(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.
γ- AMYLASE (CO1.1)

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AMYLASE (CO1.1)
Production of amylases is in economical bulk production capacity.
Microbes are also easy to manipulate to obtain enzymes of desired characteristics.
The microbial amylases meet industrial demands a large number of them are
available commercially.
“BACILLUS LECHENIFORMIS” “BACILLUS AMYLOLIQUIFACIENS” “ASPERGILLUS
NIGER” are useful applications in food, brewing, textile, detergents and
pharmaceutical industries.
In detergents production, they are applied to improve cleaning effect and are also
used for starch de-sizing in textile industry.
Mainly employed for starch liquefaction (a process of dispersion of insoluble
starch in aqueous solution) to reduce their viscosity, production of maltose,
oligosaccharide mixtures.
PRODUCTION OF AMYLASE

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AMYLASE (CO1.1)
MICROORGANISM: Bacillus spp. was isolated from soil and maintained on nutrient agar slants and subcultured for every 10 days.
INOCULUM AND FERMENTATION MEDIUM: The inoculum was prepared by addition of sterile distilled water into the freshly grown
nutrient agar slants, from this 0.5ml of cell suspension was inoculated into 100ml of sterilized fermentation medium and incubated at
35°C for 10 hrs.
The composition of fermentation medium are:
Bacteriological peptone –6gm
Magnesium sulfate –0.5gm
Potassium chlorate –0.5gm
Starch –1gm (pH -7)

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AMYLASE (CO1.1)
Extraction Of Amylase From Fermentation Medium:
After incubation the fermentation medium was harvested by centrifugation at 5000 rpm for 20 min at 4°C.The supernatant was
collected and subjected to estimate the amylase activity.
EFFECT OF PH: The fermentation medium was prepared by varying pH values(5.0, 6.0, 7.0, 8.0) for the production of amylase.
 EFFECT OF TEMPERATURE: To study the effect of temperature on amylase production, the submerged fermentation was carried
out at different temperatures (25°C, 30°C, 35°C and 40°C).

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AMYLASE (CO1.1)
Assay of Amylase:
The amylase activity is determined following the method of Bernfeld.
An assay mixture containing, enzyme extract, starch as a substrate and DNSA as coupling reagent was used.
One unit of amylase activity was defined as the number of micro moles of maltose liberated by 1ml of enzyme solution per
minute.

 Catalase is an enzyme that converts hydrogen peroxide
into water and oxygen.
 The bacteria that contain this enzyme are Usually aerobic
(need oxygen) or facultative anaerobes (can live with or
without oxygen).
CATALASE (CO1.1)

CATALASE (CO1.1)
 CATALASE TEST
The catalase test is used to detect the presence of the enzyme catalase in bacteria.
Purpose:-
 Identification for gram-positive & gram-negative organisms.
 It is a primary test used in the differentiation of staphylococci
and streptococci.
 Also valuable in differentiating aerobic and obligate anaerobic
bacteria.

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CATALASE (CO1.1)
Commercial catalases are produced from Aspergillus niger through a solid-state fermentation process
INDUSTRIAL PRODUCTION OF CATALASE

 Peroxidases or peroxide reductases commonly break up peroxides.
 Peroxidase can be used for treatment of industrial waste waters.
 For example, phenols, which are important pollutants, can be removed by enzyme-
catalyzed polymerization using horseradish peroxidase.
 Thus phenols are oxidized to phenoxy radicals, which participate in reactions where
polymers and oligomers are produced that are less toxic than phenols.
 It also can be used to convert toxic materials into less harmful substances.
 There are many investigations about the use of peroxidase in many manufacturing processes like
adhesives, computer chips, car parts, and linings of drums and cans. Other studies have shown
that peroxidases may be used successfully to polymerize anilines and phenols in organic solvent
matrices.
 Peroxidases are sometimes used as histological markers. Cytochrome c peroxidase is used as a
soluble, easily purified model for cytochrome c oxidase.
PEROXIDASE (CO1.1)

PEROXIDASE (CO1.1)
 Isolation and Identification of Bacteria
 Endospore Staining
 Pure culture preparation:
 BioChemical Tests: Mannitol Fermentation test, Methyl Red and Voges Proskauer Test (MRVP) ,Starch Hydrolysis, Gelatin Hydrolysis and
Casein Hydrolysis are performed.
 Screening of B Subtilis for Production of Peroxidase :
 Observation : Appearance of pink color shows positive.
 Production media for peroxidase:
 Enzyme extraction:
INDUSTRIAL PRODUCTION OF PEROXIDASE
 Assay of crude peroxidase enzyme

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 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
 Bacteria producing lipases include species of Pseudomonas, Achromobacter and Staphylococcus.
 Yeasts like Torulopsis and Candida are also commercially used.
LIPASES (CO1.1)

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LIPASES (CO1.1)
INDUSTRIAL PRODUCTION OF LIPASES

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LIPASES (CO1.1)
INDUSTRIAL PRODUCTION OF LIPASES
 Isolation and screening of lipase-producing microorganisms
 Production and media development for lipase (submerged culture)
 Purification and kinetic characterization of lipases
 Thermostability of lipase
 Shear tolerance of lipases

 Proteases are the second most important industrial enzymes
after amylase.
 About 500 tons of the enzymes are produced per year
 A protease (also called a peptidase or proteinase) is an enzyme
that catalyzes (increases the rate of) proteolysis, the
breakdown of proteins into smaller polypeptides or single
amino acids.
 They do this by cleaving the peptide bonds within proteins by
hydrolysis , a reaction where water breaks bonds.
PROTEASE (CO1.1)

PROTEASE (CO1.1)
INDUSTRIAL PRODUCTION OF PROTASE
 Step I: Isolation of proteolytic microbes:
•Proteolytic microbes can be isolated by observing hydrolysis in casein agar.
•After isolating the suitable strain, it necessarily increases enzyme production by optimizing process parameter like
media composition, pH, volume, moisture content (in-case of solid-state fermentation), concentration of mineral salts,
age and size of inoculum, fermentation time and temperature, organic and inorganic supplements.
•Among the various proteases, bacterial proteases are the most significant as compared with animal and fungal
proteases.
•Among bacteria, Bacillus spp are specific producer of extracellular proteases.
•For industrial utilization, the genes for formation of several proteases have been cloned- protein engineering has
been used to develop, modify Bacillus subtilapeptideases with altered amino-acid sequences.
•Corresponding changes in enzymatic properties such as substrate specificity, pH optimum and stability to bleaching

PROTEASE (CO1.1)
INDUSTRIAL PRODUCTION OF PROTASE
 Step II: Media formulation:
•Media rich in nitrogen sources such as soyabean milk, casein, gelatin and carbohydrate sources such as starch, or lactose are
generally used for protease production.
 Step III: Fermentation:
•The nature of fermentation, solid or submerged influences the growth of moss as well as enzyme production.
•For the production of alkaline proteases by using B. subtilopeptidase cultured are stored in lyophilized state or under liquid nitrogen
(for sterility).
•Initial growth is carried out in shaken flask and small fermenter at 30-37oC in 40-100 mm3.
•Process:
• Fed batch process (to keep down the concentration of NH4
+ ions and aminoacids).
• Sometimes continuous process can also be used but it is not so common.
• Aeration-1vvm, time 48-72 hrs.
 Step IV: Purification:
•Different methods can be applied for purification of enzyme like ultrafiltration.
•Chromatographic technique (ion exchange) purification by treatment with activated charcoal and H2O2.

PENICILLINASE (CO1.1)
penicillins
Story of Penicillin

Story of Penicillin

Working of Penicillin (Beta Lactam Antibiotics )

Beta Lactam Antibiotics
 β-lactam antibiotics are antibiotics that contain a beta-lactam ring in
their molecular structure.
 This includes penicillin derivatives (penams), cephalosporins and
cephamycins (cephems), monobactams, carbapenems and
carbacephems.
 Most β-lactam antibiotics work by inhibiting cell wall biosynthesis in
the bacterial organism and are the most widely used group of
antibiotics.
 Until 2003, when measured by sales, more than half of all
commercially available antibiotics in use were β-lactam compounds.
 The first β-lactam antibiotic discovered, penicillin,

Antimicrobial resistance

 The ability of various bacterial species to inactivate penicillin was first
observed by Abraham and Chain in 1940.
 Penicillinase was the first β-lactamase to be identified.
 It was first isolated by Abraham and Chain in 1940 from Gram-negative E.
coli even before penicillin entered clinical use, but penicillinase production
quickly spread to bacteria that previously did not produce it or produced it
only rarely.
Penicillinase
 Harper, Smith and Smith, and Proom have reported methods for the extraction of penicillinase from the cells and
culture filtrates of paracolon organisms.
 The importance of Penicillinase in the sterility testing of penicillin, and possibly in penicillin assay.

 Penicillinase-producing bacteria, including paracolon and coliform cultures and various organisms of the Bacillaceae.
 Cellular material was investigated by the method of Harper,
 Comparative tests were conducted with all these organisms on the following media: Difco nutrient broth,
peptone, yeast extract, yeast extract plus peptone, yeast extract plus glucose, and peptone plus glucose.
 Filtering the cultures through a Seitz filter was observed to remove most of the penicillinase activity.
 One strain of Bacillus megatherium and the NRRL 569 culture were selected as the best enzyme
producers.
 Method of Method of Assay
PRODUCTION OF PENICILLINASE

PRODUCTION OF PENICILLIN (INDUSTRIAL )

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