prodcution types, conditions, media used, sources of inoculum, advantages, characterization and process parameters.

1. PRODUCTION OF VITAMINS: Riboflavin,
ENZYMES: Amylase, Protease
Presented by,
VARUN HR
19TUST4040
2nd M.Sc biotechnology
J B Campus
Guided by,
Dr. RAJESHWARI H PATIL
Dept. of Microbiology and
Biotechnology
J B Campus

2. AMYLASE

3. HISTORY
 Its history began in 1811, when 1st starch degrading
enzyme was discovered by ‘kirchhoff’ in wheat.
 α-Amylase were named by ‘kuhn’ in 1925.
 In 1930, ‘ohlsson’ discovered another amylase, which yielded β-
Mannose & he named it β-Amylase.
 In 1990s 3-D crystal structures of each form were determined.
courtesy- PDB

4. INTRODUCTION
 Amylase are important hydrolase enzymes which have been
widely used since many decades.
 These enzymes rapidly cleave internal glycosidic linkages in
starch molecules to hydrolyse them and yield dextrins and
oligosaccharides.
 Among amylases α-amylase is in max. demand due to its wide
range of applicatios in industrial front.
 Industries find enzymes as good alternative over other chemical
catalysts due to increasing awareness on environmental issues
among consumers.
 The ubiquitous nature, case of production and broad spectrum
of applications make α-amylase an industrially important

5. TYPES OF AMYLASE
1. α-Amylase – is a hydrolase enzyme that catalyzes the
hydrolysis of internal α-1,4-glycosidic linkages in starch
to yield products like glucose and maltose. It depends on
metal cofactor.
2. β-amylase – is a exo-hydrolase enzyme, act from non-
reducing end of polysaccharide chain by hydrolysis of α-
1,4-glucan linkages to yield successive maltose units.
3. γ-amylase – cleaves α(1-6)glycosidic linkages, in
addition to cleaving the last α(1-4)glycosidic linkages at
the non reducing end of amylase and amylopectin.

6. SOURCES
 α-Amylase can be isolated from plants , animals or
microorganisms. Eg. Barley , rice plants , cassava mash waste
water.
 Microorganisms- recently, extensive research on microbial
production of Amylase, due to-
- their rapid growth, which inturn speed up production.
- Easy to handle, & require lessers space.
- Can be easily manipulated using genetic engineering- strain
improvement, mutations by which production is optimised.
- Can be tailored to cater to needs of growing industries & to
obtain enzymes with desired characteristics.

7. 1. BACTERIAL SOURCES:
- The most widely used bacterial species are, Bacillus spp.
B.amyloliquefaciens & B.licheniformis are widely used for
commercial production of enzyme.
- Halophilic amylase are produced from Halophilic bacteria
such as, Chromohalobacter spp., Halobacillus spp.,
Haloarcula hispanica, Halomonas meridiana etc.
2. FUNGAL SOURCES:
- Aspergillus spp., Aspergilus oryzae, A. niger, A.awamori
- Penicillium spp., Penicilium fellutanum, P. expansum

8. PRODUCTION METHODS
 SUBMERGED FERMENTATION
 SOLID STATE FERMENTATION

9. SUBMERGED FERMENTATION (smf)
 Smf employes free flowing liquid substrates – molases and
broth.
 Products yielded in fermentation are secreted into
fermentation broth.
 The substrates are utilized quite rapidly; hence the
substrates need to be constantly replenished.
 This method suitable for microorganisms such as bacteria
that require high moisture content for their growth.
 It is 1°ly used for the extraction of 2° metabolites that need
to be used in liquid form.

10. Advantages:
- Allows utilization of genetically modified organisms to
greater extent.
- Media sterilization and purification of end products can de
done easily.
- Control of process parameters like temperature, pH,
aeration, O2 transfer & moisture can be done conveniently.

11. SOLID STATE FERMENTATION (ssf)
 This method is used for microbes which requires less moisture
content for their growth.
 Commonly used solid substrates are bran, bagases, & paper
pulp.
 The substrates are utilised very slowly and steadily. Hence the
same substrate can be used for a longer duration, thereby
eliminating the need to constantly supply substrate to the
process.
 Main advantage of this technique - nutrient rich waste materials
can be easily recycled & used as substrates.
 Other advantages – simpler equipment, higher volumetric
productivity, higher concentration of products & lesser effluent

12. PROCESS PARAMETERS
 TEMPERATURE: 2 temperatures, Temp. for growth of microbes &
optimum temp.(45-46c & 50c).
 pH: Pyrococcus furiosus(6.5-7.5), B.amyloliquefaciens(7.0), Aspergillus
sp.(5.0-6.0).
 Duration of fermentation- it should be optimum.
 Carbon source: B.subtilis (wheat bran&banana waste), B.licheniformis
& A.niger (wheat bran), B.amyloliquefaciens (wheat bran & groundnut
oil cake in 1:1)
 Nitrogen source:
Organic – peptone, yeast extract, soyabeen meal.
inorganic -ammonium sulphate, ammonium chloride &
ammonium hydrogen phosphate.
 Moisture: fungal- less moisture, bacterial- more moisture.

13. PURIFICATION OF α AMYLASE
 The crude amylase enzyme can be precipitated &
concentrated using ammonium sulphate precipitation or
organic solvents.
 Precipitated sample is subjected to dialysis against water or
buffer for further concentration.
 Followed by any of the chromatographic techniques like ion
exchange, gel filteration & affinity chromatography for
further separation & purification of enzyme.

14. CHARACTERIZATION OF α AMYLASE
 It is done by determining the hydrolysates obtained after
enzyme action on starch by PAGE.
 Purified enzyme sample + molecular markers like
BSA(67kDa) & ovalbumin(43kDa) are run on the gel.
 Resulting bands are then stained using staining agents like
coomassie brilliant blue & silver nitrate and visualized.

15. APPLICATIONS OF AMYLASE
 In production of fructose & glucose by enzymatic
conversion of starch.
 In bakery industry.
 In detergent industry.
 Desizing of textiles.
 Paper industry.
 Fuel alcohal production.

16. PROTEASE

17. HISTORY
 Pepsin(aspartic protease) of the stomach, discovered,
characterised & named in 1825, it was crystallised in 1930.
 Trypsin & chymotrypsin(serine protease) from pancreatic
secretions were discovered in 1800s & crystallised in 1930s.
 Papain(cysteine protease) from papaya, discovered in 1800s &
pure forms were reported in 1954.
courtesy- PDB

18. INTRODUCTION
 In recent times, protease has gained considerable importance
in the world market.
 Proteases are group of proteins included in the subclass
hydrolases, within the main class enzymes.
 Serine alkaline protease(SAP) are one of the most important
groups of industrial enzymes – they account for appr. 35% of
total microbial enzyme sales.
 Serine protease is produced by various types of fermentation
techniques using microorganisms.
 Among proteases, bacterial proteases are more significant than
animal & fungal proteases.
 Bacillus is the most invigorated species producing extracellular
proteases among many bacterial species.

19. PROTEASE TYPES
1. Based on their function
a. Serine proteases
b. Aspartic proteases
c. Cysteine proteases
d. Metallo proteases
2. Based on action site
a. Exo-peptidase: Amino peptidase, Carboxyl peptidase.
b. Endo-peptidase: serine protease, aspartic protease,
cysteine/thiol protease, metalloprotease.

20. SOURCES
1. PLANT SOURCES:
- Climate and cultivation conditions govern protease
production.
- Papin, bromelain, keratinases & ficin are examples of
plant originated proteases.
2. ANIMAL SOURCES:
- Pancreatic trypsin, chymotrypsin, pepsin & renins
- Their production depends on population of slaughtering
live stock.

21. 3. PROTEASE FROM MICROBES:
- Microbial originating protease are good sources due to their
broad biochemical pathways.
- Microbial proteases have all biotechnological related
properties.
a. Bacterial – 2 groups of enzymes defined, metal needed &
serine types. eg.; Genus Bacillus.
b. Fungal- fungi type proteases may be produced by current
methods such as soli-state method.
c. Viral- viral types involve in the vital production of fatal
disease like AIDS. Eg.; serine, aspartic & cysteine.

22. PRODUCTION OF PROTEASE
 Protease can be produced by different fermentation
techniques, including solid state fermentation & submerged
fermentation.
 To obtain commercially viable enzyme production,
fermentation media has to be properly optimised.

23. MEDIUM DESIGN
 Concentration of media components are really important as they are
tools for bioprocess medium design.
 Culture medium supplies the microorganisms with all the essential
elements for microbial growth.
 Certain microorganisms are capable of synthesizing all of their cellular
constituents from carbon & nitrogen sources.
 However, most of microorganisms require some source of
micronutrients ( aminoacids, trace elements, vitamins).
 Culture conditions that promote production of enzymes like proteases
are significantly different from culture conditions promoting cell growth.
 Therefore optimization of media component is required for optimum
cell growth & product formation.

24. CELL COMPOSITION
 Most of the enzymes formed by the organisms are
produced as a result of their response to media
components, such as nutrients, growth hormones and ions.
 Qualitative & quantitative nutritional requirements of cells
need to be determined to optimize growth and product
formation.
 Protease is comprised of 53.8% carbon & 15.6% nitrogen.
 Production of protease heavily depends on the availability
of both carbon and nitrogen sources in the medium.
 Either an excess or a deficiency of C and N2 may cause
repression of the synthesis of protease.

25. PROCESS PARAMETERS
 CARBON SOURCE
- Glucose is frequently used , other sugars used are lactose,
maltose, sucrose & fructose.
- At high concentration repression of protease synthesis as observed.
Hence, carbs are added either continuously or in aliqouts
throughout fermentation.
 NITROGEN SOURCES
- Most organisms can utilize both inorganic & organic forms of
nitrogen.
- In inorganic sources, low levels of protease production is reported.
- Enzyme synthesis is repressed in Aminoacids or Ammonium ions.
- No repression was found in presence of Ammonium salts.

26.  METAL ION & SALTS
- Divalent metal ions like Ca, Co, Cu, B, Fe, Mg, Mn & Mo are
required for optimal production.
- Phosphate source like potassium phosphate at conc. 2g/l is
used, it also helps in buffering the medium.
- Excess of conc. Shows inhibition of production.
 TEMPERATURE & Ph
- Specific growth rate is gradually increased upto optimum
temperature. Beyond that rapid decrease of speccific growth
rate is observed.
- Culture pH strongly affects many enzymatic processes and
transport of several species across the cell membrane.
- Variation in pH alters acid-base equilibria & fluxes of various
nutrients, inducers and growth factors b/w abiotic and biotic
phase.

27.  AERATION & AGITATION
- During fermentation, aeration rate indirectly indicates dissolved O2
levels in fermentation broth.
- Variation in agitation speed influences the extent of mixing in shake
flasks/bioreactor & will also affect the nutrient availability.
- Optimum yields of protease was observed @200rpm for B.subtilis &
B.licheniformis.
 INOCULUM & INCUBATION TIME
- Optimum inoculum size was required for protease production.
- Increase in production using small inoculum is suggested (due to higher
surface area to volume ratio )
- if the inoculum is too small, reduced production of protease ( due to
insufficient no. of bacteria)
- Normally protease is auto degradable in nature. So protease yield can
be increased by proper incubation time.

28. PURIFICATION OF PROTEASES
 RECOVERY
- After successful fermentation, broth was kept in low
temperature(-4°c) to prevent microbial contamination as well as
to maintain enzyme activity & stability.
- The removal of cells, solids, & colloids from the fermentation
broth is the 1° step in enzyme downstream processing.
- The vaccum rotary drum filters & cold centrifuges are commonly
used.
- To prevent the clogging of filters or the losses in enzyme activity
caused by imperfect clarification, it is necessary to perform
some chemical pretreatment of the fermentation broth before
commencing separation.

29.  ISOLATION & PURIFICATION- when isolating enzymes on industrial scale for
commercial purposes, the primary consideration has been the cost of production in relation
to value of end product.
a. PRICIPITATION
- It is the most commonly used method , it also performs both purification
& concentration steps.
- It is generally affected by addition of reagents such as salts/an organic
solvent, which lowers solubility of desired proteins in an aqueous
solution.
b. CONCENTRATION
- Because the amount of enzyme present in cell-free filterate is usually
low, the removal of H2O is a 1° objective.
- So, membrane separation processes have been widely used for
downstreaming.
ULTRAFILTRATION – one such process, largely used for recovery of
enzymes and formed a preferred alternative to evaporation. This
process is inexpenssive & offers both purification, concentration &
diafilteration.

30. APPLICATIONS
 Detergent industry
 Leather industry
 Silver recovery
 Waste treatment
 Food industry

31. RIBOFLAVIN

32. HISTORY
 Riboflavin was discovered in 1920, isolated in 1933, & 1st
synthesised in 1935.
 IUPAC ID- Dimethyl-10-[(2s,3s,4R)-2,3,4,5-
tetrahydroxypentyl]benzo(g)pteridine-2,4-dione.
 Formula- C17H20N4O6, Molar mass- 376.36g/mol,
Pubchem ID- 493570, other names- vactochrome,
lactoflavin, vitamin G
2D 3D
courtesy-PubChem courtesy- PubChem

33. INTRODUCTION
 Riboflavin (vit-B2) is a crucial micronutrient that is a precursor
to coenzymes flavin mononucleotide & flavin adenine
dinucleotide, & it is required for biochemical reactions in all
living cells.
 For decades, one of the most important applications of
riboflavin has been its global use as an animal & human
nutritional supplement.
 Being well-informed of the latest research on riboflavin
production via the fermentation process is necessary for the
development of new & improved microbial strains using
biotechnology & metabolic engineering techniques to increase

34. PRODUCTION OF RIBOFLAVIN
 The production process is composed of 3 main steps;
1. Upstream processing- strain development, sterilization of
C & N2 sources, medium & inoculum preparation.
2. Bioprocess/fermentation- runs under optimal pH,
temperature, aeration & agitation rates.
3. Downstream processing- pasteurization, isolation,
purification, recrystallization & drying.
 The annual total Riboflavin market is appr. 9000t, & the
final price is appr. $15/kg –for feed grade product, $35-
50/kg –for food grade product.

35. SOURCES
 Commercial Riboflavin production is currently based on indutrial
fermentation using overproducing strains of GE
microorganisms.
 Chemical synthesis of vit-B2 from ribose is being replaced by
fermentation process because of economic & environmental
considerations.
MICROORGANISMS:
- B.subtilis, Ashbya gossypii, Eremothecium ashbyii, & Candida
famata are the riboflavin production strains.
- These strains are environmentally safe and often used in food &
feed supplements industry.
- Today, strains of A.gossypii & B.subtilis are more preferable (
fermentation is unstable in case of E.ashbyii & C.famata.)

36. PROCESS
 The synthetic industrial riboflavin process is described in detail by stahmann
et.al.(2000)
 It begins with d-ribose reacting with 3,4-xyline in methanol. This produces
riboside.
 In this step, the industrial production of d-ribose can be obtained from
glucose by Bacillus mutants lacking transketolase( a major enzyme of
pentose phosphate pathway ).
 Riboside is hydrogenated to give N-(3,4-dimethylphenyl)-D-1’-ribamine.
 This product is coupled with phenyl diazonium halogenide, = azocompund.
 Cyclo condensation with barbituric acid to give Riboflavin.
DISADVANTAGES:
- It has max. Yield(60%) from substrate, thus generates lot of waste.
- Requires organic solvents.
- Requires 25% more energy compared to single-stage fermentation route.

37. Chemical synthesis vs Microbial synthesis

38.  MEDIUM
- Medium components before inoculum preparation &
fermentation processes have to be sterilized separately by
several groups(C, N2, salts in H2O & A.A) to avoid maillard
reactions in which products can become inhibitors for riboflavin
production.
 INOCULUM, TEMPERATURE & pH
- Inoculum preparation includes use of low concentration(2-
10%v/v) inoculum broths containing young, undifferentiated
microbes.
- Fermentation of A.gossypii is performed at optimum
temperature range of 26-30°c.
- Initial pH of culture medium was appr. 6.5-7.5 in aerobic

39. PRECIPITATION & CONCENTRATION OR
PURIFICATION OR (DOWNSTREM PROCESSING)
 Down streaming begins with Pasteurization of broth to remove
all viable cells of production organism present in final product.
 Due to low solubility of riboflavin in natural aqueous solvents,
parts of fermentation product accumalates as needle-like
crystals in broth, which can be easily separated by
centrifugation/filteration.
 Crystallization is completed in the crystallizer by evaporation of
some water.
 Subsequent washing of crystals with hot dil. Acids( Hcl/H2SO4)
disrupts strain DNA.
 Further separation via decantation followed by purification &
drying (vaccum/spray drying) allows acquisition of a final
product (powder/granule) with a riboflavin content of upto 96%.

40. RIBOFLAVIN USES
 It is a primary adjuvent in cancer treatment.
 Controlled inactivation of recombinant viruses with
Riboflavin.
Functionality of Riboflavin -

41. REFERENCES
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Amylase production & applications: A review, Journal of Applied &
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of serine alkaline protease by Bacillus spp., J BiochemTech(2012)
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alkaline proteases, Journal of scientific & industrial Research, vol.61,
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42. 5. Liudmila A. Averianova, Larassia A. Balabanova, Oksama M. Son,
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