This document discusses the production of lipases and cellulases. It describes that lipases are produced by microbes like bacteria, fungi and yeast through fermentation and are used in industries like food processing, detergents, and pharmaceuticals. Cellulases are enzymes that break down cellulose and are produced by fungi and bacteria through fermentation. They have applications in food, textile, pulp and paper industries. The document provides details on lipase-producing microorganisms, fermentation conditions, purification methods, and applications of both lipases and cellulases.
Introduction :
Antibiotics are antimicrobial agents produced naturally by other microbes (usually fungi or bacteria)
The first antibiotic was discovered in 1896 by Ernest Duchesne and in 1928 "rediscovered" by Alexander Fleming from the filamentous fungus Penicilium notatum.
The antibiotic substance, named penicillin, was not purified until the 1940s (by Florey and Chain), just in time to be used at the end of the second world war.
Penicillin was the first important commercial product produced by an aerobic, submerged fermentation
Introduction :
Antibiotics are antimicrobial agents produced naturally by other microbes (usually fungi or bacteria)
The first antibiotic was discovered in 1896 by Ernest Duchesne and in 1928 "rediscovered" by Alexander Fleming from the filamentous fungus Penicilium notatum.
The antibiotic substance, named penicillin, was not purified until the 1940s (by Florey and Chain), just in time to be used at the end of the second world war.
Penicillin was the first important commercial product produced by an aerobic, submerged fermentation
The heart of the fermentation or bioprocess technology is the Fermentor or Bioreactor. A bioreactor is basically a device in which the organisms are cultivated to form the desired products. it is a containment system designed to give right environment for optimal growth and metabolic activity of the organism.
A fermentor usually refers to the containment system for the cultivation of prokaryotic cells, while a bioreactor grows the eukaryotic cells (mammalian, insect cells, etc).
The material describes components of industrial fermentation media with their respective metabolic importance for the industrial microbes. it also addresses industrial scale sterilization methods.
commercial production of cellulase enzyme and its usesCherry
Cellulose is an organic compound with the formula (C6H10O5) and is the most abundant organic polymer on Earth.
Cellulose is an important structural component of the primary cell wall of green plants, many forms of algae and the oomycetes.
The heart of the fermentation or bioprocess technology is the Fermentor or Bioreactor. A bioreactor is basically a device in which the organisms are cultivated to form the desired products. it is a containment system designed to give right environment for optimal growth and metabolic activity of the organism.
A fermentor usually refers to the containment system for the cultivation of prokaryotic cells, while a bioreactor grows the eukaryotic cells (mammalian, insect cells, etc).
The material describes components of industrial fermentation media with their respective metabolic importance for the industrial microbes. it also addresses industrial scale sterilization methods.
commercial production of cellulase enzyme and its usesCherry
Cellulose is an organic compound with the formula (C6H10O5) and is the most abundant organic polymer on Earth.
Cellulose is an important structural component of the primary cell wall of green plants, many forms of algae and the oomycetes.
Production of secondary metabolites : enzymes which involves the upstream technological process
Introduction
History
Process involved
Contribution of different micro-organisms
Flowchart
Example: Methods Production of Amyalse in industrial view
Production of Enzyme - Lipase.
INTRODUCTION: Lipases are hydrolases capable of catalyzing the hydrolysis of Triglycerols (TAGs) into Glycerol and Fatty acids (FAs).
These enzymes operate at the interfaces of Biphasic systems, which is a phenomenon known as interfacial activation.
These do not require co-factors and are easily immobilized on different matrices.
The active sites of lipases are generally characterized by amino acid triad composed of serine, histidine and aspartate.
Lipases exihibit region-selective properties and enantioselective catalytic behaviour and are considered to be the most versatile catalyst in lipid biotechnology.
These enzymes can be employed in a large number industrial processes ( production of agrochemicals, cosmetics , biodiesel etc.)
HISTORICAL BACKGROUND: In 1856, Claude Bernard first discovered a lipase in pancreatic juice as an enzyme that hydrolyzed insoluble oil droplets and converted them to soluble products.
In 1901, the presence of lipases has been observed for Bacillus prodigiosus , B.pycocyancus and B.fluorescens which represents today’s best studied lipase producing bacteria now named Serratia marcescens , Pseudomonas aeruginosa and P.fluorescens.
Lipase have traditionally been obtained from animal pancreas and are used as a digestive aid for human consumption either in crude mixture with other hydrolases (pancreatin) or as a purified grade.
Lipolase was the first commercial recombinant lipase industialized from the fungus Thermomycesl anugiwnosus and expressed in Aspergillus oryzae in 1994.
PROPERTIES: pH optima
Temperature optima and thermal inactivation
Activation and inactivation of the enzyme
Substrate specificity
SOURCES: Plant lipases:
These have been isolated from the leaves, oils, latex and seeds of oleaginous plants and cereals.
Yeast Lipases:
These include species Candida antartica, Candida rugosa, Candida utilis and Saccharomyces species. The production of Biodiesel includes lipases from Thermomycesl anuginosus.
Animal Lipases:
These include pancreatic and pregastric lipases.
Porcine and Human pancreas were the first sources of lipases used in food processing.
Bacterial Lipases: The genera Pseudomonas and Burkholderia are the most widely used for the production of bacterial lipases. P.aeruginosa produces a cystiene hydrolase solvent tolerant lipase.
Fungal Lipases:
Filamentous fungi are considered to be the best source for production of lipases. The genera includes Aspergillus, Rhizopus , Penicillium , Mucor, Geotrichum and Yarrowia etc.
PRODUCTIONTECHNOLOGY:
UpstreamProcessing:
Screening
Strain selection
Inoculum preparation
Immobilization
Fermentation :
Solid-State Fermentation
Submerged Fermentation
Downstream Processing:
Filtration
Centrifugation
Chromatography
Aqueous two phase
Raw Materials and Nutrients:Olive oil, Palm oil, Coconut oil
wheat Bran, rice bran
yeast extract, peptone
Urea, NaNO2
Sucrose , glucose , fructose
KH2PO4
MgSO4 .7 H2O
Microbial Sources:
Bacillus sp.
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Production of α-amylase using new strain of Bacillus polymyxa isolated from s...IOSR Journals
In this study, a new amylase producer strain was isolated from sweet potato tuber. This strain was able to grow at 37 °C and produce α-amylase in high quantity compared to other standard strain cultures. In the first part, cultivation in shake flask in standard medium was carried out to give complete information about the growth and production kinetics of this strain. The results clearly demonstrate that the isolated strain is able to production α-amylase in submerged culture with concentration up to 2050 kat/L after 20 h cultivation. Furthermore, medium optimization was carried out by changing the starch concentration and cell cultivation in medium of mixed carbon source (composed of starch and glucose of ratio 15:5 g/g) to enhance the production process and to increase the growth rate. The volumetric and specific α-amylase production in this optimized medium were 4550 kat/L and 1060 kat/g, respectively. Further improvement in enzyme production process was achieved by scaling up the process from shake flask to 3-L stirred tank bioreactor under non-oxygen limiting condition. The maximal volumetric and specific α-amylase productions in bioreactor batch culture were 5210 kat/L and 1095kat/g, respectively, after only 14 h cultivation
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Lipase production and purification Likhith KLIKHITHK1
Lipase (tri acyl glycerol acyl hydrolase, EC 3.1.1.3) catalyzes the hydrolysis of the carboxyl ester bonds in tri acyl glycerols to produce di acyl glycerols, mono acyl glycerols, fatty acids and glycerol under aqueous conditions and the synthesis of esters in organic solvents.
Under the controlled conditions, lipases are able to catalyze a large number of reactions. Lipases of microbial origin are of considerable commercial importance, because of the high versatility and high stability, moreover, the advantage of being readily produced in high yields.
Many microbial lipases have been commercially available in free or immobilized form. Numerous species of bacteria (Bacillus, Pseudomonas, and Burkholderia), yeasts (Candida rugosa, Yarrowia lipolytica, and Candida antarctica) and molds (Aspergillus, Trichoderma viride) produce lipases with different enzymological properties and specificities but microbes are known to be more potent lipase producer.
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2. LIPASE
• Lipase is the digestive enzyme needed to digest
fat.
• Lipase is the primary digestant used to split fats
into fatty acids and glycerol.
• The physiologic role of lipases is to hydrolyze
triglycerides into diglycerides, monoglycerides,
fatty acids, and glycerol.
• Lipase is produced in pancreas, seeds
Euphorbiaceae (Ricinus communis)
3.
4.
5. LIPASE:
• Lipases are available from many sources however,
the most suitable sources for lipase production are
microbes including bacteria, fungi and yeast.
• lower cost and shorter time
• optimal growth conditions
• Lipase production is dependent upon a number of
factors including carbon and nitrogen sources, pH,
temperature, aeration and inoculum size.
6. • Lipases are widely used in the processing of fats
and oils, detergents and degreasing formulations,
food processing, the synthesis of fine chemicals
and pharmaceuticals, paper manufacture and
production of cosmetics, beverage
• Lipase can be used to accelerate the degradation of
fatty waste
• Lipase are important biocatalysts in industrial
applications.
7. STRAINS:
• Candida lipolytica
• Asperigillus niger
• Pseudomonas, Bacillus and Lactobacillus sp
• Samples are subjected to serial dilution
• Incubation at 37°C for 24-48 hours
• Nutrient agar
• A clear zone around the colonies indicates the
production of lipase.
8. Substrates used for lipase
• Different types of agricultural wastes and oily
substrates are used for the lipase because of their
lipolytic nature.
• Rice bran
• Wheat bran
• Gingelly oil cake
• Almond meal
• Mustard oil cake
• Neem oil cake
• Groundnut oil cake
• Gingerly seed and groundnut kernel
• Olive oil as lipid substrates is suitable for the
production of acidic lipase
9. FERMENTATION
• Initially, the effect of nine factors, namely,
concentrations of glucose, dextran, olive oil, NH4Cl,
trace metals, K2HPO4, MgCl2, and CaCl2 and
inoculum density were studied
• olive oil as inducer and yeast extract as nitrogen
source
• The optimal medium composition for the lipase
production were glucose 0.1, olive oil 3.0, NH4Cl
0.5, yeast extract 0.36, K2HPO4 0.1, MgCl2 0.01,
and CaCl2 0.4 mM ,maltose
• wheat bran, rice husk, lentil husk, banana waste,
watermelon waste as carbon source
10. To maintain sufficient oxygen concentration for the
optimum cell growth and lipase activity, fermentation has
been carried out at 600 rpm and at different aeration rates.
Gas flow rate of 50.34 cm3 s−1 yielded optimum
production of lipase.
For the airlift bioreactor, the best aeration condition was
2.5 vvm, which yielded similar lipase activity after 30 h
of fermentation.
A maximum lipase activity was detected in the late
stationary phase at 200 rpm and air flow rate of 0.8 vvm
Higher lipase activities could be achieved at lower
temperature levels and higher air flow rate values. A
maximum lipase activity was obtained at 27 °C at an air
flow rate of 0.8
11. Optimization:
• Lipase production was optimized at different pH
(7 - 10), temperature (30 - 60°C) incubation
period (1-5 days) with constant shaking at 120
rpm. Bacteria were cultured in nutrient broth with
1% olive oil. During the cultivation, lipase activity
will be measured every 12 h to determine the
maximum lipase producing period.
12. Purification
The culture supernatant containing extracellular lipase
obtained from fermented broth was treated with 0.4M CaCl2
in order to precipitate fatty acids followed by centrifugation at
4°C and 12000rpm for 30 min. The supernatant was collected
in a glass beaker and to it chilled acetone was added slowly to
allow protein precipitation. The precipitates were then
harvested by centrifugation at 4°C and 12000rpm for 30 min.
The pellet thus obtained was resuspended in 34 mL of 20 mM
Tris-HCl buffer (pH 8.0) to allow the solubilization of
proteins. The unsolubilized proteins were then removed by
centrifugation at 4°C, 12000rpm for 30 min. Supernatant then
subjected to ultrafiltration and dialyzed overnight against
same buffer at 4°C. The protein content and lipase activity
were determined after each step.
13. APPLICATION:
• Yogurt and Cheese fermentation
• Convert vegetable oil to fuel
• Degrades lipids
• Recombinant lipases used in baking and laundry
• Investigate and diagnose acute pancreatitis
• Dairy industry
• Detergents and degreasing formulations
• Anti-asthma drug
• Biodiesel production
• Chemical industry
• Pharmaceutical industry, cosmetic industry,
14. Cellulase
• Cellulases break down the cellulose molecule
into monosaccharide ("simple sugars") such as
beta-glucose, or shorter polysaccharides
and oligosaccharides.
• Cellulose is commonly degraded by an enzyme
called cellulase. This enzyme is produced by
several microorganisms, commonly by bacteria
and fungi
• Bacteria which have high growth rate as
compared to fungi have good potential to be used
in cellulase production. However, the application
of bacteria in producing cellulase is not widely
used.
15. STRAINS
• Asperigillus niger
• Trichoderma koningii
Cellulase yields appear to depend upon a complex
relationship involving a variety of factors like inoculums
size, pH value, temperature, presence of inducers,
medium additives, aeration, growth time
Carbon Sources: The effect of various carbon sources
such as starch, glucose, maltose, lactose, and fructose at
the concentration of 1 to 5% was examined in the
production medium.
Nitrogen Sources: Various nitrogen sources like yeast
extract, peptone, urea, and ammonium sulphate were
examined for their effect on enzyme production by
replacing 0.5% peptone in the production medium
16. Screening and Isolation of microorganism
Cellulase-producing bacteria were isolated from soils
by the dilution pour plate or spread plate method
using CMC agar media. The plates were incubated at
45, 50, and 55°C for 24 hours. To visualize the
hydrolysis zone, the plates were flooded with an
aqueous solution of 0.1% Congo red for 15 min and
washed with 1 M NaCl . A fungalcolony isolate with
the highest activity was selected for optimization of
cellulose production.
The isolated fungal colony was subcultured and
maintained on Czapek-Dox-agar slants and stored at
4 ºC in a refrigerator, until needed.
17. Production medium contained (g/L) glucose 0.5 gm, peptone
0.75 gm, FeSO4 0.01 gm, KH2PO4 0.5 gm, and MgSO4 0.5 gm.
The inoculated medium was incubated at 37°C in shaker
incubator for 24 h. At the end of the fermentation period, the
culture medium was centrifuged at 5000 rpm for 15 min to
obtain the crude extract, which served as enzyme source.
pH
Flasks with broth containing the optimum concentration of
substrate and carbon source are taken and the pH of the broth
is adjusted to 7.0, 8.0, 9.0, 10.0, and 11.0
Temperature
Production medium at pH 7 was inoculated with overnight
grown selected bacterial strain. The broth was incubated at
different temperatures from 35, 40, 45, 50, 55, and 60°C for
24 h.
18. Submerged fermentation (SmF)
Submerged fermentation was carried out in 250 ml
Erlenmeyer flasks containing 100 ml of fermentation
medium. The composition of the medium contained the
following g/l of distilled water. L-Glutamic acid, 0.3;
NH4NO2, 1.4; K2HPO4, 2.0; CaCl2, 2.0; MgSO4, 0.3;
protease peptone, 7.5; FeSO4, 5.0; MnSO4, 1.6; ZnSO4,
1.4; tween 80, 20 % (v/v) ; coir waste, 30. The medium
was sterilized by autoclaving at 121ºC for 15 min. Each
flask was inoculated with 1ml of the above said
inoculum. The cultures were incubated on a rotary shaker
(120 rpm) at 30ºC for 72 h.
19. Solid state fermentation (SSF)
Solid state fermentation was carried out in 250 ml
Erlenmeyer flasks that contained 10 g of coir waste and
15 ml of distilled water (moistening agent). The flasks
were sterilized at 121ºC for 15 min and cooled to room
temperature. About 1ml of inoculum was added, mixed
well and incubated at 30ºC in a humidified incubator for
96 h. The flasks were periodically mixed by gentle
shaking.
20. Enzyme extraction
At the end of the fermentation the culture broth from
submerged fermentation was centrifuged at 6000 rpm
for 15 min and the supernatant was used as a source of
extracellular enzyme. In solid state fermentation (SSF)
the enzyme was extracted from the coir waste by
mixing homogenously the entire waste with (1:10 w/v)
distilled water and agitated on a rotary shaker (120
rpm) at 30 ºC with a contact time of 1h. Dampened
cheese cloth was used to filter the extract and pooled
extracts were centrifuged at 6000 rpm for 15min and
the clear supernatant was used as a source of
extracellular enzyme.
21. APPLICATION:
• Cellulase is used for commercial food
processing in coffee.
• Cellulases are widely used in textile industry
and in laundry detergents.
• They have also been used in the pulp and paper
industry for various purposes, and they are even
used for pharmaceutical applications.
• This cellulose-degrading enzyme can be used,
for example, in the formation of washing
powders, extraction of fruit and vegetable
juices, and starch processing
22.
23. • Cellulases are used in the textile industry for
cotton softening
• Used in laundry detergents for colour care,
cleaning
• Used in the food industry for mashing
• Used in the pulp and paper industries for drainage
improvement and fibre modification
• They are even used for pharmaceutical
applications