Enzymes

1. PRODUCTION OF ENZYMES
LIPASES
AND
CELLULASE
Done by,
S.Ponkalaivani

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)

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

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

25. THANK YOU