NAMES REGISTRATION

BULYABA CAROLINE 17/U/6978/CHE/PE

FANKI ANNET 17/U/6987/CHE/PE
MUTAWATA STELLA 17/U/15117/CHD/GV
CHELANGAT MAUREEN 17/U/6982/CHE/PE
NKUGWA MARK WILLIAM 17/U/7029/CHE/PE
SEGAWA JOHN 17/U/15118/CHD/GV
IROTA EMMANUEL JOSHUA 17/U/6988/CHE/PE
EGESSA ANTHONY 17/U/15116/CHD/GV
ATUHWERE ANDREW 17/U/6975/CHE/PE
AHEREZA JANITH 17/U/6967/CHE/PE
NAKATO HARRIET 17/U/7016/CHE/PE
PRODUCTION AND CHARACTERISATION OF LIPASE
ENZYME FROM LACTOBACILLUS
ACIDOPHILUS .
 PROBLEM STATEMENT
The demand for lipase enzyme increases
daily world wide, this has been majorly
due to the high cost of production of the
enzyme, due to this reason, this project
is aiming at putting lactobacillus
acidophilus as a cheaper raw material for
the production of lipase enzyme since
lactobacillus acidophilus is largely found
in milk.
 MAIN OBJECTIVE

Production and characterisation of lipase

enzyme from lactobacillus acidophillus
 SPECIFIC OBJECTIVES
• To purify and characterise the enzyme for
its maximum activity.
• To isolate, identify and screen
lactobacillus acidophilus from curd
sample
• To obtain the optimum activity by using
temperature, pH, V max, Km, activators,
and inhibitors using standard methods
 METHODOLOGY

Collection of raw material

 collection of raw materials (Cont’d)

• The raw material to be used for the

production of lipase is curd that will be
obtained from fermented milk samples from
cows in the local area of Banda (Kampala).
• The milk will be collected in a sterile
stainless steel container placed in an ice
box, brought to the laboratory and carried
the isolation of Lactobacillus acidophilus.
 Isolation of lactobacillus acidophilus from
curd sample

• The Lactobacillus acidophilus will be isolated

by serial dilution plate agar method from curd
sample.
• 1g of the curd sample will be weighed and
inoculated in 9ml sterilized distilled water and
the dilution will be made up to 10ml. The
sample will be mixed in vortex shaker ,
transferred to 1ml to a second dilution and 1ml
to sterilized Petri plates .
 Isolation of lactobacillus acidophilus from
curd sample (Cont’d)
• The sterilized nutrient agar media will be
poured in a plate agar, swirled in
clockwise and anti-clockwise direction.
The plates will be solidified and
incubated at 37oC for 24-48hrs. After
incubation ,the sample will be observed
for growth of small, circular, creamish
colonies of Lactobacillus.
 Screening of Lactobacillus acidophilus
• The isolated strains will be screened for the
production of lipase enzyme. 250 ml of Lactobacillus
will be prepared in a selective agar base media in a
conical flask, sterilized at 15 psi for 15minutes.
• The media will be cooled and poured into sterilized
Petri plates. The plates will be kept for solidification
and then the isolated strains will be streaked. The
plates will be incubated at 37°C for 24 hours. After
incubation the plates will be observed for growth of
large and whitish colonies of Lactobacillus.
 Maintenance of culture

• The isolated bacterial culture of Lactobacillus

will be maintained on a nutrient agar media
(NAM) slants and stored at 4° C in refrigerator.
• Development of the inoculums and
production of lipase from Lactobacillus in
selective media (Lactobacillus selective broth).
For the development of inoculum 1ml culture
of Lactobacillus will be transferred from stock
to 100 ml sterile nutrient broth and
Lactobacillus selective broth
 Maintenance of culture (Cont’d)
• For the production of lipase 100ml of nutrient
broth will be taken and Lactobacillus selective
broth in a 250 ml conical flask. The media will
be sterilized at 15 psi for 15 minutes, cooled
and 1% inoculum (A410= 0.5) of Lactobacillus
will be added. The flask will be incubated at 37
°C and at 150rpm for 72hrs . The lipase
production will be checked for every 24 hours.
Development of the inoculum in a Lactobacillus
selective broth

• The development of inoculum 1 ml culture of

Lactobacillus will be transferred from stock to
100 ml sterilized nutrient broth and
Lactobacillus selective broth. For the
production of lipase 100ml will be taken of
nutrient broth and Lactobacillus selective
broth in a 250 ml conical flask.
 Development of the inoculum in a
Lactobacillus selective broth (Cont’d)
• The media will be sterilized at 15 psi for 15
minutes, cooled and 1% inoculum (A410= 0.5)
of Lactobacillus will be added. The flask will be
incubated at 37 °C and at 150 rpm for 72hrs.
The lipase production will be checked for
every 24 hours.
 Lipase enzyme assay

• Lipase activity will be assayed in the

Lactobacillus broth by using p-nitro
phenol as a standard curve. The
Lactobacillus broth will be centrifuged at
10,000rpm for 10 min and collects the
supernatant.
 Lipase enzyme assay (Continued)
• To the supernatant, lipase activity will be
carried out by using 0.05 M Tris-Hydrochloric
Acid buffer, pH 8.5. To 2.9 ml of Tris
-Hydrochloric Acid buffer (0.05 M, pH 8.5),
added 60 μl of the substrate (p-NPP, 9 mM).
• The reaction mixture will be incubated at 55OC
in a water bath for 10 min in order to remove
the turbidity and 40 μl of enzyme will be
added thereafter.
 Lipase enzyme assay (Cont’d)
• The reaction mixture will again be
incubated at 55 degrees Celsius in water
bath for 10 min. The reaction will be
stopped by chilling at -40 degrees Celsius.
A standard curve of p-Nitro-phenol will
be plotted at the selected concentrations
(100-1000 μg/mL) of Absorbance 410nm
of test sample.
 Lipase enzyme assay (Cont’d)
• One unit (U) of lipase activity will be
defined as amount of enzyme required to
release one micromole of p-NPP from
the substrate (p-NPP) per minute by one
mL of the enzyme preparation.