Optimization Of Lipase Production Media
Parameters By A Newly isolated
BACILLUS LICHENIFORMS KDP From
Oil Mill Soil
(impact factor:2.958)
指導人:褚俊傑(老師)鄭秀娟(TA)
組員:鄭育軒
張弘儒
方嘉篁
Abstract
To maximize the lipase enzyme production:
lipase enzyme using various supplements along with the
optimal physical conditions for enhancing the production.
Selenium(硒) is used as a media supplement and thus
reported by us for the first time.
The maximum lipase production (16.23U/ml) was found to
be at 60μM/l.
The maximum metal ion and nickel production was found
to be 80μM/L.
The new isolate produced their maximum extracellular
lipase at pH 7.5 (18.69 U/mL) and 45°C (17.36U/mL).
The maximum lipase yield 12.43U/mL and15.38U/mL was
found to be in the medium containing 0.1mg/l urea and
1mg/l ammonium nitrate respectively.
All the experiments proved that the physicochemical
parameters and the lipase production are interdependent.
Introduction
Lipase的功用
Lipases have promising applications in organic chemical
processing, detergent formulation, synthesis of
biosurfactants, the agrochemical industry, waste water
treatment, leather (removal of lipids from animal skins),
medicine (blood triglyceride assay), paper manufacture,
nutrition, cosmetics and pharmaceutical processing.
Lipase
Lipase production by microorganisms is highly
influenced by medium components like nitrogen
sources, carbon sources…
 fatty acids
 triglycerides
 sugars
 complex polysaccharides
which can stimulate or repress lipase production.
Lipase
The most useful lipase producer genus used in industry is
Bacillus among gram-positive bacteria.
Bacillus subtilis
Bacterium Bacillus subtilis its capability to secrete
homologous and heterologous proteins in appreciable
quantities into the growth medium.
Methods(利用Extracellular lipase
assay)
 Samples of the culture medium were withdrawn and
centrifuged for 15 min at 10,000g.The supernatant was
used to estimate the extracellular lipase activity by
using an olive oil emulsion as a substrate (olive oil 25%,
0.1 M NaOH 7.5%, polyvinyl alcohol (2%) 67.5%).
 The free fatty acids released during the reaction were
then titrated with 0.05 M NaOH
 One unit of lipase activity is defined as the amount of
lipase inducing the release of 1 mmol of fatty acid per
minute at 37℃and pH 7.
Optimization of Fermentation
Parameters
Optimization of different nutrient and physical parameters
for lipase production were studied by maintaining all
factors constant except the one being studied.
 pH values
 Temperature
 Selenium and Nickel
 Urea and Ammonium nitrate
Statistical Analysis
The Pearson product moment correlation coefficient was
calculated to measure the degree of linear relationship
between the variables.
The correlation coefficient assumes a value between -1
and +1. If one variable tends to increase as the other
decreases, the correlation coefficient is negative.
Conversely, if the two variables tend to increase
together the correlation coefficient is positive.
Results And Discussions
Growth conditions affect the synthesis of lipase by
microorganisms. Carbon sources, nitrogen sources,
the presence of activators and inhibitors, incubation
temperature, pH, inoculum amount and oxygen
tension can influence lipase production.
Effect of temperature on lipase
production by B.licheniforms
KDP
Lipase production was studied in the temperature range 2560°C at an interval of 5°C.
The maximum lipase activity (17.36 U/mL) was obtained at
the temperature of 45°C .
Effect of pH on lipase
production by B.licheniforms
KDP
 Maximum biomass and lipase production was
obtained at an initial medium pH of 7.5 (18.69 U/mL).
Effect of different concentration
of Urea and Ammonium nitrate
on lipase production by
B.licheniforms KDP
The maximum lipase yield and biomass were found to be in
medium containing 0.1mg/l urea and 1mg/l ammonium
nitrate.
These experiment data indicates that the ammonia nitrate is
the better nitrogen source than urea.
Effect of different concentration
of Se on lipase production by
B.licheniforms KDP
The maximum biomass and enzyme production (16.23
U/mL) were obtained in media supplemented with
60μM/L of Se
Effect of different concentration
of Ni on lipase production by
B.licheniforms KDP
The maximum lipase production was found at 80μM/L
Ni grown culture. The perfect positive correlation (γ=
+1) was found for 60-80μM/L of Ni grown culture.
Conclusions
Ammonium nitrate was found to be a best nitrogen source
than urea. The new isolate produced their maximum
extracellular lipase at pH 7.5 (18.69 U/mL). Maximum
extracellular (17.36 U/mL) lipase activity was obtained at
45°C.
Licheniforms was a thermophilic organism and it could
tolerate higher temperature. The metal ions Se and Ni
supported maximum production of lipase at 60μM/L and
80μM/L respectively.
END