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

AbstractTo 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.

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.

Introduction

LipaseLipase production by microorganisms is highly

influenced by medium components like nitrogen sources, carbon sources…

fatty acidstriglyceridessugars complex polysaccharides which can stimulate or repress lipase production.

LipaseThe most useful lipase producer genus used in industry is

Bacillus among gram-positive bacteria.

Bacillus subtilisBacterium 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Samples of the culture medium were withdrawn and centrifuged for 15 min at 10,000g.The supernatant was used to estimate the extracellular lipase activity by using an olive oil emulsion as a substrate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 valuesTemperatureSelenium and NickelUrea and Ammonium nitrate

Statistical AnalysisThe 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 25-60°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.

ConclusionsAmmonium 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