PRODUCTION AND CHARACTERISATION OF LIPASE ENZYME FROM

LACTOBACILLUS

BACHELOR OF SCIENCE IN CHEMICAL AND PROCESS ENGINEERING

A PROJECT PROPOSAL SUBMITTED TO THE DEPARTMENT OF CHEMISTRY IN

PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE

DEGREE OF BACHELOR OF SCIENE IN CHEMICAL ENGINEERING

SUPERVISOR: Dr NYENDE DAVID

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INTRODUCTION

Enzymes are proteins that have catalytic functions indispensable to maintenance and activity of

life. All chemical reactions occurring in a living organism are dependent on the catalytic actions

of enzymes, and this is why enzymes are called Biotransformation. At present, there are about

4,000 kinds of enzymes whose actions are well known. Enzymes function are highly selective

catalysis in such a way that they selectively catalyze specific reactions (reaction specificity) and

specific materials (substrate specificity). (Garlapati et al., 2010).

Lipase is an enzyme that catalyzes the hydrolysis of fats (lipids). Lipase is produced by the

pancreas, liver, intestine, tongue, stomach, and many other cells. In particular, Candida albicans

has a large number of different lipases, possibly reflecting broad lipolitic activity, which may

contribute to the persistence and virulence of C. albicans in human tissue. (Tan et al., 2003).

Lipase can hydrolyze ester bond of long chain fatty acid which is mainly component of oil. The

sources of lipase enzyme are generally found in nature such as plants, animals, yeast, fungi and

bacteria, for example, Candida rugose, Fusarium oxysporum f. sp. Lin,Candida Antarctica,

Rhizopus oryzae, Lactobacillus spp. Bacillus stearothermophilus Burkholderia sp. Bacterial

lipases are important enzymes applications in various industries, because of friendly for

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environment, non-toxic and no harmful residues. For instant, there are widely uses in dairy

industry and pharmaceutical industry, detergent and surfactant, taste or flavor industry,

agricultural industry, chemical, cosmetic and perfume. (Loo et al., 2006).

Lactobacillus, are group of rod-shaped, Gram-positive, non-spore-forming bacteria of the family

Lactobacillaceae, Lactobacillus is a type of bacteria. These are "friendly" bacteria that normally

live in our digestive, urinary, and genital systems without causing disease. Lactobacillus is also

in some fermented foods like yogurt and in dietary supplements. Various species of

Lactobacillus are used commercially during the production of sour milks, cheeses, and yogurt,

and they have an important role in the manufacture of fermented vegetables (pickles and

sauerkraut), beverages (wine and juices), sourdough breads, and some sausages. (Boekema et al

2007).

Lactobacillus is used for treating and preventing diarrhea, including infectious types such as

rotavirus diarrhea in children and traveler’s diarrhea. It is also used to prevent and treat diarrhea

associated with using antibiotics. (Azim et al., 2001).

In the last decades, the interest in microbial lipase production has increased, because of its large

potential in industrial applications as additives for foods (flavour modification), fine chemicals

(synthesis of esters), waste water treatment (decomposition and removal of oil substances),

cosmetics (removal of lipids), pharmaceuticals (digestion of oil and fats in foods), leather

(removal of lipids from animal skins) and medicine. (Ramani et al., 2010).

Lipases are employed in situ and sometimes together with other enzymes, during the elaboration

of bread, cheese, and other foods to improve their shelf-life and their rheological properties, or to

produce aromas. Moreover, they are used ex situ to produce flavors, and to modify the structure

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or composition of acylglycerols by inter- or trans esterification, in order to obtain acylglycerols

with an increased nutritional value, or suitable for parenteral feeding ( Nadia et al., 2010).

Lipase are used in pharmaceutical and agrochemical industries for the modification or synthesis

of antibiotics, anti-inflammatory compounds, pesticides, etc., and for the production of

enantiopure compounds or the resolution of racemic mixtures (Hasan et al., 2006).

Problem Statement

Lipases are hydrolytic enzymes which have many industrial and environmental applications. For

employing the lipase for specific application its basic characteristics should be well

understood.Hence its activity, protein molecular weight determination and optimisation of

different parameters is necessary. This has fueled high demand for lipase enzyme in the world

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market, due to this reason, this project is aiming at putting lactobacillus as cheaper raw material

for the production of lipase enzyme since lactobacillus is largely found in milk which can easily

be accessed and maintained during the production of lipase enzyme. Approach: In the present

study isolation, characterisation and partial purification of lipase produced by the lactobacillus

for alkaline lipase production will be performed.

Main Objective

Production and characterisation of lipase enzyme from lactobacillus

Specific Objectives

To purify and characterise the enzyme for its maximum activity.

To isolate, indentify and screen lactobacillus from curd sample

To obtain the optimum activity by using temperature, pH, V max, Km, activators, and inhibitors

using standard methods

1.1.5 Research Questions

What are the existing methods used to produce lipase enzyme?

What are the factors that cause inefficiencies of the activity of lipase enzyme?

How effective and efficient the methods in improving the activity of lipase enzyme?

Scope

This research aims at looking at the conditions affecting the production of lipase enzyme

from lactobacillus, method and materials used in the production of lipase enzyme, factors

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affecting the activity of lipase enzyme, optimum temperature and PH for the activity of lipase

enzyme produced from lactobacillus.

Geographical Scope

This research was carried out from biology laboratory Kyambogo University

Content Scope

The researcher conducted his research on production and characterisation of lipases enzyme from

lactobacillus

Time Scope

The researcher commenced his research in the month of August, 2018 and lasted for a period of

ten months. The research work was therefore expected to end in the month of May, 2019.

Justification

I believe that this project will provide an alternative source for lipase production using

lactobacillus. The milk curd sample contain sufficient quantities of lactobacillus capable of

fermenting sugars into lactic acid, this warrant lactobacillus to used as lipase feedstock. In this

study, the enzyme will be characterized to improve the activety.

Significance

This research project is intended to be of considerable interest to policy makers as far as enzyme

issues in Uganda are concerned as it put forward a new way which can be put in place to enhance

lipase production thus alleviating on the enzyme problem in the country.

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If one is to produce lipases from the lactobacillus they have to be certain of the conditions of

production that’s why the optimum temperature, pH substrate concetration was also important

2.0 REVIEW OF LITERATURE:

This chapter basically gives information on the writings of other authors as well as what the

researcher has encountered pertaining successful achievements of production and

characterization of lipase enzyme from lactobacillus

2.1.0 Theoretical basis for the study

Lipase (Triacylglycerol acylhydrolase EC 3.1.1.3) being a serine hydrolase catalyse the

hydrolysis and synthesis of ester formed glycerol and long chain fatty acids s. It occurs in plants,

animals and microbes. Lipases from microbes are find use in diverse range of industries like

detergents, pharmaceuticals, beverages, dairy etc, which make them commercially important2.

Lipase also finds use in health foods, chemicals and pharmaceuticals for transesterification and

enantioselectivity. Recently, various strategies in the pharmaceutical and chemical industries

have used lipases in the synthesis of optically pure drugs and agrochemicals that are more

effective and produce fewer side effects compared with their race mates.

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The enantioselective and regioselective nature of lipases have been utilized for the resolution of

chiral drugs, fat modification, synthesis of cocoa butter substituents, biofuels, and for synthesis

of personal care products and flavour enhancers. Current applications include flavour

enhancement of cheese, acceleration of cheese ripening, manufacture of cheese-like products,

and lipolysis of butterfat, and cream. The free fatty acids take part in simple chemical reactions

where they initiate the synthesis of other flavour ingredients such as aceto-acetate, ß-keto acids,

methyl ketones, flavour esters, and lactones Thus, lipases are today the enzymes of choice for

Pharmacists, Biophysists, Biochemical and Process engineers, Biotechnologists, Microbiologists

and Biochemists.

Research has been carried out on plant lipases , animal lipases, and microbial lipases, particularly

bacterial and fungal. Although pancreatic lipases have been traditionally used for various

purposes, it is now well established that microbial lipases are preferred for commercial

applications due to their multifold properties, easy extraction procedures, and unlimited supply10.

This research work will open the doors for the low cost industrial production of lipase.

Similar studies and their findings

Babu Joseph, et al., studied on the lipases are a class of enzymes, which catalyze the hydrolysis

of long chain triglycerides and constitute the most important group of biocatalysts for

biotechnological applications. Cold active lipases have lately attracted attention as a result of

their increasing use in the organic synthesis of chiral intermediates. Due to their low optimum

temperature and high activity at very low temperatures, which are favourable properties for the

production of relatively frail compounds. Cold active lipases are today the enzymes of choice for

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pharmacists, biochemical and process engineers, biotechnologists, microbiologists and

biochemists. The present review describes various industrial applications of cold active microbial

lipases in the medical and pharmaceuticals, fine chemical synthesis, food Industry, domestic and

environmental applications

Akram Kashmiri, et al., worked on new strain of lipolytic Trichoderma viride was isolated from

the soil on a selective medium that contained olive oil as the only source of carbon and energy.

The isolated strain was cultivated for lipase production in shake flasks at 30±10C and the

fermentation pattern was studied. The maximum extracellular lipase activity of 7.3 U/ml and the

maximum intracellular activity of 320 U/g mycelium were noted after 48 h. Although maximum

fungal biomass was present at 13.6 g/l at 60 h but highest specific growth rate was observed

between 6 and 18 h. The extracellular lipase present in the broth was purified 134-folds with an

overall yield of 46% through purification procedure of ammonium sulphate precipitation, ion

exchange and gel permeation chromatography. The Km value of the purified enzyme for triolein

hydrolysis was found to be 1.229 mole/l

Pratyoosh shukla, et al., worked on the lipases of the Rhizopus species family are important and

versatile enzymes that are mainly used in fat and oil modification due to their strong region

specificity. In the present study 20 samples were collected randomly from different ecological

niche of Ranchi, Jharkhand, India during July 2005 to March 2006 and were primarily screened

by sprinkling method and serial dilutions techniques for isolation of lipase producing indigenous

species of fungi from Jharkhand. Out of these fungi Aspergillus niger KG-2, Rhizopus oryzae

KG-5, Rhizopus oryzae KG-10 were found to be the excellent producers of lipase. Due to

amazingly high yields of lipase from Rhizopus oryzae KG-5 it was further selected for further

physicochemical studies. It was found that Rhizopus oryzae KG-5 has maximum activity of

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48.66 I.U. and was further found as the prominent producer of lipase in liquid media (modified

lipase production media). Various physicochemical parameters such as pH, temperature, effect of

media components and time were also studied.

Orapin Bhumibhamon et al., worked on the Six isolates of lipase-producing microorganisms

were screened from two types of waste water. The bacterial isolates were KLB1, KLB2, KLB3

and showed highest lispase activity was KLB1 which later was identified as Pseudomonas sp.

Lipase of Pseudomonas sp. KLB1 was found to be an inducible enzyme with palmolein. The

model for lipase production was growth associate pattern. As regards to the physicochemical

properties, the Pseudomonas sp. KLB1 lipase had maximal activity at 50°C and pH 9. For its

stability, even though this enzyme showed the maximal stability at pH 7.0 and 37°C, its stability

was increased when incubated at pH 8.0-10 and 37, 50, 60, and 70°C. The residue activity was

76% and 76.23% at pH 10, 70°C and pH 9, 37°C. However, the lipase showed two pH stability

ranges that possible indicated two types of lipases were formed in Pseudomonas sp. KLB1. The

lipase was activated by Ca2+, K2+, and Na2+, (NH4)2S2O3 and ascorbic acid but inhibited by Zn2+,

Mn2+, Co2+, KI and EDTA. The enzyme is also more specific on the medium and long chain

triacylglycerol of vegetable oil than of animal fat. The Km and Vmax of tributyrin hydrolysis

were 110.9 m, and 2.45 m s-1, respectively whereas these kinetic parameters from palmplein

hydrolysis were 1,188.8 m, and 5.25 m s-1, respectively.

Christen, et al., Selected Rhizopus delertiar and grown in a submerged culture and produced a

lipase at 14 U/ml. It was compared with solid state fermentation with a polymeric resin

(Arribedile). Lipase was produced and simultaneously adsorbed on the support: 96 U/g initial

dry matter were obtained when dextrin was used as the carbon source against only 68 and 58 U/g

for maltose and glucose, respectively 11.

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Valeria, et al., Isolated a wild fungal strain from soybean oil and identified as Penicillium

aurantiogriseum initially presented a volumetric lipase activity of 0.4 U/ml in submerged culture

in a medium containing 0.5 % yeast extract and 1% olive oil. Studies were undertaken to

improve lipase production. The effect of nitrogen source was studied by adding casein peptone,

meat peptone, yeast extract or ammonium sulfate to a medium containing potassium nitrate and

other mineral salts. The best yield, of 13 U/ml after 72 h, was obtained with the medium

supplemented with ammonium sulfate. With the ammonium sulfate concentration increased to

double the C/N ratio from 2.5 to 5, a lipolytic activity of 18 U/ml was obtained. Olive, corn, soya

and sunflower oils were tested as carbon sources in this medium, with olive oil at 1 % giving a

lipolytic activity of 25 U/ml after 48 h, the highest yield obtained in this study. Enzyme

production was best at 29 °C, within a range tested from 26 to 32 °C. These results are promising

because this strain produces lipase in an inexpensive inorganic medium and we succeeded in

increasing the lipolytic activity 62-fold over the initial values obtained with the non-optimized

medium.

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METHODOLOGY

Collection of raw material

The raw material to be used for the production of lipase is curd sample, from local area of Banda

(Kampala). The sample will be collected in a sterile stainless steel container, brought to the

laboratory and carried the isolation of Lactobacillus.

Isolation of lactobacillus from curd sample

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The Lactobacillus is to be isolated by serial dilution plate agar method from curd sample. Weigh

1g of the curd sample and inoculate in 9ml sterile distilled water (10-1) and make the dilution up

to 10-5. Mix the sample in vortex shaker and transfer 1ml to second dilution and 1ml whto sterile

Petri plates. Pour the sterile nutrient agar media, swirl in clockwise and anti-clockwise direction.

Solidify the plates and incubated at 37Oc for 24-48 hrs. After incubation observe for the growth

of small, circular, cremish colonies of Lactobacillus.

Screening of Lactobacillus

The isolated strains will be screened for the production of lipase enzyme. Prepare 250 ml of

Lactobacillus selective agar base media in a conical flask. Sterilize at 15 psi for 15minutes. Cool

the media and pour into sterilized Petri plates. Keep the plates for solidification and then streak

the isolated strains. Incubate the plates at 37°C for 24 hours. After incubation observe the plates

for growth of large and whitish colonies of Lactobacillus.

Maintenance of culture

The isolated bacterial culture of Lactobacillus is maintained on 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 is to be transferred from stock to 100 ml sterile nutrient broth and

Lactobacillus selective broth. For the production of lipase take 100ml of nutrient broth and

Lactobacillus selective broth in a 250 ml conical flask. Sterilize the media at 15 p.s.i for 15

minutes, cool and add 1% inoculum (A410= 0.5) of Lactobacillus. Incubate the flask at 37 °C at

150rpm for 72hrs and the lipase production is checked for every 24 hours.

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Development of the inoculum and production of lipase from Lactobacillus in selective media

(Lactobacillus selective broth)

For the development of inoculum 1 ml culture of Lactobacillus is transferred from stock to 100

ml sterile nutrient broth and Lactobacillus selective broth. For the production of lipase take

100ml of nutrient broth and Lactobacillus selective broth in a 250 ml conical flask. Sterilize the

media at 15 p.s.i for 15 minutes, cool and add 1% inoculum (A410= 0.5) of Lactobacillus.

Incubat the flask at 37 °C at 150rpm for 72hrs and check the lipase production for every 24

hours.

Lipase enzyme assay

Lipase activity will be assayed in the LBS broth by using p-nitro phenol as a standard curve. The

LBS broth will be centrifuged at 10,000rpm for 10 min and collects the supernatant. To the

supernatant, lipase activity will be carried out by using 0.05 M Tris-HCl buffer, pH 8.5. To 2.9

ml of Tris-HCl buffer (0.05 M, pH 8.5), added 60 μl of the substrate (p-NPP, 9 mM). Incubate

the reaction mixture at 55OC in a water bath for 10 min in order to remove the turbidity and 40 μl

of enzyme will be added thereafter. The reaction mixture is again incubated at 55O C in water

bath for 10 min. The reaction is stopped by chilling at -40O C. A standard curve of p-Nitro

phenol is plotted at the selected concentrations (100-1000 μg/mL) of Absorbance 410nm of test

sample.one unit (U) of lipase activity is 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.

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