ABSTRACT
Isolation and optimization of Lipase producing bacteria from palm plantation soils of Michael Okpara University of Agriculture, Umudike was carried out using culture techniques. Microorganisms of four genera were isolated from the three samples collected from the areas under study. Bacillus, Staphylococcus and Pseudomonas occurred (100%) in all the samples which suggests them to be most prevalent while Enterobacteria sp occurred in only two samples (67%). Optimization was carried out using Pseudomonas which showed highest clearance zone in the isolation medium. Maximal lipase production was achieved at the optimum incubation time of 48hours (25.90µ/mol), temperature of 40oC (24.41 µ/mol) and pH of 7.0 (24.59 µ/mol) using olive oil as the standard substrate. The effect of various substrate for optimum production of lipase was also investigated and palm oil was observed to have the peak enzyme activity. (22.07 µ/mol) That is to say that the search for novel gene for enzyme production should be channeled to soil sample exploitation.
TABLE OF CONTENTS
Preliminary Pages
Title Page i
Certification ii
Dedication iii
Acknowledgement iv
Table of Content v
List of Tables vi
List of Figures viii
Abstract ix
Chapter One
1.1 Introduction 1
1.2 Aim of
Study 4
1.3 Objective 4
Chapter Two
2.0 Literature
Review 5
2.1 Lipases 5
2.2 Sources
of Lipases 6
Chapter Three
3.0 Materials
And Methods 10
3.1 Area of
Study 10
3.2 Sample
and Sampling 10
3.3 Isolation
of Lypolytic Bacteria 10
3.4.0 Characterization
of Lypolytic Bacteria Isolates 11
3.4.1 Colony
Morphology and Characteristics 11
3.4.2 Microscopic
Morphological Characteristics 11
3.4.3 Biochemical
Reaction Test 12
3.4.4 Identification
of Lypolytic Bacteria Isolate 12
3.5.0 Optimization
of Parameters for Lypolytic Bacteria Activity 12
3.5.1 Optimization
of Temperature for Lipase Activity 12
3.5.2 Optimization
of pH for Lipase Activity 13
3.5.3 Optimization
of Incubation Time 13
3.5.4 Optimization
of Substrates for Lipase Activity 13
3.6 Extraction
of Crude Enzyme 14
3.7 Determination
of Lipase Activity 14
Chapter Four
4.0 Results 15
Chapter Five
5.0 Discussion 26
5.1 Conclusion 29
5.2 Recommendation 30
References
LIST OF TABLES
Table 2.1 Bacteria and Fermentation Techniques
For Lipase Production
Presented in
Resent Literature 9
Table 4.1 Occurrence of Lypolytic Bacteria
Isolates 16
Table 4.2 Characteristics of Lypolytic Bacteria
Isolates 17
Table 4.3 Effect of Incubation Time on Lipace
Activity Using Pseudomonas (µ/mol) 18
Table
4.4 Effects
of PH for Lipase activity using Pseudomonas (µ/mol) 19
Table
4.5 Effect
of temperature On Lipase Activity using Pseudomonas (µ/mol) 20
Table
4.6 Effects of
Substrate on Lipase activity Using Pseudomonas
(µ/mol) 21
LIST OF FIGURES
FIG. 4.1 Effect
Of Incubation Time On Lipase Activity 22
FIG. 4.2 Effect
Of Various pH Ranges In Lipase Activity 23
FIG. 4.3 Effect
Of Temperature In Lipase Activity 24
FIG. 4.4 Effect
Of Various Substrates On Lipase Activity 25
CHAPTER ONE
1.1 INTRODUCTION
Lipases are by definition water soluble
enzymes which have the ability to hydrolyze triacylglycerol to release free
fatty acids and glycerol. They constitute a major group of biocatalyst that has
immense biotechnological applications. Lipases have been isolated and purified
from fungi, yeast, bacteria, plant and animal sources (Joseph, Rantekeand and Thomas,
2008). Of all these, bacterial lipases are more economical and stable (Snellman
et al., 2002).
Bacterial lipases are used extensively in
the food and dairy industry for the hydrolysis of milk, fat, cheese, ripening,
flavor enhancement and lipolysis of butter fat and cream (Falch and Falch, 1991).
Lipases are also used in detergent industry as additives in washing powder
(Jaeger et al., 1994), in textile
industry to increase fabric absorbency (Sharma and Christi 2001) for the synthesis
of biodegradable polymers or compounds (Linko et al., 1998) and different transesterification reactions (Farha, Aamer,
and Abdulhameed, 2006). In addition, the enzyme is used as a catalyst for the
production of different products used in cosmetic industry (Eugene, 1974), in
pulp and paper industry to improve quality (Bajpai, 1999) in the synthesis of
biodiesel (Noureddini et al., 2005),
in degreasing of leather (Nakamura and Nasu, 1990) and pHarmaceutical industry
(Sirishaet al., 2010).
Currently, bacterial lipases are of great
demand because of their potential industrial applications.
Lipases show stability and more activity
in higher temperature and they are usually stable in the presence of chemicals
(Castro-Ochra et al., 2005; Messias et al., 2009;Ultatree et al., 2010). High global demandfor
lipases and billion-dollar business (more than 1000 ton each year which makes
lipase the third largest group of enzymes based on total sales volume after
protease and carbohydrate) has resulted in increased number of research to
identify, isolate and introduce new lipase-producing microorganisms (Hasan et al., 2006; Shu et al., 2010; Treichel et al.,
2010).
Majority of lipases currently used in the
industry have microbial sources (Castro-Ochra et al., 2005; Hassan et al.,2006;
Nthangein et al., 2001; Adiguzel et al., 2009). Some of theadvantages of
these bacterial lipases include high and wide range conditions, stability,
simplicity, ease of mass production and gene manipulation, activity in extreme
conditions (high/low temperature and PH),no requirement for cofactors, high
specificity and low waste production, the possibility of use in continuous
operation, ease of recovery and re-use, cost benefit and low required
downstream processing (Haba et al.,
2000; Sheikhet al., 2003; Treichel et al., 2010).
One of the richest sources for
identification and isolation of lipases producing organisms is the soil. Such
studies introduce new lipases with stability in different temperature and PH,
specificity of certain fatty acids and substrates and enantioselectivity. Some
important lipase-producing genera include Pseudomonas,Bacillus,
Burkholderia etc. Lipases as mentioned earlier are used in many industrial
applications such as pharmaceuticals, food, detergent, paper and pulp, agro
chemicals, surfactants and bioremediations (Pogori et al., 2007; Sharma et al.,
2011). The main function of these enzymes is to hydrolyze triglycerides to
glycerol and free fatty acids at an oil-water interface (Kulkarim and Garde et al.,2002; Palroja and Sethar, 2002).
Lipases can be produced from various sources e.g. animals, plants and
microorganisms.
However, for industrial applications,
Lipases from microorganisms are more interesting because
1.
They
can be produced in the high yields
2.
There
are many varieties of catalytic activities which can be used in many
applications
3.
The
genetic manipulation is available (Hassan et
al., 2006)
Several studies reposted the elevation of
bacterial lipase production by addition of some metal ions such as Na+,
Ca2+, Mg2+ in the culture medium (Josephet al., 2006; Kader et al., 2007; Sharma et al.,
2009). It was explained that Ca 2+ may help the structure formation
of active enzymes leading to higher enzyme activity. However, the mechanism of
metal ions as a lipase enhancer are still not clearly understood.
Lipases in recent times have emerged as
key enzymes in swiftly growing biotechnology, owing to their multi-facetted
properties which find usage in wide varieties or array of industrial
applications such as food technology, detergent, chemical industries and
biomedical sciences (Jaeger et al.,
1994,1999; Pandey et al., 1999). The
natural substrates of lipases are long chain triacylglycerol which have very
low solubility in water and the reaction is catalyzed at the lipid-water
interface. Under micro-aqueous conditions, lipases possess the unique ability
to carry out the reverse reaction leading to esterification, alcoholysis and
acidolysis. Besides being lipolytic, lipases also possess esterolytic activity
and thus have a very diverse substrate range, although they are highly specific
as chemo-, region-, and enantioselective catalyst (Jaeger et al., 1994, 1999; Jaeger and Reetz 1998; Kazlauskas and Bornschew
1998; Pandey et al., 1999; Beisson et al., 2000; Gupta and Soin 2000;
Jaeger and Eggert, 2002).
In this study, the lipase producing
bacteria were isolated from palm plantation and soils within MOUAU. Isolate
showing the highest lipase activity was identified and its culture conditions
for maximal lipases activity was investigated.
1.2 AIM OF STUDY
The sole aim of this research work is to
isolate lipase producing bacteria from palm plantation soils collected from
areas within Michael Okpara University of Agriculture, Umudike, Abia State,
Nigeria and to determine the optimization conditions of the enzyme.
1.3 OBJECTIVES
The study has the following objectives;
·
To
isolate and identify lipase producing bacteria from palm plantation soils
·
To
determine the percentage occurrence of isolated bacteria
·
To
determine the optimal conditions of the bacteria for maximum lipase production
·
To
represent result graphically
Click “DOWNLOAD NOW” below to get the complete Projects
FOR QUICK HELP CHAT WITH US NOW!
+(234) 0814 780 1594
Buyers has the right to create
dispute within seven (7) days of purchase for 100% refund request when
you experience issue with the file received.
Dispute can only be created when
you receive a corrupt file, a wrong file or irregularities in the table of
contents and content of the file you received.
ProjectShelve.com shall either
provide the appropriate file within 48hrs or
send refund excluding your bank transaction charges. Term and
Conditions are applied.
Buyers are expected to confirm
that the material you are paying for is available on our website
ProjectShelve.com and you have selected the right material, you have also gone
through the preliminary pages and it interests you before payment. DO NOT MAKE
BANK PAYMENT IF YOUR TOPIC IS NOT ON THE WEBSITE.
In case of payment for a
material not available on ProjectShelve.com, the management of
ProjectShelve.com has the right to keep your money until you send a topic that
is available on our website within 48 hours.
You cannot change topic after
receiving material of the topic you ordered and paid for.
Login To Comment