Internship from June 2016 to August 2016

IMMOBILISATION OF LIPASE ENZYME FROM CANDIDA RUGOSA AND BIO-CATALYSIS FOR OPTIMUM REACTION IN BIODIESEL PRODUCTION Report writing of the internship at the University of Central Lancashire (Preston, UK)

By Nazih MAALOUL

Supervisors: Doctor Tapas SEN (UCLAN)

Fabien COREE (EIDD)

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Acknowledgements

I would like to thank Doctor Tapas SEN for his allowing me to work on his project and also for

his guidance and supervision throughout the project. We have had weekly meetings to discuss

my progress; he always gives me his detailed explanations and advice. Behind him, I

enormously learnt and won in confidence.

Many thanks to Amina AHMAD-MUHAMMAD, MSc Student in Instrumental Analysis at Uclan

(UK, Preston) working with me on the same project and sharing her knowledge with me

during the project. I improve my English skills talking with her.

I would like to thank Laboratory technicians, staffs of the laboratory and office team for all

the time spent together.

Many thanks to the teaching staff of the EIDD for all the knowledge which I acquired.

Particularly, Fabien CORRE who has been my supervisor.

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Contents

Acknowledgements .............................................................................................................................. 1

Abbreviations........................................................................................................................................ 3

Abstract ................................................................................................................................................ 4

Introduction .......................................................................................................................................... 5

I. Aims of research and Literature review ............................................... 6

1. Biodiesel................................................................................................................................ 6

2. Soybean oil ............................................................................................................................ 7

3. Catalyst ................................................................................................................................. 7

4. Immobilisation of enzyme ..................................................................................................... 8

5. Strategies for immobilisation ................................................................................................ 9

6. Superparamagnetic iron nanoparticles (Fe3o4) for Enzyme Immobilisation ........................ 12

7. Surface Functionalization of iron nanoparticles silica coated ............................................. 13

II. Materials and Methods ......................................................................15

1. Synthesis of nanoparticles .................................................................................................. 15

2. Preparation of solutions...................................................................................................... 15

3. Determination of the density of the nanoparticles (Silica-coated iron oxide) .................... 16

4. Enzyme immobilisation by physical adsorption .................................................................. 16

5. Surface of functionalization of nanoparticles (Enzyme immobilisation by Chemical

binding).. ..................................................................................................................................... 17

6. Hydrolysis of esters ............................................................................................................. 18

7. Characterisation .................................................................................................................. 19

III. Results and discussion .......................................................................21

1. SEM ..................................................................................................................................... 21

2. FTIR ..................................................................................................................................... 22

3. Magnetic properties of nanoparticles analysis ................................................................... 23

4. Determination of the amount of immobilised lipase enzyme on nanoparticles by Bradford

assay ........................................................................................................................................... 24

5. Hydrolysis of ester .............................................................................................................. 25

6. Transesterification reaction ................................................................................................ 27

Conclusion .......................................................................................................................................... 28

References .......................................................................................................................................... 29

Appendix ............................................................................................................................................. 31

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Abbreviations

Abbreviated form Full form

APTS 3-aminopropyl triethoxysilane

CRL Candida rugosa lipase

PBS Phosphate Buffer Saline

PNP p-nitrophenol

PNPP p-nitrophenyl palmitate

rpm Rotations per minute

SPIONs Superparamagnetic iron oxide nanoparticles

NPs nanoparticles

FNPs Functionalized nanoparticles

EDAX Energy Dispersive Analysis of X-ray

kOe kilooersted

w/v Weight per volume

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Abstract

Immobilisation of enzymes on solid supports (functionalised and non-functionalized

nanoparticles) and bio- catalysis for biodiesel production using soybean oil has been an area

of concern in this study project. Numerous researches were done on biodiesel production

from different types of vegetable oils, including palm oil, sunflower oil, corn, olive, soybean

oil, etc. In this project, transesterification reaction was accomplished with the use of lipase.

Moreover, lipase enzyme was immobilised on the surface of silica-coated superparamagnetic

iron oxide nanoparticles through physical adsorption and chemical binding processes.

Characterization of the NPs was achieved with the use of SEM and determined its magnetic

properties with VSM. The NPs were functionalized with APTS and therefore the ability of the

enzymes to be chemically bound to the surface. Free and enzymes immobilised were used in

hydrolysis of ester (PNPP) to produce an alcohol (PNP) and transesterification reaction of

soybean oil in order to determine the catalytic efficiency of the lipase enzyme for production

of biodiesel. During the process of hydrolysis, more alcohol was produced at highest rate with

free enzymes in comparison with immobilised enzyme on both materials. Furthermore, the

efficacy of enzyme immobilised on FNPs was more than that of non-FNPs. However, the

enzyme immobilised on FNPs was used in transesterification reaction of the soybean oil by

using UV-Visible spectrometer.

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Introduction

During two months I have been working at the Uclan, at The School of Forensic and

Investigative Sciences. Student at the School of Engineer of Denis Diderot, I have chosen to

do my internship abroad to discover a new country and to study in a new environment with

new team.

The discovery of the fossil fuels during the industrial revolution considerably amplified the

world development. This discovery profoundly affected agriculture, economy, policy, and

environment. We are past an artisanal society into a commercial and industrial society.

Over the years the bulk of energy demand is met through fossil fuels. However, these reserves

are limited in some part of the world. One of the most important natural resources is petrol

which has much application as the petroleum diesel. The petrol resources are soon finished

and the gases emitted are toxic. The modern researchers are in an effort to produce diesel

from renewable biological sources such as vegetable oils and animal fats called biodiesel.

It is produced through transesterification reaction of the parent oil or fat to achieve a viscosity

close to that of petro diesel. This fuel is non-toxic and bio-degradable resource and doesn’t

destroy the Ozone layer, doesn’t produce toxic gases such as SOx, CO, and CO2.

During my internship, I have for mission to immobilise lipase enzyme from Candida rugosa on

the surface of the silica coated superparamagnetic iron oxide nanoparticles through

adsorption and covalent binding processes. Furthermore, I realise kinetic study of the

hydrolysis of ester (PNPP) to produce an alcohol (PNP) in order to study the catalytic efficiency

free and immobilised lipase enzymes. Finally, for the process of bio catalysis in order to get

optimum reaction in production of biodiesel using soybean oil.

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I. Aims of research and Literature review

1. Biodiesel

Natural oil is converted into biodiesel by one process called transesterification. It’s reaction

between alcohol and ester; the products give another ester.

Biodiesel is the monoalkyl esters of vegetable oils or animal fats which are insoluble in water.

Different studies have been made using different oils as raw material, different alcohols

(methanol, ethanol, and butanol) as well as different catalysts (homogeneous catalysts such

as sodium hydroxide, potassium hydroxide, sulfuric acid and supercritical fluids, and

heterogeneous ones such as lipases).

The triglyceride molecules from the oil or fat source reacts with the alcohol which is

specifically methanol or ethanol particularly for the production of biodiesel. This reaction is

carried out in three steps. Firstly, the triglycerides react with the alcohols to produce

diglycerides, and the same reaction is repeated further to produce monoglycerides. The last

step, Monoglycerides produces glycerin. In all of the above reactions esters are produced. 3

moles of alcohol and 1 mol of oil are necessary to realise the transesterification reaction.

(Marchetti et al., 2007).

Figure 1: The reaction of Transesterification of triglycerides with alcohol

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The transesterification of triglycerides is not a new process. Since 1853, that experience has

been realised before the diesel engine has existed.

The biodiesel obtained in this process is also a mixture of mono, di- or triglycerides which the

ester could be extracted and purified though it is a tedious process

Triglycerides are molecules being a member of the category of lipids, which consist of one

glycerol molecule and three fatty acid molecules (hydroxyls groups) bound together by ester

bonds. They are the main constituent of animal fats and vegetable oil.

Figure 2: General chemical formula of triglycerides R1, R2 and R3 are Carbon chains of fatty acids

2. Soybean oil

In principle, any source of fat can be used to prepare biodiesel. Soybean oil belongs to the

category of vegetable oil. The source of biodiesel has to respect two important criteria: low

cost and wide scale of production. The soybean oil is the product of the most common

departure for the production of biodiesel. In among other sources, there are a canola oil or a

corn oil. About 90 % of the biodiesel produced in the United States is made from soya oil.

The major unsaturated fatty acids in soybean oil triglycerides are:

14% of saturated fat (10% palmitic acid and 4% stearic acid)

23% of monounsaturated fat (oleic acid)

51% of polyunsaturated fat (7% alpha linolenic acid, 51% linoleic acid).

3. Catalyst

A catalyst is a chemical specie which increases the speed of spontaneous chemical reaction.

The catalyst can be a solid, liquid or more rarely a gas. It does not appear in the equation of the

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reaction and does not influence the direction of evolution of the transformation. There are

many types of catalyst. Before the 2000s, the catalyst usually used was homogenous (reactive

and present in the same phase) but there were problems to separate at the end of the reaction

enzyme and products. So, the transesterification reaction of vegetable oils by enzymatic

catalysis has been a big development these recent years. Enzymatic catalyst present many

advantages: Firstly enzymes are biodegradable, the reaction conditions are relatively soft (low

temperature and pressure) what decreases the price regarding energy and equipment. It is

possible to work in an aqueous or non-aqueous environment. But the enzymatic catalysis

possesses inconveniences and too expensive than a basic catalyst.

Lipases, also called triacylglycerol hydrolases, are one of an essential groups of enzymes used

for bio-transformations. The physiological role of lipases is to hydrolyse the triglycerides to

diglycerides, monoglycerides, fatty acids and glycerol. Lipases have the ability to achieve

transesterification reactions. They hydrolyze all types of ester bonds in triglycerides.

Furthermore, the lipases have unique capabilities to carry out various transesterification

reactions on esters such as esterification, acidolysis and alcoholysis at a water-lipid interface

under aqueous conditions at micro-level.

For this project, Candida Rugosa Lipase (CRL) was used. Candida Rugosa (CRL) claims more

applications than any other bio-catalysts and there have been many publications concerned

with its molecular biology and structure. CRL could be used for Hydrolysis of p-nitrophenyl

esters (we will study this reaction in further), esterification synthesis of ethyl isovalerate or

transesterification of ethyl butyrate. Various parameters influence the enzymatic activity such

as temperature, pH, concentration, ionic strength.

4. Immobilisation of enzyme

The enzyme lipase could catalyse various types of transesterification reaction and also used by

numbers of researchers even though it is a cost effective process. Their cost and time-limited

stability are the main drawback in their industrial use. However, there are many methods that

could be implanted to reduce the cost by the re-usability of the expensive enzyme. It’s

necessary to immobilise enzyme on support. This is a brief definition from Production of

biodiesel using immobilised lipase — a critical review. Crit Rev Biotechnol. 2008, “enzymes

physically confined or localised in a certain defined region of space with retention of their

catalytic activities, and which can be used repeatedly and continuously”. A major goal of

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enzyme immobilisation is an increase of the enzyme life. Furthermore, immobilisation

improves the stability, catalytic activity and thus increases the biodiesel yield. The enzymes

are immobilised on the surface of certain carriers such as epoxy resins, zeolites, amorphous

and porous silica supports and magnetic nanoparticles. All of the carriers have different

methods to attach the enzyme with them. The support has to respect certain conditions. It

should have high surface areas, a high density of reactive groups (multipoint covalent

attachment).

5. Strategies for immobilisation

There are such methods as physical adsorption (non-covalent), covalent binding, entrapment,

encapsulation by which the enzyme is immobilised on the surface of the carrier. It would be

essential to be mentioned that the immobilisation strategy could be chosen effectively to

output the maximum catalytic activity and stability of the enzyme.

Adsorption could be defined as a physical phenomenon. It is the capacity of certain

organic materials to fix a molecule given to their surface. Adsorption where many weak

physical forces such as hydrophobic interactions, Vander Wall forces and dispersion

forces are involve attaching the substrate on the surface of the carrier. This technique

of immobilisation presents several advantages. It is a method very simple to operate

requiring only to put in touch the enzyme and the support in conditions of pH,

temperature and of Ionic strength known. Furthermore, it is easily reversible. However,

adsorption presents a big inconvenience because of the weak interaction between

enzyme and the support, less stable, the enzyme is desorbed from the support over

time. Moreover, few studies have reported up to 80% yield of biodiesel possible by this

approach.

Figure 3: Adsorption enzyme on a support

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The principle of covalent binding method of immobilisation is that a free functional

group of the enzyme interacts with a functional group of the reagent. Generally, the

functional groups of the reactive functions are carboxylic, thiol, hydroxyl or amine.

These groups are little reactive and must of this fact are activated to being able to

react with the groups of enzyme, not occurring in the enzyme catalysis. This

immobilisation method has advantage of fixing enzyme permanently and to increase

its stability (with regard to the method of adsorption), giving it a more important

lifetime. However, the reagent used for grafting are likely to denature the enzyme and

therefore causes loss of activity.

The principle of encapsulation is to fix enzyme in a matrix. Immobilisation is made

physically and not chemically unlike covalent binding. These matrices can be inorganic

(silica gels), polymers (polyacrylamide, polyurethane ...) or composite (carbon paste).

The main advantages of this method of immobilisation are; inexpensive, easy to

implement and can be applied to a large number of enzymes. However, the enzyme

may diffuse through the matrix and also it is only applicable for small substrates.

Furthermore, the active groups of the enzyme may react with the matrix and

therefore cause a reduction of catalytic activity.

Figure 5: Enzyme encapsulation

Figure 4: Covalent Binding for immobilisation enzyme

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The self-immobilisation strategy, also called as chemical conjugation, is an effective

method where a third molecule such as glutaraldehyde is used to immobilise the

enzyme. The molecule used, in fact, has bi-functional characteristics thus capable of

cross-linking. Therefore, it acts as a bridge between the enzyme and the carrier by

which it conjugates both materials at both ends. This method offers high enzyme

stability, thus, very good yield.

Figure 6: Resume of different methods of enzyme immobilisation

In the case of this work, Candida Rugosa lipase immobilised on iron nanoparticles coated silica,

ester hydrolysis reaction (is a chemical reaction in which a covalent bond is broken by the

action of another molecule) on two types of support: non-functionalized (adsorption) and

functionalized iron oxide nanoparticle (covalent binding) were studied.

What is the influence on the yield of the reaction when the

enzyme is immobilised relative to free enzyme?

What is the influence between functionalized (covalent

binding) and non-functionalized support (adsorption)?

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6. Superparamagnetic iron nanoparticles (Fe3o4) for Enzyme Immobilisation

Magnetite is a mixed oxide of Fe2+ and Fe3+ inverse spinel structure. The unit cell consists of

thirty-two oxygen atoms forming a face-centred-cubic (fcc) closed-packing structure, with

iron cations located at octahedral and tetrahedral sites. When the nanoparticle’s size is

reduced below a certain critical size rc, the nanoparticle presents only a single domain, until

it reaches certain level, then the magnetic anisotropy energy becomes smaller than the

thermal energy, thus nanoparticle becomes superparamagnetic (the entire nanoparticle

aligns with the applied field) at room temperature. The distinctive time between two flips is

termed as Neel relaxation time. The time taken for the measurement of magnetisation of the

nanoparticles in the absence of external magnetic field is longer than the Neel relaxation

time. Hence, their magnetisation appears to be zero averagely: this is referred to as

superparamagnetic.

Figure 7: Unit cell of spinel structure

Iron oxide magnetic nanoparticles has ability for greater performance in biocompatibility and

chemical stability and comparison with the other metallic nanoparticles. We decided to

choose the superparamagnetic nanoparticles over other carriers to immobilise the enzyme

for many reasons. Magnetic properties of nanoparticles allow easy separation from the

reaction mixture by an application of external magnetic field. It could be recovered easily, re-

usable and used for longer time which ultimately supports the economical standpoints. Also,

the large surface area exhibited by nanoparticles gives it ability to attach biological materials

such as enzymes. In addition, these nanoparticles help in the experiment being dispersed in

the buffer and not being aggregated.

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There are risks associated with the surface of naked nanoparticles oxide one of which is

affecting the magnetic ability. Thus, reaction occurs and modifies the structure of the enzyme

during its immobilisation. So we recover nanoparticles with an inert layer. Nanoparticles are

coated by an inorganic component (typically silica or carbon) to produce a core-shell

structure, or coated with an organic shell (surfactants or polymers). This increases stability in

suspension. In context silica coating was used in this studies.

Furthermore, surface modifications are very easy because of the presence of abundant silanol

groups on the layer of silica which are harmonious with many functional groups such as

amines, carboxylic group where molecules including proteins and enzymes are covalently

bonded on the functionalised silica. Silica-coated magnetic nanoparticles are readily

amended by other functional groups because of the surface being hydrophilic.

7. Surface Functionalization of iron nanoparticles silica coated

Surface functionalization is the strategy by which the surfaces of the nanoparticles are engineered

in such a way that it improves interaction with enzymes or other required materials. Amendment

of the external (surface chemistry) of any physical material improves its interactive nature with

the other material. However, addition of functional groups onto the surface of a material

through chemical method is known as functionalization. In this study, organosilanes

(aminosilanes) are the suitable chemical compounds for functionalisation of silica coated

magnetic nanoparticles. This is because of their ability to conjugate wide choice of bio

molecules such as enzymes to surfaces containing amine or carboxylic groups.

The compounds, organosilanes have two different functional groups (bi-functional) and

recognised by the chemical formula X-(CH2)n-SiRn(OR’)3-n. X symbolises the headgroup, (CH2)n

is the alkyl chain spacer group and Si(OR’)n represent anchor groups that attach the silanol

hydroxyl groups on the surface of the silica which follows alkoxyl (OR’) group hydrolysis. The

most widely and used aminosilanes are (3-aminopropyl)-triethoxysilane (APTS).

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Figure 8: Chemical structure of APTS

The following is the reaction of APTS on the surface of nanoparticles during functionalization.

The reaction was accomplished in 24 hours with organic solvent Toluene and Triton X–100 as

surfactant. The amine group from the APTS formed a chemical bond with the OH group from

the enzyme which made the enzyme immobilised to be strongly attached on the FNPs. Cross-

linking of enzymes could play an important role in the surface functionalisation of the

nanoparticles. Treatment with glutaraldehyde led to the conversion of surface amine (from

APTS) to aldehyde. Thus the yield of enzyme immobilisation on the surface modified is better.

Figure 9: The Cross-linking of enzyme onto Iron NPs magnetic coated with silica by using APTS

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II. Materials and Methods

1. Synthesis of nanoparticles

The synthesised core-shell superparamagnetic iron oxide nanoparticles coated with silica with

density 59.6 mg/mL was provided by Dr Tapas Sen, project supervisor of the study. The

nanoparticles were synthesised by co-precipitation. This is a simple method to realise, the

aggregate formation can be reduced or eliminated. Once the critical supersaturation threshold

has reached the precipitation of solid particles is made thanks to solid precursors.

Fe2+ (FeCl2·4H2O) and Fe3+ ions (FeCl3·6H2O) with ammonia solution treating under

hydrothermal conditions. A mass of 2.0 g of FeCl2·4H2O and 5.4 g of FeCl3·6H2O (molar ratio

1:2) was dissolved in 100 ml of water at final concentration of 0.3 M iron ions. Ammonium

hydroxide solution (0.7 M ; 75 mL) was added slowly, turning the solution black. The reaction

was allowed to proceed with stirring at room temperature. After the reaction mixture shall be

washed several times with water and ethanol.

Silica-coated magnetite nanoparticles were prepared via the large scale deposition of silica

onto magnetite nanoparticle from silicic acid solution at pH 10.

2. Preparation of solutions

Stock solution of PBS buffer (Phosphate-buffered saline)

This solution was prepared by dissolving 1 PBS tablet in 100 mL of distilled water. The

buffer solution prepared was used for washing and storing of the lipase enzymes.

Lipase enzymes (Candida rugosa) stock solution

A stock solution of lipase enzymes with concentration of 2600 µg/mL prepared by

dissolving 104 mg of enzymes powder in 40 mL of PBS buffer solution and refrigerated.

This solution will be used for all experiences.

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Reagent A

Reagent A was prepared by mixing the solutions prepared as follows, 0.0667g of Gum

Arabic was dissolved in 12mL of 250mM Tris-HCl buffer and 0.267g of sodium

deoxycholate with 48mL of deionized water. This solution was mixed in 1:1 mixture of

isopropanol and used for the hydrolysis of ester (PNPP). The solution was then stored in

the refrigerator for further use.

3. Determination of the density of the nanoparticles (Silica-coated iron oxide)

The initial weight 1.5 mL Eppendorf was taken and then added 1mL of the nanoparticles. After

removing the supernatant, the solid was put into 700C oven for drying at overnight. The initial

weight was the subtracted from the final weight (weights of solid and the Eppendorf tube).

The average mass of the solid was 59.7mg. Therefore the density of the nanoparticles was

calculated to be 59.7mg/mL of nanoparticles suspension.

4. Enzyme immobilisation by physical adsorption

In Eppendorf tube add 1mL suspension nanoparticles (now we know the density) and removal

the supernatant by applying external magnetic. Subsequently, 1 mL Lipase enzymes were

added into the nanoparticles at temperature room overnight by end-over-end rotation at 40

rpm during 24 hours. After, the absorbance of the solution was measured as 0.762.

The concentrations of the lipase enzymes before and after immobilisation on the non-

functionalized nanoparticles were 2600 and 455 µg/mL (from the calibration curve)

respectively. Therefore the concentration of the enzymes immobilised on the nanoparticles

Weight W1 (g) W2 (g) W3 (g)

Initial (empty Eppendorf tubes) 0.9510 0.0575 0.0607

Final (add 1 mL suspensions

nanoparticles)

1.0119 1.0090 1.0153

∆W 0.0609 0.0575 0.0607

weight (mg) 60.9 57.5 60.7

Tableau 1: Density calculation

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was 2145 µg/mL and hence the quantity was adsorbed was 35.9 µg/mg of nanoparticles

which shows consistency with the value obtained in Sen et al, 2010. The knowledge of this

result is essential because it is necessary to know exactly the amount of lipase immobilised

for hydrolysis experiment.

5. Surface of functionalization of nanoparticles (Enzyme immobilisation by Chemical binding)

The surface of the nanoparticles was modified by wetting 300mg of the nanoparticles

(corresponds to 5 mL suspension nanoparticles) in 100µg of deionised water. The resultant

materials were then functionalised by addition of 10% (w/v) APTS in 20mL toluene. Because

toluene is hydrophobic in nature, the hydrated nanoparticles were then dispersed in the

presence a surfactant, Triton and incubated overnight at 290C in an incubator. Toluene was

used as a solvent to avoid aminosilane from being hydrolysed and self-polymerised in deionised

water before condensing on the solid surface. The hydrolysis and condensation on the solid

surface for the construction of uniform layer of aminosilane during the process of

functionalisation was achieved with the adsorbed deionised water.

Subsequently, the supernatant was removed and washed the solid functionalised

nanoparticles three times in 5ml of PBS buffer solution and added 5ml of the same solution (to

maintain the same density of non-functionalised nanoparticles).

Them, the same procedure than non-functionalised is realised to determine the lipase amount

absorbed on the functionalized nanoparticles. The quantity of the enzyme immobilised on the

functionalised nanoparticle was calculated to be 40.48 µg/mg which was more than the

amount adsorbed on non-functionalised nanoparticle. This is due to the change in the surface

properties of functionalised nanoparticles than the surface of the non-functionalised as a result

of the amine groups bound on to the surface of the functionalised nanoparticles.

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Non-functionalised Functionalised

Number of

Eppendorf tubes 1 2 3 1’ 2’ 3’

Absorbance 0.720 0.774 0.794 0.622 0.678 0.659

Average 0.762 0.653

Quantity enzyme in

Eppendorf tubes

after 24 hours

455 µg/mL 184 µg/mL

Quantity enzyme

immobilised on

nanoparticles after

24 hours

2145 µg/mL 2416 µg/mL

Quantity enzyme

immobilised on NPs

35.9 µg/mg nanoparticles 40.48 µg/mg nanoparticles

Tableau 2: Resume of enzyme immobilised for both methods immobilisation

6. Hydrolysis of esters

Through this method, the effectiveness of lipase enzymes was studied. Free and immobilised

enzymes on either non or FNPs of 500 µg quantity were used in the hydrolysis of PNPP (4-

nitro phenyl palmitate). This lipase reacted in 1ml of the ester solution with the concentration

of 3.74 µmol/mL and was prepared in the 1:1 mixture of isopropanol and reagent-A (0.0667g

of the Gum Arabic + 0.267gm of the deoxycholate +12mL of the 250mM Tris-HCL + 48mL

deionised water at pH 7.8) in 1.5ml Eppendorf tube by end-over-end rotation (40 rpm) for

3hours at room temperature. The supernatant was collected and absorbance was measured

at 410 nm using UV-Visible spectrometer in 20 minutes intervals over 1 h 30 min.

The hydrolysis reaction was examined by measuring the concentration of PNP in the reaction

solution through the use of calibration curve made from range of standard solutions of PNP

prepared in 1:1 mixture of isopropanol and reagent A.

Subsequently, the nanoparticles were washed three times in 1mL of 1:1 reaction mixture.

Then the materials were hydrolysed with PNPP under the same condition as earlier discussed

for the study of catalytic efficacy and re-usability of the enzyme immobilised nanoparticles.

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In this research, reusability of the immobilised enzyme for hydrolysis of long chain esters was

demonstrated in up to three cycles.

7. Characterisation

UV-Visible Spectroscopy

Several molecules absorb visible or ultraviolet light. The absorbance of the solution is

dependent on the concentration of the solution. However, this is based on Beer’s law which

states that:

A = ε*l*c where

ε = proportionality constant known as absorptivity; l = path length; c = concentration

Furthermore, different molecules absorb radiation at different wavelengths. In this research,

UV-Visible spectrometer (WPA Lightwave II) was used for determining the concentrations of

lipase enzyme and 4-nitrophenol for bio catalysis and transesterification reaction.

Scanning electron microscopy (SEM)

Scanning electron microscopy is a very important and powerful tool in magnifying and exploring

shapes of microstructures. The silica-coated nanoparticles used in this work was characterised

with FEI QUANTA 200-SEM using 20 kV as electron acceleration voltage.

Samples were prepared by putting little and diluted nanoparticles solution on carbon pad

attached on aluminium stub, and allowed to dry overnight. Images were obtained on the

computer screen after the interaction with the atoms of the sample by the electrons. The

elements present in the sample were observed with EDAX (Genesis Spectrum SEM Quant ZAF)

after gold coating.

FTIR Analysis

The aim of any absorption spectroscopy such as FT-IR and UV-Visible, is to measure the amount

of absorbed light by a sample at each wavelength. The Fourier transform infrared spectroscopy

technique was used in this experiment. It contains three parts: source of radiation, sample and

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detector. During the process, some of the IR radiation passes through the sample (transmission)

and some of it absorbed by the sample (absorption). The absorbed radiation by the sample

produces peaks, each of which represents a certain functional group, hence an excellent tool for

qualitative analysis.

Fourier Transform Infrared Spectrometer (thermoscientific IR 200 Nicolet) was used to identify

the samples used in the analysis: CRL, non-FNPs, FNPs, enzyme immobilised on non-FNPs and

FNPs, the solid after hydrolysis of ester (PNPP) and transesterification reactions on FNPs. The

samples were analysed in solid form after drying in the oven at 50 0C overnight. FTIR spectrum

of each sample was observed.

Vibrating Sample Magnetometry (VSM)

Vibrating Sample Magnetometry (VSM) is one of the best effective implementations of

magnetometer. The sample is magnetised by presenting it in a constant external magnetic

field. The magnetised sample vibrates and introduces agitation in the external magnetic field.

At room temperature (298 K), vibrating sample magnetometer (VSM) of 7 kOe was used to

obtain the measurements of magnetisation curve and saturation magnetisation of samples.

Bare and silica-coated nanoparticles were packed into plastic tubes of ~10 mm length and

internal diameter of ~1.9 mm after being crushed.

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III. Results and discussion

1. SEM

The main technique for determining the topography, morphology, composition of a sample

can be achieved by using scanning electron microscope. Due to the problems associated with

the instrument, image obtained was not perfect.

Figure 10: The SEM image of Silica coated SPIONs (A) & (B) with EDAX (C)

A B

C

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We see that nanoparticles are aggregated. The nanoparticles’ size are polydispersity. The

nanoparticle appears to be spherical. Although larger aggregates can be present due to

sample preparation techniques involved with SEM. EDAX result displays the available

elements including Si and O, approving the existence of SiO2 on the nanoparticles. The

presence of sodium might be presence of impurities. However, the iron (Fe) and oxygen (O)

present confirms the formation of iron oxide. Hence the EDAX result approves the formation

of silica coated SPIONs indicating sodium as an impurity.

2. FTIR

The following figure represents the overlay FT-IR spectra of the samples used in the project

work.

Figure 11: FT-IR spectra of samples analysed

The respective strong absorption peaks existing at 544.66 and 545.96 cm-1 for non and FNPs

show the presence of Fe-O stretching vibrations of magnetite nanoparticles. Even though, the

Fe-O of the bulk magnetite nanoparticles commonly absorbs at ~570 cm-1. Due to the

nanoparticles finite size, the band shifts to high wave numbers. The lower absorption by the

mentioned two supports (non-FNPs) might be due some unavoidable errors during the

experiments.

0

20

40

60

80

100

120

01002003004005006007008009001000%

Tra

nsm

itta

nce

wave number cm-1

FT-IR Spectra

Non-FNPs FNPs

Immobilized enzyme on non-FNPs Immobized enzyme on FNPs

CRL After hydrolysis of PNPP

After transesterification reaction

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In FNPs, the absorption peak at 1126 cm-1 indicates the existence of Si-OH vibrational stretch.

The vibrational stretching of HC-H absorbs at 2861.70 cm-1 for the APTS modified magnetite

nanoparticle.

The existing peaks at 1632.63 and 1630.59 cm-1 confirms the presence of the bound lipase

enzymes on non-functionalized and functionalized nanoparticles respectively. Hence, this

confirms the immobilisation on the two supports.

The absorption peak at 1647.74 cm-1 signifies the presence of lipase enzyme (Candida

rugosa) as it falls within the region 1700 – 1600 cm-1. The origin of this band comes from C=O

vibrational stretch of peptide group as reported earlier by ((Natalello, Ami et al. 2005).

After hydrolysis of ester (PNPP) with immobilised enzymes on FNPs, the signals in the range;

1700 – 1750 cm-1 are apportioned to the C=O of esters and it presence confirmed by the

absorption at ~1751 cm-1. Similarly, the C-O bonds shows stretching vibration at 1001.82 cm-

1. The absorption peaks at 1345.40 and 1550 cm-1 correspond to N-O and C=C bonds

respectively. However, with the identified peaks, confirms the presence of ester (PNPP) and

alcohol (PNP).

The spectrum of the solid sample after transesterification reaction showed absorption at

1738.03 cm-1 which correspond to the C=O group of esters. Also, the peak at 553.04 cm-1

indicates the Fe-O stretch.

3. Magnetic properties of nanoparticles analysis

Figure 12: Magnetic susceptibility data of large-scale bare magnetite QBLSBM

24

The above figure displays the magnetisation data for large-scale bare magnetite QBLSBM,

prepared from large-scale oxidative hydrolysis of ferrous sulphate. The data we have been

given by Docteur Tapas. The nanoparticles are realised by co-precipitation (same protocol

than this project).

The saturation magnetisation is Ms = 67 emu/g. Due to the polydispersity nanoparticles size

distribution in the sample (25-200 nm), small hysteresis appears. The possibility of impurities

existence is due to the magnetic measurements from the QBLSBM that was synthesised

months earlier and stowed in water, and thus causes oxidation of the magnetite.

4. Determination of the amount of immobilised lipase enzyme on nanoparticles by Bradford assay

This is a spectroscopic technique for determination of the concentration of protein in solution

and is constructed based on absorbance shift of the dye (Coomassie Brilliant Blue G-250). In

state of acidity, the colour of the dye changes to blue during binding of the dye to the enzyme

analysed and the more the quantity of the analyte, the deeper the colour blue. The interaction

of the two (protein and dye) causes stability of blue colour of the dye by the protein and the

concentration of protein is achieved through the calibration curve of absorbance versus protein

concentration in µg/mL.

Bradford assay depends on interaction of dye to protein. The anionic form bound to the protein

has maximum wavelength of 595nm. The increase of the wavelength is directly proportional to

the quantity of bound dye and to the quantity (concentration) of protein. Therefore, the

amount of the dye on the protein is proportional to the quantity of the

Bradford assay was used to determine the concentration of lipase enzymes on the

nanoparticles with the use of UV-Visible spectrometer as described by Bradford assay Sen et

al, 2010.

The stock solution of the lipase enzymes was used to prepare six different diluted solutions

in PBS buffer with 1mL of Bradford reagent. The concentration of the lipase immobilised on

nanoparticles was determined from the calibration curve created from the series of standard

solutions and measured the absorbance at the wavelength of 595 nm.

25

The calibration curve is linear and consistent. So the Beer-Lambert law is respected and we

can use it to determine the amount of lipase absorbed on the nanoparticles.

5. Hydrolysis of ester

With reference to Sen et al, 2010, alcohol (PNP) was produced from ester (PNPP) in presence

of an enzyme catalyst (CRL). In this method, free and immobilised enzymes (by physical

adsorption and chemical binding) were used to compare the efficacy of the both as described

below.

Figure 13: Reaction scheme for the hydrolysis of 4-nitro phenyl palmitate

However, after completion of the reaction, the supernatant was taken and measured the

absorbance using UV-Visible spectrometer at maximum wavelength of 410 nm. The

calibration curve of PNP is shown below.

y = 0,0004x + 0,58

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

0 100 200 300 400 500 600 700 800 900

Ab

sorb

ance

(5

95

nm

)

Concentration lipase (µg/ml)

Calibration curve of Lipase

26

We observe that quantity of nitrophenol increases with time. This is logical because there is

conversion of ester to alcohol.

We can see in the graph that, free lipase enzyme showed maximum catalytic activity and

maximum nitrophenol produced; the major disadvantage is the impossibility to re-use

enzyme and is not viable in the industrial scale.

When immobilised Lipase enzyme on the nanoparticles, it is possible to re-use many times

with the nanoparticle (apply external magnetic field) after washing.

0

200

400

600

800

1000

1200

1400

1600

1800

0 20 40 60 80 1004-n

itro

ph

eno

l co

nce

ntr

atio

n (

µm

ol/

g en

zym

e)

Times (minutes)

Hydrolyse of PNPP(Amount of 4-nitrophenol created by absorbance measure)

Free enzyme

Functionalised (First cycle)

Functionalised (Second cycle)

Functionalised (Third Cycle)

Non-functionalised (First cycle)

Non functionalised (second cycle)

Non functionalised (Third cycle)

y = 0,009x - 0,0034R² = 0,9973

-0,2

0

0,2

0,4

0,6

0,8

1

1,2

0 20 40 60 80 100 120 140

Ab

sorp

tio

n 4

10 n

m

4 nitrophenol concentration (µg/mL)

Calibration curves 4-nitrophenol in 1:1 mixtures of isopropanol and reagant A

Second…

27

The yield is better when nanoparticle is functionalised. After 1,5 hours the average of

conversion hydrolysis of ester to alcohol is 1240 µmol/g enzyme. While with use enzyme

immobilised on the nanoparticle non-functionalised, the production of alcohol (nitrophenol)

decrease in successive cycles until it becomes low at third cycle. So for re-use Lipase enzyme,

it is necessary before to functionalised nanoparticles, the rate of hydrolysis conversion of

ester to alcohol is better. The main reason is with the non-functionalised nanoparticles, the

lipase is loosely bonded (physically adsorbed) during the first cycle of hydrolysis of PNPP

whereas with nanoparticle is functionalised, chemically bonded was created and allow to give

stability and strong binding between lipase and nanoparticle. So through the cycles, quantity

enzyme on nanoparticles is substantially the same.

6. Transesterification reaction

The vegetable oils solubility in aqueous ethanol depends on the system temperature and

concentration of alcohol. Alcohols are not miscible with vegetable oils at ordinary

temperature. Thus, addition of good solvent such as n-hexane rises the oils solubility and

lowering the solubility temperature. Ethanol dissolves many organic compounds as they have

similar intramolecular forces. Also non-polar bonds such as; C-C, C-H and C = O (highly polar)

makes it a versatile solvent.

Due to the time constraint, the transesterification reaction was not undertaken but used UV-

Visible spectrometer to see the reaction influence between the oil (soybean oil) which is the

ester and the alcohol (ethanol in hexane).

First step, wavescan is doing only with soybean oil and 1:1 mixture of ethanol/hexane.

Absorbance was measured as 0.033 at the wavelength maximum of 271 nm. Then, 500 µg of

immobilised enzymes were added to the solution and allowed for end-over-end rotation (40

rpm) overnight. Absorbance was measured as 1.383 at 688 nm maximum wavelength. The

shifting of the wavelength maximum resulted from the reaction that took place and produced

another ester.

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Conclusion

The use of biodiesel has become indispensable due to the tremendous environmental effects.

The interest in bio catalysis for the production of biodiesel has been a growing development

due to its benefits.

Due to the high cost of enzymes, the process of immobilisation is one of the ways of utilising

the enzymes for reusability and more stability.

Enzymes attached by physical adsorption and thus desorbed easily while in FNPs, enzymes are

strongly attached (by chemical binding).

The results obtained from FT-IR confirmed the type of the iron oxide (magnetite) and the silica.

The nanoparticles and other samples used in this research were characterised by FT-IR, VSM,

and SEM attached with EDAX techniques.

The findings of this study encourage for further research into continuous method of biodiesel

production using different enzyme. Moreover, the hydrolysis reaction should be studied at

different temperatures to examine the effect at different temperature. Effects of Other

immobilisation methods should also be investigated and reduce their fouls.

Moreover, this internship allowed me to discover a new country and to improve my English. I

learnt to live alone in a new country; it is a great responsibility which gave me a lot of

experiences. More, I had to work with my proper idea, and I could do all what I wanted and

when I wanted help, I could speak with my supervisors. So doing this internship abroad was

only benefit for me. I think what would be awaited from an engineer in nanotechnologies.

29

References

1. Ma, F. (1999). Biodiesel production: a review. Bioresource Technology. 70, p1-15. 2. Lu J, Chen Y, Wang F, Tan T. (2009). Effect of water on methanolysis of glycerol trioleate catalyzed by immobilized lipase Candida. In organic solvent system. J Mol Catal B Enzym. 56, p122-125. 3. Krawczyk, T. (1996). Biodiesel-Alternative fuel makes inroads but hurdles remain. Inform. p801-829 4. Shay, E. (1993). Diesel fuel from vegetable oils: status and opportunities. Biomass and Bioenergy, 4(4), pp.227--242. 5. Nagle, N. and Lemke, P. (1990). Production of methyl ester fuel from microalgae. Applied Biochemistry and Biotechnology, 24(1), pp.355--361. 6. Calvin, M., (1985). Fuel oils from higher plants. Ann. Proc. Phytochem. Soc. Eur. 26, p.147-160. 7. Bartholomew, D., (1981). Vegetable oil fuel. JAOCS 58, pp.286A-288A. 8. Weisz, P., Haag, W. and Rodewald, P. (1979). Catalytic production of high- grade fuel (gasoline) from biomass compounds by shape-selective catalysis. 9. Sinha, S., Agarwal, AK., Garg, S. (2008). Biodiesel development from rice bran oil: transesterification process optimization and fuel characterization. Energy Conver Manage. 49, p1248-1257. 10. Enweremadu, CC., Mbarawa, MM. (2009). Technical aspects of production and analysis of biodiesel from used cooking oil – a review. Renew Sustain Energy Rev. 13, p2205-2224. 11. Ali, Y., Hanna, MA. (1994). Alternative diesel fuels from vegetable oils. Bioresource Technology. 50, p153-163. 12. Vicente, G., Martínez, M., Aracil, J. (2004). Integrated biodiesel production: a comparison of different homogeneous catalysts systems. Bioresource Technology. 92, p297-305. 13. Sadrolhosseini, AR., Moksin, MM., Mahmood, W., Yunus, M., Mohammadi, A.,Talib, Z. (2011). Optical Characterization of Palm Oil Biodiesel Blend. Journal of Materials Science and Engineering. 5, p550-554.

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14. Gilbert, E. J. (1993). Pseudomonas lipases: Biochemical properties and molecular cloning. Enzyme and Microbial Technology, 15 (8), pp634-645. 15. Turcu, M. C. (2010). Lipase-Catalyzed Approaches Towards Secondary Alcohols: Intermediates For Enantiopure Drugs. University of Turku, Finland. 16. Heck, A. M.; Yanovski, J. A.; Calis, K. A. Orlistat;. (2000). New Lipase Inhibitor for the Management of Obesity. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy, 20 (3), 270-279. 17. Gupta, R.; Gupta, N.; Rathi, P. (2004). Bacterial Lipases: An Overview of Production, Purification and Biochemical Properties. Applied Microbiology and Biotechnology, 64 (6), 763-781. http://www.unil.ch/files/live//sites/esc/files/shared/These_Moret.pdf http://perso.numericable.fr/chimorga/Niveau_M1/protec/protec.php https://tel.archives-ouvertes.fr/tel-00675661/document https://tel.archives-ouvertes.fr/tel-00813982/document file:///C:/Users/Nazih/Downloads/VD2_DEVINEAU_STEPHANIE_04102013.pdf http://popups.ulg.ac.be/1780-4507/index.php?id=2125 http://www.easybiologyclass.com/enzyme-cell-immobilization-techniques/ http://bibli.ec-lyon.fr/exl-doc/TH_T1819_kwan.pdf https://tel.archives-ouvertes.fr/tel-00836242/file/1991_Kumaran_Satish.pdf http://www.enscm.fr/attachments/284_ENSCM_2011_JARRAR.pdf http://www1.lsbu.ac.uk/water/enztech/immethod.html file:///C:/Users/Nazih/Documents/Stage/PAULY_Matthias_2010.pdf

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Appendix

Samples Times (min)

Concentration µg/mL (with calibration curve)

Concentration µmol/g enzyme

2 10.67 152.49 Non

functionalised 40 33.37 227.93

80 51.91 476.73

Functionalised 2 10.67 152.49

40 50.56 722.31

80 86.51 1235.95

1 13.033 186.19 Free lipase 35 83.59 1194.22

75 110.67 1581.06 Tableau 3: Summary table of hydrolysis of 4 nitro phenyl palmitate using enzyme immobilised on iron-coated silica nanoparticles and free enzyme (kinetic study at RT)

Figure 14: Other SEM pictures of NPs coated-silica