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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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)
1
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.
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
20
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
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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.
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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
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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…
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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.
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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
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