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PEROXIDATION REACTIONS CATALYZED BY
SOL-GEL IMMOBILIZED CHLOROPEROXIDASE
A University Thesis Presented to the Faculty
of
California State University, East Bay
In Partial Fulfillment
of the Requirements for the Degree
Master of Science in Chemistry
By
Selina Chan
Summer 2013
Table of Contents
Table of Contents ................................................................................................... ..................ii
Abstract .................................................................................................................. .................iv
Acknowledgments ................................................................................................. .................vi
List of Figures ........................................................................................................ ............... vii
List of Tables ......................................................................................................... .................ix
List of Equations .................................................................................................... .................. x
Chapter 1 Introduction ........................................................................................... .................. 1
1. Chloroperoxidase (CPO) from Caldariomyces fumago ............................... ................. 1
2. Sol-gel encapsulation technique .................................................................... ................. 3
3. Peroxidation reactions catalyzed by CPO ..................................................... ................. 6
a) TMPD ........................................................................................................ ................. 6
b) ABTS ........................................................................................................................... 7
c) Pyrogallol ..................................................................................................................... 7
4. CPO active site............................................................................................... ................. 9
5. Practical Applications for CPO ..................................................................... ............... 10
Chapter 2 Materials and Methods ......................................................................... ................ 13
1. Materials ......................................................................................................... ............... 13
2. Instruments ..................................................................................................... ............... 15
3. Preparation of buffers .................................................................................... ............... 15
4. Methods .......................................................................................................... ............... 18
Chapter 3 Experimental Results ............................................................................ ................ 29
1. Stock solution of CPO ................................................................................... ............... 29
2. Optimal conditions for CPO in solution ....................................................... ............... 30
3. Activity of CPO immobilized in sol-gel beads ............................................. ............... 35
4. Leakage of CPO from sol-gel beads ............................................................. ............... 39
a) Leakage on CPO loading ........................................................................... ............... 40
b) Technique variation: Open versus closed sonication ............................... ............... 43
5. Reuse of CPO immobilized in sol-gel beads ................................................ ............... 45
6. Methanol or ethanol incubation..................................................................... ............... 48
Chapter 4 Discussion ............................................................................................. ................ 53
1. pH and concentration profile of CPO............................................................ ............... 53
2. Leakage of CPO from sol-gel beads ............................................................. ............... 54
3. Reusability of immobilized CPO in sol-gel beads ........................................ ............... 55
4. Effects of methanol or ethanol incubation .................................................... ............... 56
5. Most practical substrate ................................................................................ ................ 57
6. Challenging experiments .............................................................................. ................ 58
a) High activity of CPO ................................................................................. ............... 58
b) Methanol determination by GC-MS ......................................................... ............... 59
ii
7. Future experiments ....................................................................................... ................ 60
References .............................................................................................................. ................ 61
Appendix ................................................................................................................ ................ 65
iii
Abstract
Chloroperoxidase (CPO) from Caldariomyces fumago is a very versatile heme enzyme that
catalyzes different reaction types, including peroxidation reactions of various substrates.
Several of these reactions are of industrial interest. To create a re-usable biocatalyst, CPO
was immobilized in a sol-gel matrix made from tetramethoxysilane (TMOS). The catalytic
efficiency of sol-gel entrapped and free CPO was compared by measuring the initial rates
of the peroxidation reactions with the substrates pyrogallol, N,N,N’,N’-tetramethyl-pphenylenediamine (TMPD), and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
(ABTS). Hindered substrate and product diffusion inside the sol-gel host was a major cause
for loss of catalytic performance. The release of methanol during the sol-gel process might
be another factor as methanol and ethanol both cause a decline of CPO activity in solution.
In addition, some catalytic activity was lost due to enzyme leakage which was most
pronounced during the maturation phase of the gel. Depending on the substrate the
catalytic efficiency of the immobilized CPO ranged between 4% (for ABTS), 8% (for
TMPD), and 13 % (for pyrogallol) in comparison to free CPO in solution. The reusability
of the CPO sol-gel beads was tested in up to 10 reaction cycles. A further decline in
catalytic efficiency was detected for all three substrates, most notably for ABTS. Our
findings are of importance for the optimization of protein sol-gel hybrid materials.
iv
v
PEROXIDATION REACTIONS CATALYZED BY
SOL-GEL IMMOBILIZED CHLOROPEROXIDASE
By
Selina Chan
Approved:
Date:
d12tVJ1/~~~zDr. Danika Leduc
0<3/.27
Dr. Ann McPartland
If 3
Acknowledgments
With the support from my advisor Dr. Monika Sommerhalter, I was able to
accomplish my thesis. From getting familiar with the lab, writing research grants, to
presenting a poster at an international conference, Dr. Sommerhalter was there to guide me
through every step of the way. I am grateful she has always motivated me to think and act
independently in her lab. What makes Dr. Sommerhalter most admirable is her
commitment to all of her students. Each of our projects is unique to her and she constantly
proposes new ideas to engage us more in the research. I would like to give my utmost
gratitude to Dr. Sommerhalter for the teamwork she and I achieved for the Immobilization
of Chloroperoxidase. It was truly an honor to work with her.
I would like to show appreciation to my thesis committee members, Dr. Tony
Masiello and Anne Kotchevar whom gave productive feedback for my thesis.
I would like to thank Dr. Michael Cheng and James Hudson of Chevron Energy
Technology Center, Mass Spectroscopy Lab for allowing me to use their Gas
Chromatography Mass Spectrometry. I had a great experience working with Dr. Cheng
whom always challenged me to think more intellectually.
The Master’s program at Cal State East Bay has become a valuable asset to my life.
Throughout these two and a half years, I have met many friends whom supported me
through my research. I would also like to give thanks to Man Hon Tsang, Tuan Le, Shuang
Huang, Mei Fu, and Anni Mai.
vi
List of Figures
Figure 1 Electron micrograph of the sooty mold Caldariomyces fumago. ............................. 1
Figure 2 SEM of the Caldariomyces fungus. ........................................................................... 2
Figure 3 Mechanism for hydrolysis and condensation in the sol-gel process ........................ 4
Figure 4 Schematic representation of shrinkage using two different drying techniques........ 5
Figure 5 Structure of Guiaicol .................................................................................................. 9
Figure 6 CPO active site. ........................................................................................................ 10
Figure 7 Oxidation reaction from alcohols to aldehydes ....................................................... 11
Figure 8 Schematic representation of the hydrolysis and condensation steps. ..................... 20
Figure 9 Immobilized CPO in sol-gel beads on a ParafilmTM.. ............................................. 20
Figure 10 Illustration of the materials needed for each kinetic assay in solution. ................ 21
Figure 11 A snapshot of the Bradford assay calibration curve. ............................................ 30
Figure 12 CPO in solution assay at varied pH using substrate TMPD ................................. 32
Figure 13 CPO in solution assay using substrate TMPD at varied concentrations .............. 33
Figure 14 CPO in solution assay using substrate ABTS at varied concentrations ............... 34
Figure 15 CPO in solution assay using substrate pyrogallol at varied concentrations ......... 35
Figure 16 Assay for immobilized CPO in sol-gel bead using TMPD. ................................. 36
Figure 17 TMPD assay with immobilized CPO at 3mM TMPD at λ=563nm. .................... 37
Figure 18 Assay for immobilized CPO in sol-gel bead using ABTS. .................................. 38
Figure 19 Assay with immobilized CPO at 3mM ABTS at λ=414nm ................................. 39
Figure 20 Activity of CPO in solution versus the amount of CPO. ...................................... 41
vii
Figure 21 Total percent leakage from the immobilized CPO ............................................... 42
Figure 22 Activity of three immobilized beads with CPO .................................................... 43
Figure 23 Percent leakage for open and closed sonication .................................................... 44
Figure 24 Catalytic effect of varied sol-gel immobilized technique. .................................... 45
Figure 25 CPO sol-gel beads after assay, washing, and storage ........................................... 46
Figure 26 Reusability of CPO sol-gel beads were analyzed with ABTS and TMPD. ......... 47
Figure 27 Reusability of CPO sol-gel beads was analyzed with pyrogallol. ........................ 48
Figure 28 Methanol standard curve by GC-MS .................................................................... 50
Figure 29 CPO Activity after incubation with methanol and ethanol. .................................. 52
Figure 30 Leakage data from a graduate student, Tuan Le. .................................................. 55
Figure 31 Assay with immobilized CPO at 1mM TMPD at λ=563nm ................................ 67
Figure 32 Assay with immobilized CPO at 10 mM TMPD at λ=563nm ............................. 68
Figure 33 Assay with immobilized CPO at 30 mM TMPD at λ=563nm ............................. 68
Figure 34 Assay with immobilized CPO at 1 mM ABTS at λ=414nm ................................ 69
Figure 35 Assay with immobilized CPO at 10 mM ABTS at λ=414nm .............................. 70
Figure 36 Assay with immobilized CPO at 30 mM ABTS with λ=414nm .......................... 70
viii
List of Tables
Table 1 Preparation of 0.1M citric acid – 0.2M Na2HPO4 buffer. ........................................ 16
Table 2 Volumes used for kinetic assays with substrate TMPD/ABTS. .............................. 23
Table 3 Volumes used for kinetic assays with substrate pyrogallol. .................................... 23
Table 4 Results of kinetic solution assays with varied concentrations of CPO .................... 41
Table 5 Methanol content measured by GC-MS ................................................................... 50
Table 6 Summary of optimal conditions for the peroxidation of three substrates. ............... 54
Table 7 Summary table of the total leakage for different CPO concentrations ................... 54
Table 8 Comparison of the catalytic performance for three substrates.. ............................... 57
ix
List of Equations
Equation 1 TMPD peroxidation reaction ................................................................................. 7
Equation 2 ABTS peroxidation reaction .................................................................................. 7
Equation 3 Pyrogallol peroxidation reaction. .......................................................................... 8
Equation 4 Beer-Lambert's law .............................................................................................. 25
Equation 5 Hydrolysis of TMOS ........................................................................................... 48
x
1
Chapter 1
Introduction
1. Chloroperoxidase (CPO) from Caldariomyces fumago
Figure 1 Electron micrograph of the sooty mold Caldariomyces fumago.
The bar in the lower left corner corresponds to 40µm in length (Symposium, Watling and Frankland).
Chloroperoxidase (CPO) from Caldariomyces fumago was first isolated by Morris
and Hager (Morris and Hager) and found to be a heavily glycosylated, secreted enzyme.
Caldariomyces fumago, also called Leptoxyphium fumago, is a sooty mold (Olejnik,
Ingrouille and Faull). There are about 200 species of tropical and sub-tropical sooty
mold. Sooty mold is often found on the surfaces of plant leaves with a dense texture
similar to a soot-like material (Symposium, Watling and Frankland). This plant
infestation is a nutrient source for insects (Hughes, et al. 1976). Crystalline CPO was
shown to contain Ferriprotoporphyrin (IX) as a prosthetic group (Sundaramoorthy, Terner
and Poulos ("The Crystal Structure of Chloroperoxidase: A Heme Peroxidase--Cytochrome
P450 Functional Hybrid"). The Ferriprotoporphyrin IX (also called heme) is coordinated by
2
a cysteine ligand. CPO is a very versatile heme-thiolate protein that can catalyze many
reactions including peroxidation, sulfonation, halogenation, oxidation, chlorination,
bromination, and epoxidation reactions (Wagenknecht and Woggon; Shaw, Beckwith and
Hager). An example for a peroxidation halogenation reaction is the biosynthesis of
caldariomycin (2,2-dichloro-1,3-cyclopentane-dione) (Hager et al.).
Figure 2 SEM of the Caldariomyces fungus.
This is the top view of the Caldariomyces fungus. The top stains are the conidial coremium. The
enzyme is secreted with oxalic acid crystals and polymer which is not visually apparent in the image.
The SEM is taken by a PhD student, Irene Oleijnik and Dr. Jane Nicklin-Faull from the University of
London, Birkbeck.
High levels of CPO are secreted by the Caldariomyces fungus (Shaw and Hager).
CPO was successfully purified using a fast and efficient biphasic extraction method with a
yield of ~70.4% and an activity of 2900 U/mg (Yazbik and Ansorge-Schumacher). The
activity of an enzyme is typically given in Units (U) per total protein (in milligram).
Chloroperoxidase is very similar to other glycoproteins such as the plant
peroxidases from horseradish and Japanese radish when comparing amino acid
composition, carbohydrate content, molecular weight, and spectral properties. CPO,
3
horseradish peroxidase, and Japanese radish peroxidase have a carbohydrate content of
30%, 18%, and 28%, respectively. CPO contains only arabinose whereas Japanese radish
peroxidase contains mannose and xylose (Morris and Hager). All of these glycoproteins
have the same unusual amino acid composition dominated by the four amino acid residues
aspartic acid, glutamic acid, serine, and proline. A specialty of CPO is the halogenations
activity; it can perform chlorination and bromination reactions whereas the other two
peroxidases cannot. As a monomer, CPO has a molecular weight of 42 kDa. The extinction
coefficient is 91 mM-1 cm-1 at 400 nm. (Hollenberg et al.)
2. Sol-gel encapsulation technique
The synthesis of silica gels from alkoxides has been discovered in 1846 by
Ebelman (Brinker and Scherer). Over the years, the sol-gel technique was enhanced and
silica sol-gels have been extensively studied. Silica sol-gels can be prepared in multiple
forms with a broad range of pore diameters (Han et al.) and surface functionalities (Menaa
et al.). The most practical use is that an enzyme can be immobilized by forming a porous
network around the enzyme (Gupta and Chaudhury). The enzyme does not need to be
bound to the gel by ionic or covalent bonds. These interactions may interfere with the
enzyme’s function. A very common precursor for the formation of silica sol-gels is
tetramethoxysilane or TMOS (Vollet et al.). The R-group in this silica alkoxide with the
formula Si (OR)4 is a methyl-group. TMOS has a low solubility in water. However, this
problem can be solved by vigorously mixing the components with a vortex mixer or using
sonication for alkoxisilanes (Donatti and Vollet). The reactions of the
4
alkoysilanes during hydrolysis and condensation are slow and therefore can be better
catalyzed in acidic solutions. The proton, (H+), with the strong positive charge can attack
the O(δ-) from the alkoxy OR groups. Catalysis using basic (OH-) conditions is also
possible. The silicon and oxygen are bound by a single bond, Si—O—Si, to give a
polymeric compound which forms a gel-like matrix. In contrast, crystalline silicate is
produced by silicon with polymeric anions as stable end products (Noll, 1968).
Figure 3 Mechanism for hydrolysis and condensation in the sol-gel process was taken from the
publication: The Sol-gel Encapsulation by Enzymes by Pierre. (Pierre).
During the gelation and the drying of the sol-gel, the pores insides the sol-gel walls
shrink. The dry gels are called xerogels. An alternative technique is supercritical drying
which prevents shrinkage. The supercritical drying technique employs liquefied CO2 and
a pressure chamber. First the liquefied CO2 replaces the solvent in the pores of the silica
gel and then the pressure is released to remove CO2 as a gas. The pore size of the gels
that were treated with the supercritical drying technique remains large; and these gels are
5
called aerogels. The Guinness Book of Records awarded a silica aerogel to be the world
lightest solid with a density of 1.9kg/m3 (Pierre, 2004).
Figure 4 Schematic representation of shrinkage using two different drying techniques.
This scheme was inspired by a similar drawing from a review article written by Pierre. (Pierre)
6
It is assumed that during gelation, the gel’s shrinkage comes to a stop when the
rigidity of the network reaches equilibrium with the tensile capillary pressure in the liquid.
As the gel shrinks, the pore radius decreases which causes the tension in the liquid to
increase along with the rigidity of the network. The pH value and the concentration of
buffers added during the condensation reactions will influence the gelation time (Gupta
and Chaudhury). When the surface of the sol-gel is covered by silanol groups, the
reaction is found to be irreversible (Nilsen et al. 1997).
3. Peroxidation reactions catalyzed by CPO
Several substrates that are transformed to colorful products by one or two step
electron oxidations catalyzed by CPO were chosen in this study. The color change
greatly facilitates the detection of CPO activity. All reactions were well characterized in
previous studies (Kadnikova and Kostić; Manoj and Hager).
a) TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine also known as Wurster’s blue was
first discovered by a German chemist, Casimir Wurster (Pearson and Gelormini). TMPD
was also used in experiments involving nicotine addiction in rats. A discovery was made to
select a nicotine antagonist to inhibit nicotine evoked dopamine release in rats. The
alkylation of the pyridine nitrogen atom can transform nicotine from a potent agonist into a
potent antagonist. As a result, TMPD completely inhibited nicotine-evoked dopamine
release meaning that TMPD acts as a selective nicotine receptor antagonist (Dwoskin et
al.). In the TMPD peroxidation reaction, the TMPD loses two electrons in one oxidation
step yielding a purple product.
7
Equation 1 TMPD peroxidation reaction.
b) ABTS, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) is a commonly
commercial reagent used to study percarboxylic acids (Pinkernell, Luke and Karst). It can
also react with bromine and chlorine species (Pinkernell et al.). ABTS performs a oneelectron oxidation step in the presence of CPO and hydrogen peroxide to form ABTS.+
which is a stable, green colored radical (Scott et al.). ABTS can also be used for
experiments testing the total antioxidant capacity in alcoholic drinks (Milardovic,
Kereković and Rumenjak) and analysis of (Fe(IV)) in water (Lee, Yoon and von Gunten).
Equation 2 ABTS peroxidation reaction.
c) Pyrogallol, 1,2,3-trihydroxybenzene is a catechin compound used as a strong reducing
agent. The single electron oxidation with hydrogen peroxide transforms pyrogallol into
purpurogallin. The formation of purpurogallin can be utilized as a inhibitor in toll-like
receptors, TLR1/TLR2 complexes (Cheng et al.). Pyrogallol is a component present in
8
Emblica officinals that can be used to inhibit cell growth on human cancer cells (Yang et
al.). Pyrogallol was also found in the analysis of three commercial hair-gel products that
contain pyrogallol for hair straightening and permanent dyeing features (Mazzei et al.).
Oxidation of pyrogallol to purpurogallin can also be performed by catalase (Tauber).
Equation 3 Pyrogallol peroxidation reaction.
The substrates pyrogallol and H2O2 interact with CPO to produce
purpurogallin, in color yellow.
Two other substrates that are often used in peroxidation reactions with CPO are
guiaicol and monochlorodimedon (MCD). Guiaicol, 2-methoxyphenol, was first
discovered by an Italian chemist (Duffey, Aldrich and Blum) and is found in roasted coffee
(Dorfner et al.). It is a phenolic compound that is formed by the spoilage of apples
identified as Alicylobacillus (Corli Witthuhn et al.). The enzymatic method used to detect
guaiacol is with CPO and hydrogen peroxide forming 3,3’-dimethoxy-4,4’biphenoquinone, a brownish color component, at a wavelength of 420nm (Bahçeci and
Acar). Guiaicol was used in the very beginning of my experiments. However due to the
smell of this chemical compound and its similarity in the structure with pyrogallol, this
compound was omitted from my experiments.
9
Figure 5 Structure of Guiaicol.
The substrate, MCD can form dichlorodimeon (Asplund, Christiansen and Grimvall;
Tzialla et al.). This substrate was donated to the lab by Bio-Research Product, Inc but due
to time constraints, experiments were not performed for this study.
4. CPO active site
CPO contains a proximal heme-thiolate ligand similar to P450 and a polar distal
pocket like a peroxidase. Many peroxidases operate with a histidine residue, however; CPO
is special in that it utilizes glutamate as an acid-base catalyst. The opening above the heme
creates the substrate binding site that allows organic substrates to move toward the
activated oxoferryl oxygen atom bound to heme iron center. The proximal ligand, Cys29
and the hydrogen bond between His 105 and Glu183 are undisturbed by the ligand binding.
The Cys29 sulfur is 3.6Å from the peptide of residue 31 and 32’s amide groups. The
amide-sulfur hydrogen bond stabilizes the iron proteins in the cysteine-ligand loop
(Sundaramoorthy, Terner and Poulos).
10
Figure 6 CPO active site.
Figure made with Pymol was inspired by a publication that has a similar illustration.
(Sundaramoorthy, Terner and Poulos "Stereochemistry of the Chloroperoxidase Active Site:
Crystallographic and Molecular-Modeling Studies")
5. Practical Applications for CPO
a) Chlorination of organochlorine industrial compounds
Organic pollutants are compounds that are generated in the production of pesticides,
plasticizers, paint, hydraulic and heat transfer fluids, additives for cutting oils, and
textile auxiliaries. CPO is the one of the halogenase enzymes that can chlorinate
organic pollutants to reduce toxicity. CPO is able to chlorinate 17 out of 20 aromatic
hydrocarbons (PAHs) with hydrogen peroxides and chloride ions (Vazquez-Duhalt,
Ayala et al. 2001). CPO chlorination was used to reduce the toxicity of aromatic groups
in fulvic acid which is found in soil and water (Niedan, Pavasars and Öberg).
11
b) Oxidation of amino alcohols to amino aldehydes
Several studies have been done to oxidized alcohols to aldehydes using CPO in acidic
conditions (Kiljunen and Kanerva).
Figure 7 Oxidation reaction from alcohols to aldehydes.
A study was completed with immobilized CPO as a biocatalyst to oxidize amino
alcohols to amino aldehydes using monoaminoethyl-N-aminoethyl (MANA) agarose gels.
The retained activity for immobilized CPO versus CPO in solution was 77%. When amino
alcohols are converted to amino aldehydes, they can be used as substrates for the aldol
addition of dehydroxyacetone phosphate which can produce aminopolyols. The
aminopolyol is then cyclated or isomerized to iminocyclitol. Iminocyclitols can act as an
inhibitor of glycosidases and glycosyltranferase for therapeutic use. (Pešić et al.)
c) Sulfonation of organosulfur in petroleum products
The need of sulfur removal in diesel, crude oil, and light gas oils has been a legal
requirement as well as an experimental challenge for oil companies. The conventional
way of de-sulfurization is expensive and inefficient for petroleum products. Therefore,
to reduce sulfur content in gasoline, an enzymatic oxidation was studied to reduce the
sulfur content.
12
Diesel fuel containing sulfur was treated with CPO oxidization coupled with
distillation to successfully remove a sixth of the sulfur content. CPO catalyzed the
oxidization of sulfoxides to sulfones in the presence of 20mM KCl and 1mM hydrogen
peroxide. The activity of the CPO was not influenced by the oxidation of the organosulfur
compounds. The authors of this study confirmed that CPO was solely responsible for the
oxidation of the organosulfur compounds. (Ayala, Robledo, et al.)
Asphaltenes is present in heavy crude oils containing 50% carbon in the form of
aromatic structures and is very difficult to break down. CPO is able to transform
asphaltenes to be more reactive and therefore less coke is generated (Ayala, HernandezLopez, et al.). CPO was also successfully immobilized using mesoporous materials to
remove organic sulfur compounds in petroleum fractions by Terres and Montiel (Terres et
al.).
13
Chapter 2
Materials and Methods
1. Materials
a) Chemicals from Sigma – Aldrich
i.
Chlorperoxidase from Caldariomyces fumago, aqueous suspension brown, >10000
U/mL (Lot # BCBD6999V, lot result 11200U/mL)
(CAS #: 9055-20-3)
ii.
Guaiacol or 2-methoxyphenol. C7H8O2. Lot # 082K3687. FW: 124.14 g/mol (CAS
#: 90-05-1)
iii.
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) diammonium salt
~98% or ABTS. C18H18N4O6S4. FW: 514.62 g/mol (CAS #: 30931-67-0)
iv.
Pyrogallol or 1,2,3-Trihydroxybenzene. C6H6O3. FW: 126.11g/mol.
(CAS #: 87-66-1)
v.
N,N,N’,N’-Tetramethyl-p-phenylenediamine dihydrochloride or TMPD. C10H16N2
2HCl. FW: 237.17g/mol (CAS# 637-01-4)
vi.
Potassium phosphate monobasic. H2KO4P Lot# 090M011V. FW136.09 g/mol.
(CAS#7778-77-0)
b) Fisher Scientific
i.
Hydrogen Peroxide, ACS certified: H2O2. 30 wt%. Lot # 010980
( CAS# 7722-84-1)
14
ii.
Sodium phosphate dibasic: HNa2O4P H2O. FW268.07g/mol. Lot# 067403.
(CAS#7782-85-6)
iii.
Acetonitrile or Methyl Cyanide. CH3CN. FW41.05. Lot# 973738.
(CAS# 75-05-8)
iv.
Sodium phosphate dibasic. Na2HPO4. FW141.96. Lot#773869.
(CAS#7558-79-4)
v.
Citric Acid, Anhydrous. FW192.13. Lot#113478. (CAS#77-92-9)
c) Chemicals from Acros Organics
i.
Methanol, for HPLC. CH4O FW32.04g/mol. Lot#1285891. (CAS# 67-56-1)
d) Chemicals from Pharmco-Aaper
i.
Ethyl Alcohol 190 Proof, ACS/USP Grade. CH3CH2OH. FW46.07g/mol.
Lot#01006251. (CAS# 64-17-5)
e) Chemicals from Fluka Analytical
i.
Tetramethyl-orthosilicate. C4H12O4Si. FW152.22g/mol. Lot#87682.
(CAS#681-84-5)
f) Chemicals obtain from chemical stockroom.
i.
Hydrochloric acid
15
2. Instruments
a) Milton Roy Company Spectronic 20D
b) Orion Research Digital Ionalyzer/501
c) Thermo Scientific Nanodrop 2000c
d) Hewlett Packard 8452A Diode Array Spectrophotometer
e) Hewlett Packard 5973 Gas Chromatography-Mass Spectrometry instrument
(Chevron)
3. Preparation of buffers
All water was deionized using a Milli-Q apparatus from Millipore. All buffer
solutions were stored at 4oC.
a) Preparation of 0.1M citric acid – 0.2M sodium monophosphate buffer.
The storage and assay buffer used to cast and store the beads is a 0.1M citric acid –
0.2M phosphate buffer. A solution of 0.1 M citric acid was prepared by weighing
out 19.213 gram of citric acid and dissolving it in 1 liter of dH2O. The 0.2M sodium
monophosphate buffer was prepared by weighing out 28.392 grams and dissolved
in 1 liter of dH2O.
Using the 0.1M citric acid and 0.2M sodium phosphate solutions, different
volumes were mixed to obtain buffers at various pH values. The pH was verified
with a pH meter to be within ±0.2 before use.
16
pH
2.6
3
3.4
3.8
4.2
4.6
5
5.4
5.8
6.2
6.6
7
7.4
0.1M Citric Acid – 0.2M Na2HPO4 Buffer
x mL 0.1 M Citic Acid
y mL 0.2M Na2HPO4
89.10
10.90
79.45
20.55
71.50
28.50
64.50
35.50
58.60
41.40
53.25
46.75
48.50
51.50
44.25
55.75
39.55
60.45
33.90
66.10
27.25
72.75
17.65
82.35
9.15
90.85
Table 1 Preparation of 0.1M citric acid – 0.2M Na2HPO4 buffer.
The volumes used to make pH 2.6 – 7.4 are listed. (Dawson, 1986)
b) Preparation of 0.2M potassium phosphate buffer, pH 6.0.
21.05 grams of potassium monobasic phosphate and 6.60 grams of sodium dibasic
phosphate were dissolved in 1 liter of deionized water yielding a pH value of 6.0.
(Dawson, 1986)
c) Preparation of 0.01M hydrochloric acid.
Concentrated hydrochloric acid was diluted to 0.01M with deionized water.
d) Preparation of substrates
The substrates were mainly prepared as to 60mM solutions, further dilutions were
made during the assays as needed.
17
i.
Preparation of 60mM TMPD in 60% v/v acetonitrile
Acetonitrile was diluted to 60% by volume with water. Acetonitrile was use to
dilute TMPD because TMPD is insoluble in water. The preparation of 60mM
TMPD (MW: 237.1g/mol) was using 10 milliliters of 60% acetonitrile to
dissolve 0.1432 gram of TMPD.
(
ii.
)(
)(
)
Preparation of 60mM ABTS
The preparation of 60mM ABTS (548.68 g/mol) was to dissolve 0.329 gram of
ABTS in 10 milliliters of water.
(
iii.
)(
)(
)
Preparation of 60mM and 600mM of pyrogallol in water
The preparation of 60mM pyrogallol (126.11g/mol) was to dissolve 0.0757
gram of pyrogallol in 10 milliliters of water.
(
)(
)(
)
The preparation of 600mM pyrogallol was to dissolve 0.757 gram of pyrogallol
in 10 milliliters of water.
(
)(
)(
)
18
e) Preparation of 0.8M hydrogen peroxide
A calculation of the preparation of 0.8M hydrogen peroxide is located in Appendix
A.
f) Preparation of the enzyme stock solution
The CPO stock solution from Sigma-Aldrich is delivered as a dark red/brown
suspension. This suspension was centrifuged in a microcentrifuge at 15000 rpm for
10 minutes. The supernatant was removed and used in all experiments. The brown
pellet was discarded. The supernatant was first diluted to 1/11 dilution with 0.2M
potassium phosphate buffer at pH 6.0. Then it was separated in six aliquots using 1
mL plastic tubes. When needed an aliquot was further diluted to 1/100, 1/500, and
1/1000 with the same buffer. The CPO dilutions were stored at 4oC.
4. Methods
a) Determination of concentration for the CPO stock solution
The stock CPO solution was determined using two techniques: UV-Vis and
Bradford assay. The supernatant solution was first measured with the UV-Vis
Nanodrop instrument in a cuvette. 50µL of 1/11 diluted CPO solution CPO was
diluted with 2950µL of 0.2M potassium phosphate buffer at pH 6.0 and analyzed in
the UV-Vis. The calculated CPO concentration with the corrected dilution factor
was 10.67 mg/mL. After the supernatant solution was separated into six aliquots,
the concentrations were confirmed by the UV-Vis Nanodrop instrument using the
pedestal. 2 µL of the 1/11 diluted CPO solution was placed onto the pedestal. The
19
Soret absorbance was determined at 400nm and calculated with a molar extinction
coefficient of 91200 M-1cm-1 (Liu and Wang). The six aliquots had an average CPO
concentration was calculated with the corrected dilution factor to be 8.99±0.71
mg/mL. The first two aliquots of the supernatant solution were measured with the
Bradford assay using bovine serum albumin (BSA) yielding 8.16±0.04 mg/mL. The
calculations for the concentration of the supernatant are summarized in the
Appendix B. Most of the experiments were done with the first two aliquots.
b) Immobilization of CPO in sol-gel beads
The method for the immobilization of CPO in sol-gel beads was very similar to an
approach used by researchers at North Dakota State University (Smith et al.). The
sol-gel mixture was prepared by combining 470µL of TMOS, 100µL of dH2O, and
9.4µL of HCl. The sol-gel mixture was then sonicated in an ice/water mixture for
30 minutes. The hydrolysis reaction is catalyzed under acidic conditions and the
sonication assists in homogenizing the sol gel solution. After sonication, 280µL of
the sol-gel solution was used to immobilize 1000 µL of 1/1000 diluted CPO with
960 µL of 0.1M citric—0.2M phosphate buffer at pH 6.0. The condensation
reaction is triggered by a change in pH with the 0.1M citric– 0.2M phosphate buffer
at pH 6.0. Figure 8 provides a schematic diagram of the sol-gel preparation and
immobilization process. The solution containing the sol-gel mixture, the
condensation/casting buffer, and the CPO was then distributed with a pipette in
volumes of 50µl per bead onto a ParafilmTM. As time progressed, the droplets of the
immobilization solution hardened into sol-gel beads. The sol-gel beads were left at
20
room temperature for 1-1.5 hours to dry Figure 9. The beads were transferred in
groups of three to be stored in a test tube with a total volume of 3mL of 0.1 M
citric – 0.2M phosphate buffer at pH 4.2 at 4oC.
470µl
TMOS
100µl
dH2O
9.4µl
0.01M HCl
Sol-Gel
Solution
Hydrolysis, HCl
280µl sol
gel solution
960µl buffer
1000µl
diluted CPO
Condensation
Immobilized
CPO
Figure 8 Schematic representation of the hydrolysis and condensation steps to immobilize CPO in sol-gel beads.
Figure 9 Immobilized CPO in sol-gel beads on a ParafilmTM. The volume of one bead is 50µl.
21
c) Peroxidation reactions
Assays were performed with different substrates and different conditions to
optimize the reaction for CPO in solution. The total time of each kinetic assay was
60 seconds; the absorbance was taken in measurements at 2 second intervals. The
wavelength used to monitor the absorbance depended on the substrate of the assay.
Each assay was measured in a total volume of 3mL with 2.65mM H2O2 and various
concentrations of CPO and substrate (Figure 10).
Substrate
Diluted
CPO
2mM
H2O2
Buffer solution,
optimal pH
Spectrophotometer
Figure 10 Illustration of the materials needed for each kinetic assay in solution.
0.1M citric-0.2M phosphate buffer at various pH values, with different concentrations
of substrate, diluted CPO and 2.65mMH2O2 is used for spectrophotometer analysis.
i.
Peroxidation reaction with TMPD
In the TMPD peroxidation reaction, the TMPD loses two electrons in one
oxidation step. This reaction is monitored at 563nm where the activity of the
enzyme turns the solution purple. TMPD is dissolved in acetonitrile since it is
insoluble in water.
22
ii.
Peroxidation reaction with ABTS
ABTS acts as an electron donor for the reduction of peroxo species such as
H2O2. The peroxidation reaction with ABTS is monitored at 414nm. The
product of the reaction is green.
iii.
Peroxidation reaction from pyrogallol to purpurogallin
Pyrogallol is a strong reducing agent. The peroxidation reaction for pyrogallol
is monitored at 420nm. When oxidizing from pyrogallol to purpurogallin the
solution turns yellow.
d) Kinetic Colorimetric Assays for CPO in solution
i.
Determination of optimal pH values and substrate concentrations
The pH was varied from 2.6 to 7.4 with increments of pH 0.4 in the TMPD
peroxidation assay. A total of 13 data points were analyzed. Data for the other
substrates was already available in our laboratory. Seven different
concentrations were studied for the substrates TMPD and ABTS ranging from
1mM to 10mM. Pyrogallol concentrations were varied in a wider range with 12
different concentrations from 1mM to 85mM. Examples on how the assays
were laid out are summarized in Table 2 and
Table 3. All experiments included a blank sample containing only buffer,
substrate and H2O2. The blank values were then subtracted from the actual
measurements to minimize any interference and to correct for the un-catalyzed
background reaction. All experiments were performed in triplicate. The
23
average values are reported on the graphs in the result section and the error bars
correspond to the standard deviation.
TMPD/ABTS
0.5mM
1mM
2mM
3mM
4mM
5mM
7mM
10mM
15mM
pH
2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6
2.6
Buffer
(µl)
2808
2783
2733
2583
2633
2583
2483
2333
2083
60mM
TMPD/ABTS
(µl)
25
50
100
150
200
250
350
500
750
60mM
H2O2 (µl)
100
100
100
100
100
100
100
100
100
1/1000 CPO
(µl)
67
67
67
67
67
67
67
67
67
Table 2 Volumes used for kinetic assays with substrate TMPD/ABTS at varied concentrations.
Assays were done using 2.65mM H2O2 at λ=563 nm, ε=12470 M-1cm-1 for
TMPD and λ=414 nm, ε=36000 M-1cm-1 for ABTS.
Pyrogallol
1mM
5mM
10mM
15mM
20mM
25mM
30mM
35mM
pH
4.2
4.2
4.2
4.2
4.2
4.2
4.2
4.2
Buffer
(µl)
2783
2583
2333
2083
1833
1583
1333
1083
Pyrogallol
45mM
55mM
pH
4.2
4.2
Buffer
(uL)
2608
2558
600mM pyrogallol
(uL)
225
275
60mM
H2O2 (µl)
100
100
100
100
100
100
100
100
60mM
H2O2
(uL)
100
100
75mM
85mM
4.2
4.2
2458
2408
375
425
100
100
60mM pyrogallol (µl)
50
250
500
750
1000
1250
1500
1750
1/1000
CPO
(µl)
67
67
67
67
67
67
67
67
1/1000
CPO
(uL)
67
67
67
67
Table 3 Volumes used for kinetic assays with substrate pyrogallol at varied concentrations.
Assays were done using 2.65mM H2O2 at λ=420 nm, ε=2640 M-1cm-1.
24
ii.
Determination of leakage of CPO from CPO sol-gel beads
After the beads were stored, the buffer was exchanged with fresh buffer at
different time intervals to test for enzyme leakage. Leakage was tested with
several variables. Different concentrations of CPO were loaded into the beads at
dilutions of 1/100, 1/500, and 1/1000. The different preparations were
exchanged at 15 minutes, 1 day, 2 days, 3 days, 4 days, and 6 days. Another
variable that was analyzed for leakage was the use of open and closed
sonication vessels during the hydrolysis process. The variation between open
and closed sonication was examined to see if there was any difference in the
leakage or the final catalytic performance of the CPO sol-gel beads. The
buffers on top of the beads prepared via open or closed sonication were
exchanged at 15 minutes, 1 hour, 1 day, and 2 days. All kinetic assays were
performed as described above with one exception: Only H2O2 and pyrogallol,
but no additional CPO was added to the exchanged storage buffer in order to
determine how much CPO leaked into the storage buffer.
iii.
Methanol or ethanol incubations of CPO in solution
A study was done to test whether the activity of CPO is altered when incubated
with methanol or ethanol. The incubation samples were prepared as follows:
0.8µg/mL to 1 µg/mL CPO was put into a 0.1M citric- 0.2M phosphate buffer
solution pH 4.2 and different concentrations of 1%, 7%, 9%, and 11% of
methanol or ethanol were put into the capped test tubes. Over 2 hours, 1 day,
25
and 23 days, aliquots were taken out of the test tube to measure the activity of
CPO with the pyrogallol assay.
iv.
Calculations for Kinetic Colorimetric Assays
The absorbance given by the spectrophotometer was analyzed as a rate of
absorption over time and then converted to IU with the Beer-Lambert’s law.
Equation 4 Beer-Lambert's law.
where ε=extinction coefficient for pyrogallol is 2640M-1cm-1 at 420nm,
A=absorbance, l=pathlength of cuvette in cm, C=concentration of CPO in M.
The number given by the spectrophotometer was in absorbance/second. This
was first converted to absorbance/minute by multiplying with 60.
Multiply by 60
The Beer-Lambert’s law is reversed to solve for the concentration change.
Where, =extinction coefficient of substrate (M-1cm-1), l= 1 cm
The unit of the rate was cross eliminated to yield M/minute. This unit is then
converted to µM/minute. Since M = mol/L, µM was converted to µmol by
multiplying with the total assay volume of 0.003L. The final unit is then
26
µmol/minutes which correspond to the unit for enzymatic activity which is
often called International Unit (IU).
Multiply by 106
to convert to µM.
Multiply by total
volume: 0.003 L
IU ÷ mL = IU/mL
(
)(
)
Divide
by
the
amount of enzyme
used in the assay:
0.1µL or 0.067 µL.
Divide by CPO stock
solution (8.99 mg)
from UV-Vis.
e) Kinetic Colorimetric Assays for CPO in sol-gel beads
The enzymatic activity was measured with three beads in 0.1M citric – 0.2M phosphate
buffer, pH 2.6-4.2. Before measuring the activity of CPO immobilized in the beads, the
storage buffer was exchange with fresh buffer. The bead assays were measured for five
minutes in intervals of 30 seconds. Within the 30 seconds the beads were agitated for
20 seconds and left to stabilize in the spectrophotometer for 10 seconds before reading
the absorbance. Agitation was achieved by pipetting the solution up and down in the
test tube. The beads assays were performed with the same parameters as the free CPO
on the same day to access the relative catalytic performance of the immobilized
27
enzyme. The assays containing free CPO were conducted with an amount of CPO in
three sol-gel beads containing 67 µl of 1/1000 diluted CPO.
i.
Optimal conditions at varied concentrations of substrate
Four different concentrations of TMPD or ABTS were used in activity assays with
CPO sol-gel beads and free CPO: 1mM, 3mM, 10mM, and 30mM. Hydrogen
peroxide concentration remained constant at 2.65 mM. The buffer was 0.1M
citric – 0.2M phosphate buffer, pH 2.6.
ii.
Reusability of the CPO sol-gel beads
The reusability of the beads was analyzed with the peroxidation reaction of the
substrates TMPD, ABTS, and pyrogallol. In this case the absorbance data for the
beads was compared to free CPO with a 1/50,000 dilution. The high dilution factor
was due to the high sensitivity of these assays. A correction factor was applied
afterwards. After conducting the first assay, the buffer was exchanged with fresh
buffer to wash the beads 2-3 times. The beads were then stored in 2mL of 0.1M
citric – 0.2M phosphate buffer, pH 4.2 at 4oC until the next measurement. When
measuring the reusability of beads, the beads were left in the same test tube and the
buffer was exchanged accordingly. The experiment was done in triplicates.
28
iii.
Determination of total methanol content by GC-MS
These experiments were carried out at Chevron in the Mass Spectroscopy Lab. An
HP 5973 GC-MS instrument with a mass selective detector and a DB1 boiling point
column was used for the methanol analysis. A one-microliter syringe was used to
draw 0.1 uL of sol-gel matrix into the GC column. The GC splits gas with the solgel matrix in a 200:1 ratio. Only a small amount of sol-gel matrix was injected in
the GC column so that there was minimal water damage to the column. The
parameters for the analysis were chosen as follows: A starting temperature of 10°C
was held for 4 minutes, then ramped up at 10°C per minute to 320°C and held at
320°C for 20 minutes. Since one sample may contain many different compounds
with different boiling points, the parameters were set constant for the methanol
calibration standards and for unknown samples. Methanol has a low boiling point
of 64.7°C and will elute out of the column in less than 1 minute.
29
Chapter 3
Experimental Results
1. Stock solution of CPO
a) UV-Vis
i.
UV-Vis by cuvette
The concentration of the CPO was analyzed by using a cuvette and found to be
10.66mg/ml in CPO.
ii.
UV-Vis by pedestal
The diluted supernatant solution was separated into six aliquots. The absorbance at
400nm of each aliquot was determined using the pedestal. According to these
measurements the CPO stock solution contains 8.99±0.71 mg/ml.
b) Bradford Assay
The supernatant solution was also analyzed with the Bradford assay yielding 8.19±0.04
mg/ml. A standard curve prepared with bovine serum albumin (BSA) in the range of 25
µg/ml to 2000 µg/ml was used.
30
Figure 11 A snapshot of the Bradford assay calibration curve.
The Bradford calibration curve extends from 25µg/ml to 2000µg/ml. The dilution of the CPO
supernatant falls within the range of the calibration curve. The standards and samples were completed
in duplicates.
The results of two different assays, UV-Vis by pedestal and Bradford assay, differ
by only 8%. In the Bradford assay only the first aliquot was measured in duplicates.
However, for the UV-Vis, all six aliquots were analyzed. The UV-Vis result obtained by
pedestal was chosen for all subsequent calculations in my research.
2. Optimal conditions for CPO in solution
CPO can catalyze peroxidation reactions for various substrates. In this work the
substrates TMPD, ABTS, and pyrogallol were used. Two important experimental
parameters were examined to obtain optimal conditions for these CPO catalyzed
peroxidation reactions: pH and concentration of the substrate. Standard deviations are
shown as error bars in the graphs. The progress of the reaction was monitored with a UVVIS spectrophotometer.
31
a) pH variance
The pH dependence of the peroxidation reactions with the substrate TMPD is
presented in Figure 12. The pH profile of the two substrates, ABTS and pyrogallol,
had already been investigated by other students in the laboratory. The peroxidation
reaction with TMPD shows a similar pH profile in agreement with published data
by Manoj and Hager (Manoj and Hager). The decrease in activity at pH 7.0-7.4 is
caused by a structural change in the active site of CPO (Blanke et al.). CPO is
known to perform best in acidic conditions and is known to be stable in an acidic
environment (Zhang et al.). For my research I wanted to choose substrates that will
yield high CPO activity values. Once CPO is entrapped in a sol-gel bead, there will
be more obstacles, such as hindered substrate diffusion, to achieve a fast reaction.
From the data below, optimal pH for TMPD is 2.6 – 3.4.
32
45
40
Activity (IU/mg)
35
30
25
20
15
10
5
0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
pH
Figure 12 CPO in solution assay at varied pH using substrate TMPD.
The CPO catalyzed peroxidation reaction of TMPD with 2.65mM H2O2, 2mM TMPD, 3µg/mL of CPO
and 0.1M citric – 0.2M phosphate buffer in total volume of 3mL. The buffer used for each experiment
was prepared by mixing 0.1 M citric-0.2 M phosphate to achieve a desired pH value. All measurements
were completed in triplicates with background subtraction.
b) Concentration Variance
As shown in Figure 13, the activity of CPO was at the highest for 4 mM TMPD.
The activity after 5 mM TMPD slowly declines. Although the activity is low at
15 mM TMPD, the errors are fairly minimal. The data was fitted with a substrate
inhibition model yielding enzyme kinetic parameters of vmax = 1240 +/- 829 IU/mg,
Km = 2.4 +/- 2.4 mM, and Ki = 3.6 +/- 3.6 mM.
33
7.0E+02
Activity (IU/mg)
6.0E+02
5.0E+02
4.0E+02
3.0E+02
2.0E+02
1.0E+02
0.0E+00
0
2
4
6
8
10
12
14
Concentration (mM)
Figure 13 CPO in solution assay using substrate TMPD at varied concentrations.
The CPO catalyzed peroxidation reaction of TMPD with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1 M
citric-0.2 M phosphate buffer and various concentrations of TMPD at pH 2.6. The various
concentrations of TMPD were obtained by adjusting the amount of substrate and 0.1 M citric-0.2 M
phosphate buffer in each assay. All measurements were completed in triplicates with background
subtraction.
The same experiment was performed with the ABTS peroxidation assay at pH 2.6
to obtain the optimal substrate concentration. ABTS shows a drop in activity that is
caused by substrate inhibition after 5 mM. The optimal concentration of ABTS was
determined to be 3- 5 mM.
34
1000
900
Activity (IU/mg)
800
700
600
500
400
300
200
100
0
0
1
2
3
4
5
6
7
8
9
10
Concentration (mM)
Figure 14 CPO in solution assay using substrate ABTS at varied concentrations.
The CPO catalyzed peroxidation reaction of ABTS with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1 M citric0.2 M phosphate buffer and various concentrations of ABTS at pH 2.6. The various concentrations of
ABTS were obtained by adjusting the amount of substrate and 0.1 M citric-0.2 M phosphate buffer in
each assay. All measurements were completed in triplicates with background subtraction.
The peroxidation reaction with the third substrate, pyrogallol, was investigated at
pH 4.2. Pyrogallol performed with high activity at multiple concentrations and followed a
trend that almost obeyed the Michaelis Menten equation. In contrast to pyrogallol, the
peroxidation reactions with the other two substrates showed strong substrate inhibition.
Similar observations were made by Manoj et al. (Blanke et al.). The data shown in Figure
15 was fitted with the program EnzFitter and the Vmax value was determined to be 4316 ±
1708IU/mg and the Km and the Ki values were determined to be 51 ± 27 mM and 58 +/39 mM. The optimal values for pH and concentration of pyrogallol in solution are pH 4.2
and 35 mM, respectively.
35
1.8E+03
1.6E+03
Actiivty (IU/mg)
1.4E+03
1.2E+03
1.0E+03
8.0E+02
6.0E+02
4.0E+02
2.0E+02
0.0E+00
0
10
20
30
40
50
60
70
80
90
100
Concentration (mM)
Figure 15 CPO in solution assay using substrate pyrogallol at varied concentrations.
The CPO catalyzed peroxidation reaction of pyrogallol with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1M
citric-0.2M phosphate buffer and various concentrations of pyrogallol at pH 4.2. The various
concentrations of pyrogallol were obtained by adjusting the amount of substrate and 0.1M citric-0.2M
phosphate buffer in each assay. All measurements were completed in triplicates with background
subtraction.
3. Activity of CPO immobilized in sol-gel beads
The activity for the peroxidation of TMPD and ABTS by CPO immobilized in solgel beads was determined by analyzing the immobilized CPO with colorimetric assays.
Fresh 0.1M citric- 0.2M phosphate buffer, pH 2.6 was exchange with the storage 0.1M
citric- 0.2M phosphate buffer before every assay. Absorbance measurements were taken
manually every thirty seconds for five minutes. Within the thirty seconds the solution and
beads where mixed by pipeting the solution up and down for twenty seconds and placing
the test tube in the UV-Vis spectrometer for ten seconds before the measurement was
taken.
36
TMPD activity assay was reviewed in the previous sub-chapter to be pH 2.6 and 35 mM for the pH and concentration, respectively. The same pH was used for the CPO solgel bead analysis with variations in the substrate concentration to determine the optimal
conditions of the immobilized CPO.
The immobilized CPO was analyzed with four different concentrations. The data
shows that there is not much change for the first three concentrations ranging from 1-10
mM, however the concentration at 30 mM shows a slightly higher activity.
60
Activity (IU/mg CPO)
50
40
30
20
10
0
0
5
10
15
20
mM TMPD
25
30
35
Figure 16 Assay for immobilized CPO in sol-gel bead using TMPD as the substrate with varied
concentrations.
The CPO catalyzed peroxidation reaction of TMPD with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1M citric0.2M phosphate buffer and various concentrations of TMPD at pH 2.6. The various concentrations of
TMPD were obtained by adjusting the amount of substrate and 0.1M citric-0.2M phosphate buffer in
each assay. All measurements were completed in triplicates with background subtraction.
The following data shows one example of the TMPD at the concentration of 3 mM. The
data shows a consistency and linearity with the triplicates. The slope was taken from 90 to
37
300 seconds since the data points recorded at 30 to 90 seconds exhibit a slow ramping
curve. The other graphs for the other concentrations can be found in the Appendix C.
0.3
Absorbance
0.25
y = 0.00103x - 0.02214
R² = 0.99817
0.2
0.15
y = 0.00098x - 0.02265
R² = 0.98951
0.1
y = 0.00100x - 0.02499
R² = 0.99863
0.05
0
0
50
100
150
200
250
300
Time (seconds)
Figure 17 TMPD assay with immobilized CPO at 3mM TMPD at λ=563nm.
The CPO catalyzed peroxidation reaction of TMPD with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1M citric0.2M phosphate buffer and various concentrations of TMPD at pH 2.6.
The same type of experiment was also performed for the substrate, ABTS. The
activity of CPO using ABTS as the peroxidation substrate was low for high and low
concentrations (at 1 mM and 30 mM), but the data shows a slightly higher activity for
concentration of 3 mM and 10mM. The error bars for the two concentrations, 3 mM and 10
mM, show that there is less error at 3mM.
38
45
Activity (IU/mg CPO)
40
35
30
25
20
15
10
5
0
0
5
10
15
20
25
30
35
Concentration (mM)
Figure 18 Assay for immobilized CPO in sol-gel bead using ABTS as the substrate with varied
concentrations.
The CPO catalyzed peroxidation reaction of ABTS with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1M citric0.2M phosphate buffer and various concentrations of ABTS at pH 2.6. The various concentrations of
ABTS were obtained by adjusting the amount of substrate and 0.1M citric-0.2M phosphate buffer in
each assay.
Figure 18 shows an example assay for one concentration of ABTS (3mM) used to
determine the activity of immobilized CPO. As shown for TMPD, 3 mM is displayed
below as comparison. The slope was taken for the first three to four data points before the
reaction levels off to obtain the fastest initial reaction rate. The variation of the slopes is
larger for TMPD than for the triplicates of ABTS. The other three concentrations are
shown in the Appendix D.
39
1.6
1.4
y = 0.0068x + 0.0665
R² = 0.9989
Absorbance
1.2
1
y = 0.003x + 0.1745
R² = 0.9247
0.8
0.6
y = 0.006x + 0.096
R² = 0.9997
0.4
0.2
0
0
50
100
150
200
250
300
Time (seconds)
Figure 19 Assay with immobilized CPO at 3mM ABTS at λ=414nm.
The CPO catalyzed peroxidation reaction of ABTS with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1M citric0.2M phosphate buffer and various concentrations of TMPD at pH 2.6.
4. Leakage of CPO from sol-gel beads prepared with varied CPO
concentrations
One of the obstacles in enzyme immobilization is the possibility that enzyme leaks
from the sol-gel matrix. During the maturation of the bead, the enzyme may not be
immobilized inside the silicate cage. It may be attached to the surface or even have leaked
out of the silicate cage. The pore size distribution of silica sol-gel beads after maturation
was measured by a graduate student, Tuan Le, using the physisorption analyzer
(ASAP2020, Micromeritics). An average pore size of a typical bead was found to be 20 Å
to 30 Å in diameter. The size of CPO is 46 Å x 53 Å x 60 Å. (Abuto et al. 2005). These
results support the hypothesis that CPO is large enough to stay immobilized inside the
beads.
40
Experiments were performed to test the amount of leakage for different
concentrations of immobilized CPO over a course of 6 days. A set of experiments to
simulate the scenario if CPO leaked out of the beads in amounts of 100%, 10%, 5%, and
1% was performed as well. The beads were stored at 4oC and later analyzed in 0.1M citric
– 0.2M phosphate buffer at pH 4.2 with the substrates 35mM pyrogallol and 2.65mM
H2O2. The storage buffers were exchanged at 1 hour, 1 day, 2 days, 3 days, 4 days, and 6
days. All experiments were performed in triplicates. The buffer exchange done at the first
hour was to wash out any CPO that may be lingering on the surface of the silicate beads
and to remove methanol released during the hydrolysis and condensation steps. It was
found that at the first hour, the CPO on the surface may not have been completely washed
out. The first day has the highest CPO activity in the storage buffer since there was not
enough time for the sol-gel matrix to form a tight mesh. The sol-gel matrix matures over
the first three days and continues to form tighter and tighter pores (Nisen et al. 1997).
a) Leakage on CPO loading
The different concentrations of CPO with dilutions 1/100 (6µg/mL), 1/500 (1.2
µg/mL), and 1/1000 (0.6µg/mL) of CPO were immobilized in the bead. The solution
assays conducted on the same day were analyzed for using 1/100, 1/500, and 1/1000
CPO at 100%, 10%, 5%, and 1%. The three concentrations were analyzed as different
percentages and converted to mg of CPO. The results are summarized in both
Table 4 and Figure 20. Plotting the different CPO concentrations (or the percentages)
versus the activity shows linearity with a correlation coefficient for the linear regression
41
of 0.9994. The linearity also confirmed that the leakage data is valid with respect to
providing a measure for CPO loss.
Dilution
Percentage
1/100
CPO
1/500
CPO
1/1000
CPO
100%
10%
5%
1%
100%
10%
5%
1%
100%
10%
5%
Activity
(IU)
2.5E+00
2.7E-01
1.0E-01
1.5E-02
5.5E-01
4.0E-02
1.4E-02
3.7E-04
2.2E-01
1.3E-02
4.1E-03
mg of
CPO
6.0E-03
6.0E-04
3.0E-04
6.0E-05
1.2E-03
1.2E-04
6.0E-05
1.2E-05
6.0E-04
6.0E-05
3.0E-05
Table 4 Results of kinetic solution assays with varied concentrations of CPO.
3.0
2.5
Activity (IU)
2.0
y = 425.12x - 0.0074
R² = 0.9994
1.5
1.0
0.5
0.0
0.0000
-0.5
0.0010
0.0020
0.0030
0.0040
0.0050
Amount of CPO (mg)
Figure 20 Activity of CPO in solution versus the amount of CPO.
The data shows the linearity of activity versus amount of CPO.
0.0060
0.0070
42
The results of the six buffer exchanges were examined. At the 1/100 CPO, the
total leakage was 51% percent. As the concentration of the immobilized CPO decreases,
the CPO sol-gel beads show less leakage. At 1/500 CPO, the total leakage decreases to
39% and at 1/1000, the total leakage was only at 22%. Standards were analyzed under the
same conditions for 1/100, 1/500, and 1/1000 dilution. Since the linearity had a
regression of R2=0.999 for the standards as shown in Figure 20, the dilution should not
matter. The dilution can be a reference point to calculate the leakage with the assumption
that 100% was the true value. As time elapses, the activity of CPO in the exchanged
storage buffer also decreases. This data proves that the bead successfully immobilizes the
enzyme with minimal leakage after the third day of buffer exchange.
100
Activity (IU/mg of CPO)
80
60
1/100
40
1/500
1/1000
20
0
-20
1 hour
leakage
1 day
leakage
2 day
leakage
3 day
leakage
4 day
leakage
6 day
leakage
Figure 21 Total percent leakage from the immobilized CPO with varied concentrations over six days
duration.
The comparison of CPO loading using 6µg/mL, 1.2 µg/mL, and 0.6µg/mL of CPO was used for 1/100,
1/500, and 1/1000, respectively. Leakage data was normalized from the 100% leakage for each dilution.
Total leakage for 1/100, 1/500, and 1/1000 was 51%, 39%, 22%, respectively. All measurements were
completed in triplicates with background subtraction.
43
After the six buffer exchanges, the beads were analyzed with the pyrogallol assay
on three different days as shown in Figure 22. The relative activity and reusability reported
in IU/mg CPO is higher for the CPO sol-gel beads with lower CPO concentration. All
CPO sol-gel beads still show activity even after two months of storage.
140
Activity (IU/mg of CPO)
120
100
80
1/100
60
1/500
1/1000
40
20
0
8/3/2012
8/14/2012
10/10/2012
Figure 22 Activity of three immobilized beads with CPO analyzed with the substrate pyrogallol after
six exchanges.
The comparison of CPO loading using 6µg/mL, 1.2 µg/mL, and 0.6µg/mL of CPO was used for 1/100,
1/500, and 1/1000, respectively. Each assay was analyzed with 35mM pyrogallol, 2.65mM H 2O2, and
0.1M citric – 0.2M phosphate buffer at pH 4.2. All measurements were completed in triplicates.
b) Technique variation: Open versus closed sonication during hydrolysis
An experiment with the open and closed sonication during the hydrolysis step was done
in addition to the CPO loading to test if there was a difference in the leakage or activity.
The data shows that with the open and closed sonication and the same buffer
exchanges, the percent leakage is very similar for the first three exchanges whereas the
2nd day of the buffer exchange there is a decrease with the closed sonication. This data
44
shows that there is no significant difference in the open versus closed sonication
method. In addition, a set of bead assays were stored in the buffer for one week without
exchange. The leakage was analyzed and found to be 21%. There also is no significant
difference with respect to the catalytic activity of the sol-gel immobilized CPO if the
technique is varied as shown in
Figure 24.
25%
Percent Leakage
20%
Open
15%
Closed
10%
No exchange
for one week
5%
0%
15min
1 hour
1 day
2 day
No exchange
for one week
Time of buffer exchange
Figure 23 Percent leakage for open and closed sonication.
The CPO catalyzed peroxidation reaction of pyrogallol with 2.65mM H 2O2, 0.2ug/mL of CPO, 0.1M
citric-0.2M phosphate buffer at pH 4.2. All measurements were completed in triplicates with
background subtraction.
45
200
180
Activity (IU/mg)
160
140
120
100
80
60
40
20
0
Closed sonication
Open sonication
Closed sonication with no
buffer exchange for one week
Figure 24 Catalytic effect of varied sol-gel immobilized technique.
5. Reuse of CPO immobilized in sol-gel beads
The ability to reuse the immobilized enzyme is an important asset. For many
applications in the environment and in industry, reusing an enzyme is a cost effective
production method. The reusability was tested with three substrates (TMPD, ABTS, and
pyrogallol) to see which substrate was best. After each analysis, the beads were washed
twice with 0.1M citric – 0.2M phosphate buffer at pH 4.2. No detergents were used in the
washing process. Despite extensive washing the substrates and products are still retained
inside the sol-gel matrix.
46
Figure 25 CPO sol-gel beads after assay, washing, and storage.
Beads are colored after reactions performed with the three substrates: Pyrogallol(right),
ABTS(middle), TMPD(right). The beads were washed and stored at 0.1M citric – 0.2M phosphate
buffer, pH 4.2.
When the set of beads are analyzed multiple times, the beads dissociates into smaller pieces
each time. The dissociation of the beads might facilitate CPO and substrate interaction.
The following data shows the first use as 100% activity. All measurements were calculated
relative to the first value to show the percent decrease after each use. The activities of the
beads were analyzed with three substrates over a seven to ten day period. The same beads
were analyzed for activity each time in triplicates.
a) Reusability for substrates, TMPD and ABTS
The TMPD assay from the first analysis to the second analysis decreased by 72%. The
activity remained less than 20% of the initial value for the next 8 days. The ABTS
assay was observed for reusability ten times. After each use, the activity of the beads
decreased in significant increments. The physical appearances of the beads were dyed
with the product of the ABTS assay the first 4-5 times; then the product seemed to
wash away leaving the beads clear. This is correlated with less activity in the beads.
Relative to the activity of the first use, the second use has decreased by 31%. Figure 26
47
below shows the percent decrease in activity relative to the first use. Some beads were
re-used after one day and others were stored for several days. It was noticed that if the
beads were allowed to rest in the storage buffer for a longer period of time, the activity
of the bead was higher than if it was analyzed daily. The longer incubation time in the
storage buffer may have helped to wash out the substrate and allowed new substrate to
interact more freely with the CPO in the sol-gel bead.
Activity (IU/mg of CPO)
80
70
60
ABTS
TMPD
50
40
30
20
10
0
Figure 26 Reusability of CPO sol-gel beads were analyzed with ABTS and TMPD.
The reaction was analyzed under optimized conditions with 3mM ABTS/TMPD and 2.65 mM H2O2 at
pH 2.6.
b) Reusability of sol-gel bead with pyrogallol assay
The next substrate was pyrogallol. A set of three beads were analyzed over a six day
period. The pyrogallol assay shows about the same activity with low reusability
compared with the TMPD and ABTS peroxidation assays. Pyrogallol was used at a
higher concentration of 35mM versus the TMPD and ABTS substrates which were
48
used at a concentration of 3mM. The higher concentration of the substrate may have
clogged the pores of the silicate cage causing a lower activity during re-use.
20
18
Activity (IU/mg of CPO)
16
14
12
10
8
6
4
2
0
11/16/2012
11/19/2012
11/20/2012
11/21/2012
11/27/2012
11/28/2012
Figure 27 Reusability of CPO sol-gel beads was analyzed with pyrogallol.
The reaction was analyzed under optimized conditions with 35mM pyrogallol and 2.65 mM H2O2 at
pH 4.2.
If the bead were to be analyzed 5-10 times, all three substrates will fall below 20% of its
original activity. This shows a low recovery in the activity of the reusability.
6. Methanol or ethanol incubation
The generation of sol gels requires two reactions: hydrolysis and condensation.
During the hydrolysis process, alcohol is released as a byproduct.
Si(OR)4 + H2O → HO-Si(OR)3 + R-OH
Equation 5 Hydrolysis of TMOS.
49
Since the reactant for this process is tetramethylorthosilicate (TMOS), methanol is
formed as a byproduct. The theoretical amount of methanol in the sol-gel bead is calculated
in Appendix E.
In theory, after the hydrolysis reaction, 88% v/v of methanol is produced. Methanol
is further diluted during the condensation reaction to 11% v/v. However, these theoretical
values can only provide an estimate for the upper limit: up to 11% v/v methanol may or
may not be entrapped within the sol gel. It is not certain how much methanol has
evaporated during the process and how much methanol is still left to hinder the CPO
activity. Therefore a determination of the total methanol amount was attempted by Gas
Chromatography - Mass Spectroscopy (GC-MS).
A methanol standard curve was completed with the following settings. Four
different concentrations were used for the calibration curve: 10%, 30%, 50%, and 70% v/v
methanol in water with 0% as the baseline. 90% v/v methanol was initially also analyzed
but found to be saturated. The standard curve in Figure 28 has a regression line with a
correlation coefficient R2 of 0.981 based on the peak area and methanol percentage.
50
2.0E+08
y = 2.44E+06x - 4.34E+06
R² = 9.81E-01
1.8E+08
1.6E+08
Peak Area
1.4E+08
1.2E+08
1.0E+08
8.0E+07
6.0E+07
4.0E+07
2.0E+07
0.0E+00
-5
5
15
25
35
45
55
65
75
%v/v Methanol
Figure 28 Methanol standard curve by GC-MS.
The sol-gel matrix was analyzed with the same settings as the calibration. A
comparison between an open and closed sonication was made. During the hydrolysis step,
the sol-gel matrix was sonicated. Other researchers have stated that some methanol
evaporates during the sonication stage (Smith et al.). To further study possible methanol
evaporation, the sonication was done with an open versus closed tube. The assumption is
that the open sonication tube will have more methanol evaporation causing a lower
methanol concentration. After the hydrolysis phase, both the open and closed sonication
tube contained over 70% v/v methanol which was the upper limit for the calibration curve.
After the condensation phase, the methanol content in the open sonication tube was
measured to be 19.5% v/v, whereas the closed sonication tube had a measured methanol
content of 17% v/v. These values are very similar. This measurement shows that the
sonication with the open versus closed tube does not impact the methanol concentration
during the hydrolysis. The results are summarized in Table 5.
51
Peak
%
Area
MeOH
Stages
Hydrolysis Open Sonication
1.4E+08 Saturated
Hydrolysis Closed Sonication
3.0E+08 Saturated
Condensation Open Sonication
5.2E+07
19.5
Condensation Closed Sonication
4.6E+07
17
Table 5 Methanol content measured by GC-MS.
The next experiment further investigates if methanol (or ethanol) will reduce the
activity of CPO. Based on the findings, the alcohol content was 20% v/v which is higher
than the theoretical value of 11% v/v. The GC-MS measurement performed after the
condensation phase might be hampered by gelation and solidification of the sample.
a) Reduction of CPO activity caused by incubation with methanol and ethanol
There are two very similar reactants that can be used to prepare sol gel beads: TMOS
and tetraethylorthosilicate (TEOS). When preparing the sol gel solution, TMOS will
produce methanol whereas TEOS causes the release of ethanol. The following
experiment probes the detrimental effect of each alcohol on CPO activity. CPO was
incubated with seven different concentrations of methanol or ethanol. Kinetic assays
were done with the CPO methanol/ethanol solution after different incubation times.
These incubation times were chosen to mimic the buffer exchange times with the CPO
immobilized beads. The same conditions were used for all kinetic assays: 35mM
pyrogallol and 2.65 mM hydrogen peroxide were used as substrate and co-substrate in a
0.1 M citric – 0.2M phosphate- buffer, pH 4.2. Figure 29 below shows that the activity
52
of CPO will decrease with the increase of alcohol concentration. The activity will also
decrease as the alcohol incubation time elapses. A comparison between the alcohol
effect on CPO activity shows that ethanol weakens the activity of CPO more than
methanol.
7E+03
Methanol: 2 hours
Methanol: 1 day
Methanol: 23 days
Ethanol: 2 hours
Ethanol: 1 day
Ethanol: 23 days
6E+03
Activity (IU)
5E+03
4E+03
3E+03
2E+03
1E+03
0E+00
0%
5%
10%
15%
20%
% v/v Methanol/Ethanol
Figure 29 CPO Activity after incubation with methanol and ethanol.
Kinetic assays with final concentration of 35mM pyrogallol and 2.65 mM H2O2 were used to analyze CPO
activity. The activity of methanol and ethanol baseline (0%wt) was determined with 1µg/mL. The incubation of
the methanol and ethanol were completed with 0.8µg/ml CPO.
53
Chapter 4
Discussion
1. pH and concentration profile of CPO
It was noticed that all three substrates, TMPD, ABTS, and pyrogallol, react well
with CPO in acidic conditions. TMPD and ABTS have a narrow range for the optimal
concentration due to substrate inhibition but the substrate pyrogallol has a wide range of
optimal concentrations which can possibly extend to 85mM. The co-substrate hydrogen
peroxide shows an inhibition at high concentrations. The data from Manoj demonstrates
that 1 mM hydrogen peroxide shows a much higher activity than 10 mM hydrogen
peroxide for the substrates TMPD and ABTS (Manoj et al.). The hydrogen peroxide
concentration used throughout my research was 2.65mM; this represents a reasonable
amount of hydrogen peroxide with respect to the literature data. Manoj also investigated the
substrate inhibition for the pyrogallol and ABTS peroxidation assay. Substrate inhibition
starts to occur at 100mM and 10mM for pyrogallol and ABTS, respectively. This data
correlates well with the findings from my research in which substrate inhibition is apparent
at concentrations higher than 5mM ABTS.
Table 6 summarizes the optimal conditions of the three different substrates for
peroxidation reactions catalyzed by CPO in solution.
54
Substrate
TMPD
ABTS
Pyrogallol
pH
2.6 – 3.4
2.6
4.2
2mM-3mM
30mM-35mM
Concentration 3mM – 4mM
Table 6 Summary of optimal conditions for the peroxidation of TMPD, ABTS, and pyrogallol
catalyzed by CPO in solution.
2. Leakage of CPO from sol-gel beads
a) CPO leakage from sol-gel beads
The higher the CPO concentration, the more leakage occurred. The data in the result
section also showed that the higher the loading the smaller deviation in the data.
Table 7 summarizes the results for different concentrations of immobilized CPO and
different conditions for monitoring CPO leakage. It is also found that if the original
buffer was left with the beads for one week and no exchange was done, the leakage will
total to 21% for 1/1000 CPO dilution.
CPO dilution
1/100
1/500
1/1000
1/1000
Percent Leakage
51%
39%
22%
21%
Condition
Six buffer exchanges
Six buffer exchanges
Six buffer exchanges
No buffer exchange
Table 7 Summary table of the total leakage for different CPO concentrations used in the sol-gel
immobilization procedure.
The trend for the leakage was the highest on the first day and as time progressed to the
sixth day, the leakage was negligible. The high percent leakage on the first day may be due
to some CPO on the surface of the sol-gel bead. On the first buffer exchange, the buffer
55
“washes out” the CPO that is attached to the surface of the sol-gel bead. The percent
leakage drastically declined by each day. The data from a graduate student Tuan Le also
confirms that after the third day of buffer exchange the effect of the leakage is minimal
(Figure 30).
7%
CPO Leakage
6%
pH 4.5
5%
pH 5.5
pH 6.5
4%
3%
2%
1%
0%
1
2
3
4
5
6
Storage days
7
8
9
10
Figure 30 Leakage data from a graduate student, Tuan Le.
The assay was completed with the 1/1000 CPO with 42mM pyrogallol and 8mM hydrogen peroxide
with 8 beads. These beads were prepared with buffers at the different pH values of 4.5, 5.5, and 6.5.
3. Reusability of immobilized CPO in sol-gel beads
The reusability of the immobilized CPO in the sol-gel beads was compared between
three different substrates. The immobilized beads had broken apart after 3-4 uses; therefore,
differences might occur as the system was no longer in the same condition as in the first
use. Since the beads broke apart, a hypothesis was made that the substrate may be able to
access the active sites more conveniently. However, the activity of the beads declined after
the re-use indicating that the hypothesis with the substrates entering the actives sites more
56
easily was false. On the six day, the activity of the bead was close to 0. An observation was
that the activity of the beads was measured on three consecutive days; on the third day, the
colored products still remained in the silicate cage causing a barrier between the CPO and
new substrates to bind. A set of beads was left in storage buffer for four days recovered
some activity. For the immobilization of CPO, it is best to choose the lowest possible
concentration that still yields high activity. Since the substrate has to travel through the
silicate cage to interact with the active site, high concentrations of substrate may get
clogged in the silicate cage causing a lower or no activity especially for reusability studies.
4. Effects of methanol or ethanol incubation
The methanol/ethanol incubation was done to investigate which alcohol will have a
more detrimental effect on the enzyme CPO. The data shows a decline in activity as the
CPO was incubated with the alcohol during a longer period of time. The incubation data
also shows that ethanol will damage the CPO to a greater extent causing less activity for the
CPO than in methanol. The data from the publication and lab experimental analysis
confirms our choice of using TMOS instead of TEOS for the immobilization of CPO in a
sol-gel. The maximum theoretical value calculated for methanol is 11% v/v. Based on the
incubation data, if a total of 11% v/v methanol were present inside the beads; this would
cause a great damage to the CPO and possibly hinder its activity.
The GC-MS data on the detection of methanol after condensation was found to be
roughly 20% for both the open and closed sonication from the calibration curve. The
calibration curve was generated with methanol in water. This result shows that the method
used to prepare and analyze the sample may not have been suitable for GC-MS, since the
57
experimental value was higher than the maximum theoretical value. Since condensation
happens right after hydrolysis, the addition of water may have caused the solution to
solidify between sampling. This data also shows that the open and close sonication does not
significantly affect the methanol concentration.
5. Most practical substrate
3 mM
3 mM ABTS
35mM Pyrogallol
TMPD
Free CPO (IU/mg)
417±58
777±39
1335±39
Immobilized CPO
(IU/mg)
Relative Catalytic
Efficiency (%)
Reusability after 5 uses
(% recovered)
24±0.7
33±0.6
164±10
6%
4%
12%
8%
10%
16%
Table 8 Comparison of the catalytic performance for three substrates. All assays were completed with
2.65mM H2O2.
After working with all three substrates and evaluating reactions with CPO, it was
found that each substrate has its own set of optimum conditions. Pyrogallol worked well at
pH 4.2 which was conveniently also the pH value of the storage buffer. Therefore,
pyrogallol was chosen as the main substrate to complete most of the studies of this thesis
including leakage, methanol or ethanol incubation, effect of CPO loading and variation in
technique with respect to open and closed sonication. Pyogallol also had a wide optimum
concentration range which showed slight substrate inhibition only after 40mM. The
experiments done with variation in technique during the preparation of the bead with the
open or closed sonication did not show any deviation in the leakage and methanol analysis.
58
The catalytic efficiency tested with the immobilize CPO versus CPO in solution showed
that the TMPD and ABTS has low catalytic efficiency at 4-6% only where the pyrogallol
has 12% at optimal conditions. Pyrogallol also presented a good data set for the reusability
analysis. Due to the high concentration of the pyrogallol, it was expected that the substrates
used for the peroxidation reactions were not completely washed out and may interfere with
the reusability. However, the data shows that after five reuses, the reusability maintained at
16% of the original activity which was higher than the other two substrates. TMPD
appeared to be entrapped in the sol-gel bead but the analysis of TMPD still produced some
activity up the 10th use. For the ABTS substrate, however, after the fifth use the beads were
clear showing that almost no activity was produced after that. Overall, for most scenarios,
pyrogallol would be the ideal substrate to use since it has a wide optimal range and can
work in the same pH range that is optimum for CPO storage. The advantage is that it has
the best catalytic efficiency and reusability which was not shown in TMPD or ABTS.
6. Challenging experiments
a) High activity of CPO
One of the challenges for this experiment was analyzing the activity of free CPO with the
Milton Roy Company Spectronic 20D. Since the CPO was diluted to 1/100, 1/500, and
1/1000 CPO for the immobilization procedure, if the same concentration of free CPO were
analyzed on the spectrophotometer, its absorbance increase in the activity assay would be
too high to detect. The diluted 1/50,000 CPO solution was analyzed with the
spectrophotometer and the slope was multiplied by 50 to obtain the same activity as
expected for a 1/1,000 dilution of CPO. Yet data for the 1/50,000 CPO dilution was found
59
to yield better activity for high substrate concentrations with less substrate inhibition than
more concentrated CPO solutions. If a 1/50,000 CPO dilution were used to immobilize
CPO in the sol gel, the hindrance with the silicate cage and the low concentration of the
CPO will produce a situation that is so unfavorable that the activity would be too low for
detection. Therefore, the comparison with the free and immobilized CPO cannot be made
with the same conditions. For further experiments it is important to analyze the free and
immobilized CPO with the same concentration. Perhaps a different spectrophotometer can
be used with stirring bar in the cuvette and timed kinetic assays of five minutes.
b) Methanol determination by GC-MS
The methanol determination did not work because of the heterogeneous mixture that was
generated while sampling. There were several other techniques that were tried for the
methanol determination. The first attempt was with deuterium-labeled methanol that was
used to confirm whether the GC-MS can detect deuterium labeled methanol. The methanol
peak area in the sample was subtracted by the deuterium standard (methanol free) to obtain
the methanol concentration. However, the peak for the deuterium did not show a clean
standard therefore this method was no longer used. The second attempt was to use GC-MS
by headspace sampling assuming that liquid and gas were in equilibrium. When headspace
measurements were completed with the standard calibration curve, it was found that the
liquid and gas phase were not in equilibrium and therefore the values were not aligned with
the calibration curve. Also, it was difficult to obtain a liquid and gas phase during the
condensation reaction. The last attempt was with the liquid injection method which was
presented in my results section. Although the experiment did not work since the
60
experimental value was higher than the theoretical value, I think that further experiments
can be analyzed with a similar method. For example, an increase in the amount of solution
with the same ratio of materials during the condensation might help with the homogeneity
of the sample.
7. Future experiments
Ideally, the experiment sol-gel immobilization of CPO can be used for chlorination
in waste water, oxidation of amino alcohols, or sulfonation in gasolines. Further
experiments can bring this experiment to a commercial application. One experiment that is
valuable to improve the catalytic efficiency of the immobilize CPO versus free CPO. To
improve the catalytic effect, an alternative option to immobilize the CPO by using a crosslink technique which makes the CPO more accessible to the substrates and co-substrates.
Another experiment that can be beneficial is further research on the reusability of the bead.
An ideal scenario is that the bead can be reused for ten times with minimal activity loss;
this might be with help of detergents or different storage buffers.
61
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Appendix
Appendix A – Hydrogen Peroxide
The H2O2 solution was made with the 30% w/w H2O2 bottle from Fisher Scientific. To
obtain the molarity, the mole and milliliters of the 30% w/w H2O2 were first determined by
dividing by the molecular weight of H2O2 (34 g/mol) and dividing by the density of H2O2
(1.1 g/mL), respectively.
30
34g mol
100g
1 1 g mL
0
mol H2O2
90 91mL
0 mol
90 01 L
0 0909 L
9 71 M
1365 µL of 9.71M H2O2 was taken to dilute the solution to 1.32M H2O2 with 10 milliliters
of water. A further dilution was made with 600µL of 1.32M H2O2 to obtain 0.8M H2O2
with 10 milliliters of water. All assays in this study were completed with final
concentration of 2.65mM H2O2.
66
Appendix B – Calculations for the stock CPO solution
1. UV-VIS Nanodrop spectrophotometer using the pedestal/cuvette
The absorbance is the 1/11 diluted CPO was taken at 400nm. Using Beer’s Law, the
concentration was calculated with the extinction coefficient as 91 mM-1cm-1, l as 1mm.
The concentration (mM) was then converted to mol/L by using the molecular weight of
CPO (MW= 42000 g/mol). The concentration was multiplied by 11 because of the 1/11
dilution. The final concentration has unit of mg/ml.
2. Bradford assay
The concentration was read from the spectrophotometer in units of µg/ml. The
concentration was multiplied by the dilution factor, 11, and divided by 1000 to obtain
the final concentration unit of mg/ml.
67
Appendix C – TMPD sol-gel immobilized assay
The slopes for the TMPD sol-gel immobilized assays are shown for the concentrations
1mM, 10mM, and 30mM. The slopes were all calculated with 90 to 300 seconds due to the
first 90 seconds that shows a slow reaction. The slopes of all three concentrations for
TMPD are very parallel. A sign of hindrance for TMPD at high concentration is the
decrease in slope for 10mM and 30mM.
0.4
0.35
y = 0.0009x - 0.0446
R² = 0.9944
Absorbance
0.3
0.25
0.2
y = 0.0011x - 0.07
R² = 0.9967
0.15
0.1
y = 0.0014x - 0.0651
R² = 0.9942
0.05
0
0
50
100
150
200
250
300
Time (seconds)
Figure 31 Assay with immobilized CPO at 1mM TMPD at λ=563nm.
The CPO catalyzed peroxidation reaction of TMPD with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1M citric0.2M phosphate buffer and various concentrations of TMPD at pH 2.6.
Absorbance
68
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
y = 0.0011x + 0.0849
R² = 0.9993
y = 0.0012x + 0.0976
R² = 0.998
y = 0.0009x + 0.1094
R² = 0.9959
0
50
100
150
200
250
300
Time (seconds)
Figure 32 Assay with immobilized CPO at 10 mM TMPD at λ=563nm.
The CPO catalyzed peroxidation reaction of TMPD with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1M citric0.2M phosphate buffer and various concentrations of TMPD at pH 2.6.
1.2
Absorbance
1
0.8
y = 0.002x + 0.3329
R² = 0.9978
y = 0.0024x + 0.3696
R² = 0.9953
y = 0.0022x + 0.3681
R² = 0.9971
0.6
0.4
0.2
0
0
50
100
150
200
250
300
Time (seconds)
Figure 33 Assay with immobilized CPO at 30 mM TMPD at λ=563nm.
The CPO catalyzed peroxidation reaction of TMPD with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1M citric0.2M phosphate buffer and various concentrations of TMPD at pH 2.6.
69
Appendix D – ABTS sol-gel immobilized assay
The three concentrations for ABTS are shown below. ABTS showed hinderence at high
concentrations such as 10 mM and 30 mM. The slope at 10 mM and 30 mM was much
smaller than 1 mM and 3 mM. With the concentration of 30 mM, only four points from the
graph was taken for the slope since the activity shows a plateau after.
1.6
1.4
y = 0.0017x + 0.065
R² = 0.9892
Absorbance
1.2
1
y = 0.0039x + 0.2627
R² = 0.9684
0.8
y = 0.0025x + 0.073
R² = 0.9988
0.6
0.4
0.2
0
0
50
100
150
200
250
300
Time (seconds)
Figure 34 Assay with immobilized CPO at 1 mM ABTS at λ=414nm.
The CPO catalyzed peroxidation reaction of ABTS with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1M citric0.2M phosphate buffer and various concentrations of ABTS at pH 2.6.
70
2
1.8
1.6
y = 0.0075x + 0.284
R² = 0.9981
Absorbance
1.4
y = 0.0086x + 0.3145
R² = 0.9824
1.2
1
y = 0.0079x + 0.4665
R² = 0.9687
0.8
0.6
0.4
0.2
0
0
50
100
150
200
250
300
Time (seconds)
Figure 35 Assay with immobilized CPO at 10 mM ABTS at λ=414nm.
The CPO catalyzed peroxidation reaction of ABTS with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1M citric0.2M phosphate buffer and various concentrations of ABTS at pH 2.6.
Absorbance
2
1.5
y = 0.005x + 0.995
R² = 0.9921
1
y = 0.0051x + 1.05
R² = 0.9792
0.5
y = 0.0058x + 1.04
R² = 0.8756
0
0
50
100
150
200
250
300
Time (seconds)
Figure 36 Assay with immobilized CPO at 30 mM ABTS with λ=414nm.
The CPO catalyzed peroxidation reaction of ABTS with 2.65mM H2O2, 0.2ug/mL of CPO, 0.1M citric0.2M phosphate buffer and various concentrations of ABTS at pH 2.6.
71
Appendix E – Methanol
In theory, after the hydrolysis reaction, 88% v/v of methanol is produced. Methanol is
further diluted during the condensation reaction to 11% v/v total methanol.
Calculation for methanol in sol gel solution:
To obtain the number of moles of methanol (abbreviated MeOH) in the sol gel solution
complete hydrolysis of TMOS was assumed. TMOS has four methyl groups, a molecular
weight of 152.22 g/mol and a density of 1.023 g/mL. The volume of TMOS used to prepare
the sol-gel solution was 0.47mL.
0 47mL 1 023g
mol
4 (
)(
)
mL
152 22g
0 01264 mol MeOH
The number of moles of MeOH was then converted to mL by multiplication with the
molecular weight and division with the density of MeOH.
0 01264 mol MeOH (
32g 1 mL
)(
)
mol 0 791g
0 51mL MeOH
The amount of MeOH in mL is then divided by the total volume of sol gel solution and
converted into a percentage.
0 51 mL MeOH
0 57 mL total solution
v
v
MeOH
72
Calculation of methanol in encapsulation solution:
The sol gel was sonicated for thirty minutes on ice. During the sonication some methanol
may have evaporated, but the theoretical values calculated here do not take into account
loss of methanol via evaporation. The theoretical values provide an upper limit for the
methanol content and were calculated based on a dilution effect using the different volumes
of the sol gel solution and the final encapsulation solution. From the sol gel solution, 280 µl
was taken to make the encapsulation solution. The final volume of the encapsulation
solution was 2.24 mL. The methanol content of 88.22 % v/v is therefore further diluted to
11.03 % v/v.
0 2 0 ul sol gel
(0 0126 mol)(0 579 uL total solution)
0
0 2 mL
2 24 mL
11v v
0 0061 mol MeOH
MeOH