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PURIFICATION AND CRYSTALLIZATION OF O- AMINO PHENOL OXIDASE (AMINOX)
Atoyebi Omolara Mustophat
Master’s thesis Department of Chemistry
organic Chemistry 626/2019
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PURIFICATION AND CRYSTALLIZATION OF O- AMINO PHENOL OXIDASE (AMINOX)
ATOYEBI Omolara Atoyebi
Supervisors: Prof. Juha Rouvinen, Dr. Chiara Rutanen
UNIVERSITY OF EASTERN FINLAND, DEPARTMENT OF CHEMISTRY
JOENSUU, 2019
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ABSTRACT
Coupled binuclear copper (CBC) enzymes have a conservative type 3 copper site, Each coordinated by 3 – histidine, catechol oxidase, tyrosinase and o–amino phenol oxidase (Aminox) were CBC enzyme that function by catalyzing the oxidation of various mono and diphenol compounds to the corresponding quinone. Aminox function is to catalyze amino phenol to quinonimines and few previous studies have been carried out on the enzyme.
The project focused on the purification and crystallization of Aminox EC 1.10.3.4 which was produced in the University of Eastern Finland. The starting protein concentration and activity was determined using UV spectrometer and different purification techniques like affinity chromatography, ion exchange chromatography and hydrophobic interaction chromatography were employed for purifying the enzyme. All purification method employed did not yield excellent separation. The result of the activity measurement of starting samples and purified fractions using colorimeter revealed the presence of Aminox through color change and the purity check was carried out using SDS-PAGE
Characteristic study indicated that, at pH 7 the enzyme showed optimum activity and stability.
Result shows more than 50% of activity was retained in the pH range of 5 to 8. Mass spectrometer was used for the analysis to ascertain the presence of Aminox in the purified fraction. The purified protein was further crystalized using the hanging drop diffusion method and the crystals gotten were subjected to measure X-ray diffraction data using X- ray diffractometer.
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TABLE OF CONTENTS
1. INTRODUCTION ... 1
1.1 COPPER CONTAINING PROTEINS ... 1
1.2 TYPE 3 BINUCLEAR COPPER CENTER ... 2
1.3 THE MOST WELL CHARACTERISED COUPLED BINUCLEAR COPPER (CBC) ENZYME ... 4
1.3.1 TYROSINASE ... 4
1.3.2 CATECHOL OXIDASE ... 9
1.4 AVAILABLE STRUCTURE OF CBC ENZYMES... 10
2.0 MATERIALS AND METHOD ... 14
2.1 MATERIAL ... 14
2.2 PROTEIN CONCENTRATION ... 14
2.3 BRADFORD PROTEIN ASSAY (BSA) ... 14
2.4ENZYME ACTIVITY ... 15
2.5 COLORIMETRY ANALYSIS ... 16
2.6 PURIFICATION PROCEDURE USING ÄKTA ... 16
2.6.1 AFFINITY CHROMATOGRAPHY ... 17
2.6.2 ION EXCHANGE CHROMATOGRAPHY ... 17
2.7 SDS PAGE ... 18
2.8 CRYSTALLIZATION PROCESS ... 19
2.8.1 SAMPLE PREPARATION ... 19
2.8.2 HANGING DROP DIFFUSION METHOD ... 19
2.8.3 CRYO- SOLUTIONS AND DATA COLLECTION ... 20
2.9 MASS SPECTROMETRY ... 21
3.0 RESULT AND DISCUSSION ... 22
3.1 ACTIVITY MEASUREMENT OF AMINOX ... 22
3.2 pH STABILITY ... 23
3.3 PURIFICATION OF AMINOX ... 25
3.4 ION EXCHANGE CHROMATOGRAM ... 27
3.5 HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC) ... 28
3.6 ANALYSIS OF AMINOX IN SDS- PAGE ... 28
3.7 MASS SPECTROMETRY ... 29
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3.8 PROTEIN CRYSTALLIZATION ... 30
3.9 X-RAY DIFFRACTION ... 31
4.0 CONCLUSION ... 33
5.0 REFERENCES ... 34
6.0 APPENDIX A ... 39
7.0 APPENDIX B ... 40
8.0 APPENDIX C ... 41
9.0 APPENDIX D ... 41
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LIST OF ABBREVIATION
Aminox o-aminophenoleoxidase
CBC coupled binuclear copper
CO catechol oxidase
Tyr Tyrosinase
AC Affinity chromatography
His Histidine
IEX Ion exchange
HIC– Hydrophobic interaction
SDS-page Sodium dodecyl sulfate polyacrylamide gel electrophoresis
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1. INTRODUCTION
1.1 COPPER CONTAINING PROTEINS
Proteins are polypeptides with biological structure made up of amino acid chains and these polypeptides are formed as a result of the condensation reaction between longer chains of amino acid. The amino acid sequence is defined by the sequence of gene encoded in a genetic code. Enzymes are protein that functions as catalyst in a living cell, it catalyzes and regulates biological reaction in all living organism. Copper enzymes are metalloenzymes with copper ion at the active site held to the amino acid side chains by coordinated covalent bond. It functions as co-enzyme by imparting activities to the enzyme.
The existence of copper ion in either reduced (Cu+) or oxidized (Cu2+) state makes it an essential cofactor in a diversity of biological redox reactions [1-3]. Copper protein serves as electron or dioxygen transporter and its corresponding copper enzymes function as oxidases, mono and di oxygenase decomposing enzymes, it also acts as nitrogen oxide (NOx) Reductases
[4]. O2 reactive center in the copper enzymes can be present as mono, di or tri-nuclear.
Copper – binding protein are present in 3- domain of life and are grouped into classes on the basis of the active site structural geometry, visibility and spectroscopic properties [5-8]. Protein containing type 1 copper site is majorly a blue copper protein that are engaged in electron transfer (e.g. halocyanin) coordinated in a distorted tetrahedral center, Enzymes containing only Type 2 copper site are non – blue copper protein which form part of the oxido - reductase family (e.g. galactose oxidase) with a square planar or tetragonal geometry around copper having either nitrogen or oxygen as ligands. Type 3 copper site are binuclear copper protein family usually coordinated by three histidine with binding ligands such as oxygen or hydroxyl ion, it comprises of gene encoding tyrosinases, tyrosinases – related proteins, catechol oxidase and hemocyanin [1, 2, 9]. o-amino phenol oxidase (Aminox) is classified as a binuclear copper protein family with a magnetically coupled binuclear active site that binds di-oxygen in a symmetric side-on manner [10]. In general, the type 3 copper catalyzed the oxidation of
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substituted phenol in the presence of oxygen as an electron acceptor which is later reduced to water in the reaction. Each enzyme catalyzed specific substrate which is used in identifying the enzyme.
Figure 1: Classification of Cu sites. From left to right: Type 1, Type 2, Type 3 [11]. Type 1mcopper centers have a distorted tetrahedral shape coordinated by two histidine, one cysteine and one weak ligand (oxygen or nitrogen). Type 2 copper ions are tetragonal or square planner with nitrogen or oxygen ligands, Type 3 copper are coordinated by 3 histidine bridged by oxygen.
1.2 TYPE 3 BINUCLEAR COPPER CENTER
All type-3 copper proteins have a conserved pair of copper-binding sites, called CuA and CuB, Type3 binuclear copper protein are characterized by two closely spaced antiferromagnetically coupled copper ions. All type 3 copper protein family e.g. tyrosinase, catechol oxidase , o- amino oxidase and hemocyanin have different sequence identity but yet share a common active site and differs in functions, while Tyrosinase (Tyr), Catechol oxidase (CO) and Aminox oxidase function as an enzyme, hemocyanin are oxygen carrier and storage protein [12-14]. The Cu(A)-binding site is characterized by a H1(n)-H2(8)-H3 motif and the Cu(B)-binding site is characterized by a H1(3)-H2(n)-H3 motif, where n represent variable number of residues between histidine. The active site comprises of two histidine coordinated copper which can be oxygenated or deoxygenated. The structure of the oxy-form at high resolution as revealed by X-ray crystal structure (fig 2) confirms that one dioxygen molecule is bound in a side on bridging coordination between the two copper atoms [15]. The binding of oxygen to the copper atom induced change in valency in which the atom is present as Cu (I) in the deoxy form but changes to Cu (II) upon oxygenation, this change result to the appearance of blue colour upon oxygenation of type 3 copper protein [16]. The active site of the oxy-form of tyrosinases,
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catechol oxidase and haemocyanin exhibit similar spectroscopy properties which implies that hemocyanin should also function as a catalyst but their activity differences (enzymatic function and oxygen transport) was ascribed to the difference in shape and accessibility of the substrate binding cavities [17].
Members of Type 3 class can be synthesized in its Pro-form or inactive form where the di- copper active centers are covered by themselves or other protein [18]. Experimental determined structure of catechol oxidase and hemocyanin gives a clearer picture of the structural difference by the presence or absence of a protein domain blocking the entrance of the active site in their respective inactive pro-enzyme form [19]. The inactive proenzyme of TYR or CO is activated by the removal of amino acid that blocks the entrance passage to the active site while hemocyanin is a silent inactive enzyme and can be activated if the amino acid blocking the entrance is also removed.
Figure 2: Dioxygen binding and coordination of substrate at the active site of type 3 Streptomyces tyrosinase. (a) shows the initial configuration after hydroxyl approach to the active site, based on copper ion crystal structure of Streptomyces tyrosinase; (b) shows the shift of the substrate to CuA; Coppers: blue, histidine residues: green, dioxygen molecule: red, monophenolic substrate: cyan (its oxygen black), equatorial coordination of CuA and CuB:
yellow frame, axial coordination of CuA and CuB: yellow lines; trans-axial coordination of CuA: grey dot.[12]
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1.3 THE MOST WELL CHARACTERISED COUPLED BINUCLEAR COPPER (CBC) ENZYME
Availability of the CBC crystals structure highly increases in the past decades as more than 70 structures of both Try and CO was deposited in the Protein data bank [20]. Although the structure of the CBC protein has been in existence way back as 1989 when the first crystal structure of hemocyanin surfaced in the RSCB PDB [21].the first crystal structure of catechol oxidase and tyrosinase was solved in the year 1989 and 2007 respectively [22, 23]. However, crystal structure of o-aminophenol oxidase which is also categorized as CBC enzyme is yet to be available. The origin, structure and potential application of the most well characterized CBC enzymes are discussed below.
1.3.1 TYROSINASE
Over the years, tyrosinases has been isolated and studied from numerous species of plant, animal and fungi but few of them have been well characterized. The first isolated Fungai tyrosinase was from edible mushroom and the first bacterial tyrosinases was from Streptomyces glaucescens [24, 25]. The study of different species shows the diversity in tyrosinase tissue distribution, location of cell and structural properties. It was established that tyrosinase protein structure common to all kind of species is yet to be found and enzyme found in all the species differ in activation characteristic even though the Type 3 binuclear copper center is common to all the species [26,27].
1.3.1.1 CLASSIFICATION AND BIOCHEMICAL PROPERTIES
Tyrosinase is by far the well-studied binuclear copper enzymes among others. It catalyzes the reaction of both the o-hydroxylation of monophenols and the two-electron oxidation of o- diphenols to their corresponding o-quinones, the oxidation of o-diphenol is much more rapid than the hydroxylation of monophenol. The oxygen incorporated into the phenolic substrate to form o-diquinone was derived from molecular oxygen as confirmed by labelling studies and two electrons was donated by the substrate to reduce the second oxygen to H2O [32].
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Figure 3: monophenolase and diphenolase activity of tyrosinase [33].
Agaricus bisporus, fungi Neurospora crassaTrichoderma reesei and Streptomyces glausescens are the only species where the best-characterized tyrosinases were derived from. Agaricus bisporus is a tetramer with two different unit while Neurospora crassa and Streptomyces glausescens, Trichoderma reesei are monomer protein as shown in table 1. Tyrosinase catalyzed the first step of melanin production in plant and animal tissue, the o‐quinones produced from Figure 3 yield several unstable intermediates by self-polymerization to give melanin [30, 31]. Melanin is the key factor in many essential biological functions such as pigmentation, primary immune response and host defense. If activity is not controlled in the synthesis of melanin it leads to increased melanin synthesis. Tyrosinase function in food and vegetable processing and it is also very essential in storing processed food.
In humans, the production of anti-tyrosinases causes an auto-immune disorder that produces a white color in patches of the skin. Type I oculocutaneous albinism, a hereditary disorder that affects one in every 20,000 people is also caused by the production of an impaired tyrosinase gene as a result of mutation [28]. Tyrosinase exist in human as extracellular transmembrane enzyme and function in tyrosinase of other origin, example plant and fungi exist as intracellular enzyme [29].
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Table 1: Biochemical and structural properties of CBC [33]
Catechol oxidase Tyrosinase O-aminophenol
oxidase Distribution Plant, fungi, insect
bacteria
Mammal, Plant, animal, fungi, insect, bacteria
Bacteria
Enzyme Classification
EC 1. 10. 3. 1 EC 1. 14. 18. 1 EC 1. 10. 3. 4
Physiological role Sclerotization, wound healing, pigment formation
Sclerotization, wound healing, pigment formation
Sclerotization, wound healing, pigment formation Location in cell Mainly intracellular Mainly intracellular Mainly intracellular
MW 30-60 KDa 30-50 KDa 30 – 50 KDa
Substrate O - diphenol O - diphenol O - aminophenol
Oxidation product O - quinones O - quinones O - quinonimines
Protein family PF00264 PF00264 PF00264
Copper center Type 3 Type 3 Type 3
EPR signal No No No
Structure Monomer Monomer, dimer,
tetramer
Not Available
7 1.3.1.2 Substrate specificity
Tyrosinase oxidizes a wider range of para substituted mono and diphenolic compounds. L- tyrosine and L -DOPA are commonly used to measure tyrosinase activity because it is a specific monophenolic and diphenolic substrate of the enzyme, it can also be used to distinguish tyrosinase and catechol oxidase [22, 34].Tyrosinase has the capability to oxidize various aromatic amines and ortho-amino phenols [35-39]. Larger compound containing tyrosyl such as catechin, peptides and protein can also be oxidized by tyrosinase [38, 40-41]. Tyrosinase, catechol oxidase and o-amino phenol oxidase are sometimes classified as polyphenol oxidase without being able to differentiate between the two enzymes, this is due to the overlap of their substrate specificities [42.].
1.3.1.3 Reaction mechanism
The active site of the tyrosinase exist in three forms with difference in their binuclear copper structure. These forms are met, oxy and deoxy tyrosinases. Met-tyrosinase contain two tetragonal Cu (II) ions anti-ferromagnetically coupled through internal bridge and is also referred to as the resting form of tyrosinase. In the oxy-form, each of the two tetragonal copper atom is coordinated by two strong equatorial and one axial NHis ‘ligands’ and the two-copper atom is bridged exogenously by oxygen molecule bound as a peroxide. Met-tyrosinase can be converted to the oxy-form by the addition of peroxide which decays back to the Met-form by the loss of the peroxide added [43-45]. Formation of oxy-form is also possible by the reduction of deoxy-tyrosinase followed by the reversible binding of dioxygen [46]. These three forms of the tyrosinase active site led to the structural model of the reaction mechanisms proposed for tyrosinase activity.
Oxidation of monophenol requires the presence of the oxy- form and binds axially to one copper of the oxy-tyrosinase (monophenolase activity), rearrangement through the reaction intermediate leads to o-hydroxylation of monophenols by bound of peroxide which generates o-diphenol and then oxidizes to the corresponding o-quinone (fig 4). Diphenols (diphenolase activity) can be oxidized by both the met and oxy - form.
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Figure 4: Monophenolase and diphenolase catalytic cycle for tyrosinase and catechol oxidase.
The catechol oxidase only exhibits the diphenolase activity while tyrosinase exhibit both [43].
1.3.1.4 POTENTIAL APPLICATION
Oxidoreductases ability to polymerize compounds makes them of great interest for many industrial applications in biotechnology, medicine, food processing, textile, and pulp and paper industries [47-48]. Tyrosinase is an oxido-reductase not commercially available for industrial purpose but commercially available for research purpose. Several potential applications of tyrosinases in food and non-food related has been reported, its activity is also causing the undesirable browning in food and vegetables, hence the method to control tyrosinase activity in food industry are constantly being search [49, 50].
9 1.3.2 CATECHOL OXIDASE
Catechol oxidase is a type 3 copper enzyme that catalyzes a two-electron transfer reaction during oxidation of o-diphenol to the corresponding o-quinone. The enzyme lacks monophenolase activity which is a distinctive attribute to differentiate catechol oxidase from tyrosinase. Although, recent research with aurone synthase (a plant catechol oxidase) gives evidence that catechol oxidase possesses hydroxylases activity towards its natural monophenolic substrate [54]. Report on bacterial or mammalian origin of catechol oxidase is not yet found but the few characterized catechol oxidase so far originated from plant and fungi.
In plant, catechol oxidase plays a major role in enzymatic browning by aiding the oxidation of catechol to o-quinines which polymerizes non-enzymatically to form the melanin that gives damage fruit dark brown color [55].
Figure 5: Reaction of P- substituted diphenol to the corresponding quinone [51].
1.3.2.1 BIOCHEMICAL PROPERTIES
Catechol oxidase belongs to the same family as tyrosinase. The most characterized are plant origin with optima pH in the neutral basic range, which includes catechol from Ipomea batatas, Populus nigra and Lycopus europaeus. The distribution, physiological role and other related properties of catechol oxidase are summarized in the table above. (Table 1)
10 1.3.2.2 SUBSTRATE SPECIFICITY
The commonly used substrate for diphenolase activity are the L-DOPA and caffeic acid which in turn is used in analyzing the activity of catechol oxidases. Unlike tyrosinases, catechol oxidases can only catalyze the oxidation of o-diphenol to o-quinones. Spectroscopy study revealed the surrounding of the two copper ions at the active side are in the oxy, deoxy and met form. The specificity of catechol oxidase towards substrate was studied for the purpose of understanding the reaction of catechol oxidase and the ability to point out the differences to tyrosinase [52,53].
1.3.2.3 POTENTIAL APPLICATION
Catechol oxidase is used in food, non-food and also medical application. it is used for quantitative determination of L-DOPA, which is use for the treatment of Parkinson. Catechol oxidase plays a key role in enzymatic browning of fruits and vegetables.
1.4 AVAILABLE STRUCTURE OF CBC ENZYMES
The monomeric structure of T3 copper enzyme are typically formed by two or three domains with different folding motifs: An N-terminal domain, a central catalytic domain and a C- terminal domain. Tyrosinases from animal and plant have all the three domains whereas bacterial tyrosinases only have the central catalytic domain associated with an exogenous protein which act as the C-terminal like domain. The function of the C-terminal domain is to block the active site of the tyrosinase, rendering the enzyme inactive in the secretory pathway.
Cleavage of the peptides from the N and C terminal domains usually convert the tyrosinase to its active form, in case of bacterial tyrosinase the exogenous protein that acts like the C terminal is removed [43].
Establishment of the crystal structure of the CBC enzyme is important for understanding biological process at molecular level. Several crystal structures of tyrosinases has been established, from Streptomyces castaneoglobisporus, from Citrobacter freundii, from Bacillus megaterium, from Junglas regia, from Burkholderia thailandensis and from Agaricus bisporus. Crystal structure of plant catechol oxidase from Aspergillus oryzae, Ipomoea batatas,
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Vitis vinifera and from Coreopsis grandiflora has also been established. Structure of o-amino phenol oxidase is yet to be established.
Assessment of the three dimensional structure of tyrosinase and catechol oxidase shows that the central catalytic domain consist of α-helical structure, in which the bi-nuclear copper site is surrounded by bundle of four α-helices (α2, α3, α6, α7) which is conserved for the formation of the active site, the bi-nuclear copper site is lodged in the bottom of a large concative and serves as putative substrate binding center [22-23, 56].
Figure 6: (A) Active site of Bacillus megaterium and its blocker residue val218 in lime green.
(B) Active site of tyrosinase Streptomyces castaneoglobisporus and its blocker residue Gly204.
(C) Active site of tyrosinase Aspergillus oryzae and its blocker V359. Copper atom presented as brown sphere and the active site residues are presented as stick [57].
The first structure of Bacillus megaterium was solved in 2011 with resolution of 2.0 -23 Å, the enzyme appear as a dimer in its asymmetric unit and found to be active [58]. The structure of Streptomyces castaneoglobisporus was solved at high resolution as a complex with caddle protein which is made up of six-stranded β-sheet and a single α-helices and the structure suggested that the caddle protein covers the hydrophobic molecular surface of tyrosinase and interferes with the binding of substrate to the active site [23]. The overall monomeric structure of Bacillus megaterium is similar to the previously determined tyrosinase from Streptomyces castaneoglobisporus except for the absence of the caddle protein.
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A B
Figure 7: (A) over all dimer structure of tyrosinase Bacillus megaterium. (B) Over all structure of tyrosinase complex with ORF378, the tyrosinase is in blue and the caddle protein is red.
The first three dimensional structure established for catechol oxidase was from Ipomoea batatas with dimension 55 x 45 x 45 Å. the overall structure of the enzyme is similar to that of tyrosinase in Streptomyces castaneoglobisporus as it has mainly α-helices and short parallel sheet of beta strand also present in the N and C terminus of the central domain with an active site.
Figure 8: (A) Overall ellipsoidal shape of catechol oxidase from Vitis vinifera. (B) Three- dimensional structure of catechol oxidase from Ipomoea batatas.
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The difference in catalytic activity of catechol oxidase and tyrosinase is based on the observation of a wide vacant space present just above the active site of tyrosinase and one of the six His ligand for one of the copper ions is highly flexible. Hence the lack of catechol oxidase ability to hydroxylate monophenolase.
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2.0 Materials and method
2.1 MATERIAL
The Aminox gene was designed by a collaborator in Chinese academy of Agricultural sciences (CAAS) China. The gene encoding for aminox was inserted in pET30a vector digested with EcoRI and NoET generating vector pET30a (+)-GIF. During clonation a stop codon was inserted before the C–terminal Histag.
Aminox was produced at University of Eastern Finland (UEF) from the transformed cell provided by CAAS.
2.2 PROTEIN CONCENTRATION
The protein concentration was determined using the bio photometer Eppendorf with 8.5mm cuvette path length. The instrument is designed to give absorbance values at wavelength 260 nm, 280 nm, and 320 nm and the ratio of the values give clarity of the sample purity. The protein buffer solution was used as a blank before actual sample measurement, so to get the exact absorbance of the sample without the buffer interference. 100µl of the buffer solution was used to blank the instrument after which 100µl of the sample was scanned through the Eppendorf and absorbance value displayed at 280nm was recorded. This absorbance was recorded as the protein concentration., a pre-programmed method from the instrument.
2.3 BRADFORD PROTEIN ASSAY (BSA)
Bradford protein assay was used to measure the total protein concentration of aminox crude sample using Bouvine Serum Albumin (BSA) as the standard solution. This method is based on maximum absorbance shift of the dye, Coomassie Brilliant Blue G-250. The dye binds with the protein being assayed and the reaction depends on the amino acid content of the measured protein.
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The BSA standard dilution with the protein buffer solution was prepared in accordance with Bio-Rad method. 50µl of the standards and samples were pipetted into a disposable tube and the Bradford reagent were added alongside into the tube. After thorough mixing by vortex, the mixture changes to yellowish to blue color. When the dye finally binds to the protein, a change in absorption maxima of the dye from 465 nm to 595 nm occur. Experiment takes place at room temperature and the absorbance is read at 565nm using a spectrophotometer at 750 nm. The standard cure for BSA is presented in Appendix B.
2.4 ENZYME ACTIVITY
All spectrophotometry assays were carried out using UV-Vis 1600 model spectrophotometer and disposable cuvettes. Polyphenol oxidase activity were determined with 5mM of the sample as the enzyme and 20 mM 2-aminophenol as the substrate which were dissolved to final concentrations in 0.1 M Sodium phosphate pH 7.0 at room temperature. In the presence of oxygen, the enzyme catalyzes the substrate to the corresponding o-quinonimine which spontaneously dimerizes to phenoxazinone derivatives. The formation of phenoxazinone derivatives was measured by recording the increase in absorbance at 433 nm (detectable wavelength of the product) with extinction co-efficient of 9600 M-1cm-1.
The reference cuvette used as blank had 300µl of the substrate, other cuvette had the same composition except for the enzymatic samples added. 50µl enzymatic sample of various dilution factor (1:1, 1:2, 1:5,1:10) were added to the substrate and quickly mixed with a pipette.
The measurement started as fast as possible and were performed within 10s interval for 5minutes. The linear part of the absorbance curves was taken into consideration as the sample activity concentration was calculated using the equation below:
𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑛𝑘𝑎𝑡
𝑚𝑙 ) = 𝐴 . 𝑉𝑡𝑜𝑡𝑎𝑙(𝑚𝑙). 𝑘
(𝑀−1 . 𝑐𝑚−1) . 𝑡(𝑠) . 𝑉𝑛 (𝑚𝑙) . 𝑙(𝑐𝑚) . 10−3 ( 𝑙
𝑚𝑙) . 109 𝑛/𝑚𝑜𝑙 Where:
𝐴 difference in intensity of absorbance in the linear part of the curve molar extinction coefficient
𝑡 reaction time
16 𝑉𝑡𝑜𝑡𝑎𝑙 total volume of the reaction mixture 𝑉𝑛 volume of enzyme
𝑘 dilution factor 𝑙 light path of cuvette
2.5 COLORIMETRY ANALYSIS
o-aminophenol oxidase activity in the crude and purified samples was monitored quantitatively using 2-amino phenol as the substrate. 2µl of crude samples and purified fraction was pipetted into the colorimetry microplate, then 2µl of the substrate was added directly on top and mixed thoroughly. The substrate was also used as reference to monitor the colour difference. Change in colour in the protein assay indicate the presence of Aminox.
2.6 PURIFICATION PROCEDURE USING ÄKTA
The crude protein sample was stored in –200C, the protein aliquot was left to equilibrate at +40C and kept at room temperature before purification procedures. The column used depends on the choice of chromatography techniques employed. The chromatography techniques used for this project were Ion – exchange chromatography (IEX), Hydrophobic chromatography (HIC) and Affinity chromatography. All purification procedures were carried out in ÄKTA purifier.
The sample was buffer exchanged using PD10 miditrap G-25 column. The column was equilibrated three times with 5 ml of buffer binding buffer and binding buffer used depends on the specific binding buffer needed for each purification procedure. The buffer used in this project were sodium phosphate, HEPES, MES and Tris HCl. The supernatant protein concentration was 19 mg/ml which was suitable for PD-10 column with no need for dilution.
After equilibrating the column with the binding buffer, 2.5 ml of the crude sample was added to the pd-10 column and ejected
with 3.5ml of the appropriate buffer needed in the Äkta system. The eluted sample went through 0.45nm pore size membrane filter.
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2.6.1 AFFINITY CHROMATOGRAPHY
The column used in this application was HisTrap HP (5 ml column volume) precharged with 2.5 ml CuSO4 for the first trial and with NiSO4 for the second trial. The nickel and copper matrix serve as a ligand to bind with the Histag peptide present in the sample. It was gathered that Histrap purification is most efficient when preloaded with nickel, this was not considered first because the protein sample contains copper metal which could easily be replaced with nickel present in the column through ion exchange. The procedure used for both nickel and copper charged column remain the same.
1ml of the protein sample buffer exchanged into 20 mM sodium phosphate was injected into the pre-equilibrated Histrap column. The binding buffer comprises 20 mM sodium phosphate, 0.5 M NaCl and 20 mM imidazole pH 7.4. The His-tag protein was trapped in the column by forming complex with the metal ion present and the non - tagged host cell proteins were washed out with the binding buffer until the UV280 was near zero. The elution buffer made up of binding buffer with 200 mM imidazole concentration was used to extract the bound protein from the column and collected through the fractionator. The system identifies the fractionator column that contains the target protein (His-tag) and the eluted fractions were concentrated using centrifugal filter 5 KDa MWCO, rpm 5000 for 1hr at 5minutes interval.
Another purification run was carried out with His-trap HP (5 ml column volume). The binding buffer and elution buffer had the same composition with the previous run except for the addition of 8 M urea.
2.6.2 ION EXCHANGE CHROMATOGRAPHY
Even though the predicted pI for the target protein is 6.5, strong cation exchange chromatography (CIEX) of the protein sample was carried out at pH 6.0 and pH 9.0 for effective evaluation. The column used for this process is Resource S (6ml CV). I ml of protein sample in 50 mM MES was injected through sterile syringe into the pre-equilibrated IEC column. The elution gradient started with 0 – 50 % in CV, step gradient 100 % and 5 CV 100
% and bound proteins were collected with 1 M NaCl through the fraction collector. The
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fractions were collected into separate pools according to protein activity shown on the chromatogram.
Anion exchange chromatography was also performed on the protein sample at pH 6.5 and pH 9.0 using bis –Tris and tris HCl as their respective buffer. The procedure was same with CIEX and the elution gradient was also from NaCl
2.7 SDS PAGE
Sodium dodecyl sulfate – polyacrylamide (SDS – PAGE) is an electrophoresis method that allows protein separation in respect to their molecular masses upon the application of an electric field. The method was done to analyze the relative abundance of purified protein. The SDS –PAGE run was carried out alongside with the PhastGel TM gradient 8-25 and 4-10 % gradient gels, PhastGel TM SDS buffer strips and Low Molecular weight (LMW 14.4 - 97.0) marker. The Low Molecular Weight (LMW) marker was used as reference to determine the average molecular weight of the sample.
LMW marker and protein samples (crude, injected, unbound and purified) were diluted by volume ratio based on their initial concentration with the SDS buffer to give 0.5 mg/ml final concentration required for the run. 1 µl bromophenol was added to the samples and standard and kept boiling for 5 minutes at 100 0C to denature proteins (from tertiary structure to linear poly peptides). the sample and standard were immediately centrifuged after boiling. 2 µl of the LMW marker and sample-sample buffer mixture were carefully charged on the gel with a comb, making sure the comb was properly fit in a lower position. After the electrophoretic run, the gel was immediately place in the second chamber of the system for silver staining. The staining took about 40 minutes to completion. The image of the gel was taken, and the gel was stored in the laboratory SDS-PAGE record book.
The procedures for preparation of reagents used for this electrophoresis method are given are given in appendix C.
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2.8 CRYSTALLIZATION PROCESS
Crystallization is a process in which crystal are formed from a solution.[15] the solution which need to be brought into supersaturated state before crystals growth depends on many factors such as the precipitant concentration, type of salt present in the reservoir solution and precipitant pH. The crystallization technique used was hanging drop vapour diffusion method in libro – style 24 - well plate and 3D structure Eco screen (MD1-13-ECO, Molecular Dimensions) was used for the screening.
2.8.1 SAMPLE PREPARATION
Fraction 1 from the purification in Histrap column charged with NiSO4 from Expression 3 at 260C was tried for crystallization. The sample concentration was 2.7 mg/ml which was buffer exchanged using pd-10 from Sodium phosphate to HEPES buffer and concentrated using vivaspin (5 KDa MW cut off) to about 5 mg/ml (concentration required for crystallization is between 5 mg/ml - 25 mg/ml)
2.8.2 HANGING DROP DIFFUSION METHOD
The first 24 - well crystal growth plate was set up by adding 500 µl of reservoir solution (primary screening reagent in order 1-1, 1-2, 1-3...1-24), protein drops were made as hanging drop on cover slips by adding 2µl of the protein sample plus 2µl of reservoir solution under the laminar flow hood. The protein drops were incubated for 24 hours, afterwards the coverslips from the first plate were transferred to a second plate prepared with solution 2-1, 2-2....2-24.
The plate was daily examined for crystal growth under the microscope (Olympus SZX 12) and the image of the observed crystals were taken.
The reservoir solution used has different concentration of polymer, salt and buffer solution and the composition of reservoir solution in each well is reported in Appendix E.
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2.8.3 CRYO- SOLUTIONS AND DATA COLLECTION
Cryo-solutions were prepared using different concentration of glycerol and ethylene glycol as cryoprotective agents, this were antifreeze, used to protect the crystal from freezing (ice formation) and improve diffraction quality. The cryo solution dilution used were given below Cryo 1 – 0.2 M magnesium acetate 4H2O, 0.1 M MES pH 6.5, 21 % v/v mpD ((±)-2-Methyl- 2,4-pentanediol). and 20% glycerol
Cryo 2 – 0.2 M magnesium acetate 4H2O, 0.1 M MES pH 6.5, 21 % v/v mpD and 20%
polyethylene glycol
Cryo 3 – 0.2 M magnesium acetate 4H2O, 0.1 M MES pH 6.5, 21 % v/v mpD and 10% glycerol Cryo 4 – 0.2 M magnesium acetate 4H2O, 0.1 M MES pH 6.5, 21 % v/v mpD and 10%
polyethylene glycol
The in-house X-ray diffractometer (MAR345 MarResearch imaging plate detector on goniometer MARresearch Network beamline and a FR591 rotating anode generator) was used for diffraction pattern collection. Cryoprotective ability of the solutions prepared above was checked by soaking a clean loop into the solution for few seconds, the soaked loop was mounted on the goniometer and later exposed to x-ray irradiation. The mounted loop was observed through the diffractometer screen to ensure ice ring weren't formed around it and the cryo solution was pellucid.
The crystal from row b column 1 well with crystallization condition 0.2 M Magnesium acetate tetrahydrate and 0.1 M MES from the 3D Eco screen was used for the diffraction study. The cover slip used in growing the crystal was flipped and work was done under the microscope to monitor the process, 2 µl of the reservoir solution was pipetted on the crystal to prevent drying off, 0.5 µl of the suitable cryo solution was pipetted on one side of the same cover slip that housed the crystal to be mounted, 0.5 µl of the reservoir solution was also added to the cryo- solution and properly stirred with the loop. Crystal was picked and plunged into the Cryo- solution and waited for 5 secs for the crystal to be soaked in the solution, after which the loop was mounted on the goniometer under liquid nitrogen stream and temperature of 100 k. The X-ray data was collected at detector distance of 150 mm and exposure time of 10 minutes.
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2.9 MASS SPECTROMETRY
In a typical MS procedure for protein sample, the injected protein solution in the ionization chamber became ionized by electron bombardment which enabled sample splitting into charged fragments.[25] Mass spectrometry measurements were carried out on a 12T SolarixTM XR Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometer (Bruker Daltonics, Bremery Germany) with electrospray ionization (ESI) (Apollo-IITMion source) operated in positive – ion mode. In native condition, 0.5 ml purified sample from AIEX expression was buffer exchanged with pd -10 in aluminum acetate buffer while in denaturation the sample was mixed in water, acetonitrile and acetic acid as 50:50:1.
Prior to sample injection into the ion source, ammonium acetate was used to flush out the residual sample from previous experiments. Parameters were set to TOF of 1.4 ms, flow rate between 120 –200 µl/hr, 100 – 500 average scan, 0.1 accumulation time, 386 – 5000 m/z range and 500 – 1000 QI mass, then the sample solutions were infused into the ion – source through a capillary. Data was collected using the FTMS control 2.0 software.
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3.0 RESULT AND DISCUSSION
3.1 ACTIVITY MEASUREMENT OF AMINOX
Qualitative and quantitative activity measurement was considered in order to determine the presence of Aminox in the crude sample. Fig 9a shows the absorbance at which the activity was measured by scanning the mixture without enzyme (substrate) and sample mixture with enzyme over a time range of 0 - 20 minute. At wavelength 435 using the UV spectrophotometer, the peak converged and increases with respect to retention time of the sample until equilibrium. Figure 9b represent the linear product between absorbance and time at 435 nm, the absorbances were highlighted and used in calculating the protein activities.
Activity calculation 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑛𝑘𝑎𝑡
𝑚𝑙 ) = 𝐴 . 𝑉𝑡𝑜𝑡𝑎𝑙(𝑚𝑙). 𝑘
(𝑀−1 . 𝑐𝑚−1) . 𝑡(𝑠) . 𝑉𝑛 (𝑚𝑙) . 𝑙(𝑐𝑚) . 10−3 ( 𝑙
𝑚𝑙) . 109 𝑛/𝑚𝑜𝑙
𝐴 = 1.42 , = 9600 , 𝑉𝑡𝑜𝑡𝑎𝑙 = 0.45 , 𝑉𝑛 = 0.4 , 𝑘 = 1, 𝑡 = 300 , 𝑙 = 1 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 0.5546
Figure 9a: convergence of peaks at 435 nm, a plot of retention time against wavelength. Wavelength scan is from 200 to 900
-0.5 0 0.5 1 1.5 2 2.5 3 3.5 4
300 350 400 450 500
time
wavelenght
blank time 0 sample time 0 blank time 5 sample time 5 blank time 10
23
Figure 9b: linear graph of absorbance versus time at 435 nm
3.2 pH STABILITY
The optimum pH at which the protein is stabilized as evidence from figure 10 is pH 7, this was carried out by incubating the enzyme in several buffer of different pH. The incubated enzyme placed in UV spectrophotometer was monitored at various pH ranging from 3 to 10 and stability was recorded for I hr and 6 days of enzyme incubation. pH 3, 4 and 5 has a wave-like slop and pH 6, 7, 8 has a linear slope that gradually increases with time while pH 9 and 10 is also non- linear slope.
0 0.5 1 1.5 2 2.5 3 3.5
0 5 10 15 20 25
absorbance
time (min)
Abs 435 nm vs time
24
Figure 10a: Stability of Aminox incubated for 1 hr at different pH ranging from 3 to 10
Figure 10b: Stability of Aminox incubated for 6 hrs at different pH ranging from 3 to 10.
It can be seen from the figure below that the enzymes retained more than 50 % of its activity in the range of pH 5-8. The bell-shaped curve from Figure 11 has it highest point at pH 7 and Aminox activity found to be maxima at pH 7 which is close to neutrality.
25
Figure 11: plot of relative activity against pH
3.3 Purification of Aminox
Affinity chromatography
The first purification carried out on the crude sample from expression 1 was the affinity chromatography using histidine - tagged column with a gradient step protocol. From the chromatograms below, the gradient elution gives two peaks, the first peak signifies the unbound protein with little or no affinity to the column while the later peak corresponds to the his-tagged protein which could be the predicted o-amino phenol oxidase. In figure 11a, both the unbound (1st peak) and bound (2nd peak) portion were active when tested using colorimetric method. The fractions that falls within the second peak from pool A6 to b10 were collected. From the chromatogram (Fig 11a) the ratio of the unbound protein is higher than the purified protein, which opposed the expected result. It was assumed that some of the Histag protein was present in the unbound, so fig 11b is the chromatogram of the second His-tag purification with the addition of urea. The function of the urea is for denaturation of the protein leaving the Histag sequence readily available for bonding to the column. Result shows that none of the eluate was active. The protein sample was then subjected to other purification techniques because the concentration of the purified sample from affinity chromatography was not enough for crystallization process.
26 (A)
(B)
Figure 12: (A) Column: HisTrap, 5ml CV, Binding buffer: Sodium phosphate, 0.5M NaCl, 20mM Imidazole pH 7.4, Elution buffer: Binding buffer + 500mM Imidazole pH 7.4. (B) Column: HisTrap, 5ml CV, Binding buffer: Sodium phosphate, 0.5M NaCl, 20mM Imidazole, 8 M urea, pH 7.4, Elution buffer: Binding buffer + 200mM Imidazole pH 7.4. The blue curve from the chromatogram represent absorbance at A280 which monitors the purification process.
27
3.4 ION EXCHANGE CHROMATOGRAM
Figure 13a and 13b of the chromatogram below represent the anion and cation exchange respectively. Eluted fraction from the His-tag purification was subjected to IEX for efficient result. In figure 14, the elute bound protein peak is insignificant as the fractions corresponding to the peak were tested using colorimetric and no colour change, which implies that no optimal sample separation. Fig 14b has three peaks and the second peak was found to be active using colorimetric method. fraction C2 to C8 was collected.
-
Figure 13a: Column - Resource Q, 6ml CV Binding buffer: 50mM MES buffer pH 9.0 Elution buffer: 1 M NaCl in MES buffer pH 9.0
Figure 13b: Column - Resource S, 6ml CV, Binding buffer - 50mM Tris HCl pH 6.0 Elution buffer: Binding buffer + 1M NaCl pH 6.0
28
3.5 HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC)
The starting sample of the HIC was active fractions collected from CIEX. The chromatogram followed the same trend and no broad peak for the bound protein
Figure 14: Binding buffer - bis Tris and elution buffer – bis tris + 1.0 NaCl pH 6.5
3.6 ANALYSIS OF AMINOX IN SDS- PAGE
The analysis of the purified sample on SDS-page gel was barely impossible to interpret due to low concentration of purified sample. In figure 15, Lane 1 (LMW) is a standard of known molecular weight ranging from 97.0 to 14.4 and the purity of the sample can be check by its matching bands. Lane 2 (injected) and lane 3 (unbound) has a matching band with lane 1 at about 40 KDa which is the weight of Aminox. Lane 4 is the eluate from purification using Histag and considered indistinct because it has no visible band this may be because the purified sample was too diluted from Åkta. Lane 2, 3,4,7 activity was checked quantitively using colorimetric method were all found to be active while Lane 6 and 8.
29
Figure 15: SDS page of Aminox of two Histag purification expression. 1- LMW 1st purification 2-injected, 3-unbound, 4 – fraction 1,
2nd purification 5- injected, 6-unbound, 7- fraction 1, 8-fraction 2
3.7 MASS SPECTROMETRY
Figure 16 represent the denatured and native form of the Aminox. Spectroscopy analysis was done on the sample to confirm the presence of Aminox in the purified fraction. Poor sample quality led to high noise level background of the spectra. The sample quality is degraded due to the present of polymeric substances that are difficult to get rid and its high peaks eventually suppressed the protein in the spectra. Mass difference between major peaks are not typical for anything peptide and 4+ charge is not protein or peptide. so, the ionic distribution is because the sample is basic
30
Figure 16: Red represent Aminox in denaturation condition and orange is for Aminox in native condition
3.8 PROTEIN CRYSTALLIZATION
The crystal growth was monitored daily for two weeks and observable changes were recorded.
Fig 17 were images from 0.2 M MgCl.6H2O salt and 0.1 Tris buffer (plate C5), 0.2 M Mg (CH3COO)2.3H2O salt and 0.1 MES (plate B1) and 0.2 M ammonium sulphate (plate D2) respectively. Crystals gotten from these conditions doesn’t have a well-defined orientation and the last one looks like a sperolytes. Fig 18 is the picture gotten from 0.2 M zinc acetate dehydrate and 0.1 MES (plate B3) and 2.0 M Ammonium sulfate and 0.1 M sodium HEPES (plate C2), observable changes occur in those images from Fig 18 after about 3 months of monitoring the medium, crystals from figure 17 b was subjected to x-ray diffraction.
31
C5 B1 D2
Figure 17: C5, salt conc.- 0.2 M Mgcl.6H2O, buffer conc- 0.1 Tris, B1, salt Conc.- 0.2 M Mg (CH3COO)2.3H2O, buffer Conc. - 0.1 MES, D3, salt Conc.- 0.2 M ammonium sulphate
B3 C2
Figure 18: B3, salt conc. - 0.2 M zinc acetate dihydrate, Buffer conc. - 0.1 MES (plate b3), C2.
Salt conc.- 2.0 M Ammonium sulfate, Buffer conc. - 0.1 M sodium HEPES
3.9 X-RAY DIFFRACTION
Crystals used for x-ray diffraction were obtained from plate B1 and B4. cryo-loop of different cryoprotectant (glycerol and polyethylene glycol PEG) mounted on goniometer at temperature of 100 K gave diffraction images and 10% PEG dilution has the best image (clear with no diffraction) was used to soak the crystal to be examine for data collection. The mounted crystal was rotated at 90o for symmetrical reflection impact with 10 seconds exposure time. In Figure 20 diffraction pattern is not of a typical protein but more like a salt. B4 crystal was after tested
32
in izit dye and the crystal dissolved when the dye was added, B1 crystal could not be broken when tried with a needle. There diffraction image is not typical for protein and the crystal us suggested to be a salt crystal
Figure 19: Diffraction image collected from Aminox crystal with 1 oscillation and 10s exposure time using detector with wavelength 1.514 and average intensity of 37.1
33
4.0 CONCLUSION
The goal of this work is to successfully purify and crystallize o-aminophenol oxidase from the crude sample produced with Enchiridia coli cells transformed with a plasmid coding for the Aminox enzyme. The concentration and activity of the crude protein sample was measured to be 16.20 mg/ml and 0.71 respectively. The crude protein concentration was quite good to start it but comprises of different protein that made up the amount and Histag should provide an easy purification by direct binding to the Histidine present in Aminox
The first purification technique employed was Histag (Fig. 12a) and give a tiny peak which was found active qualitatively but the concentration value 0.158 gotten from bio photometer is very low and the unbound fraction was also active. It was assumed that the histidine is folded and not readily available to be trapped in the copper matrix column and there for eluted with the unbound. In Fig 12b 8M urea was added to the buffer with the aim to denature the protein and makes the histidine open but the chromatogram is similar to the one without urea and none of the fractions was active. Suggestion was made that the activity found in fig 12a may be due to imidazole plus copper reaction. Ion exchange and hydrophobic interaction chromatography was as well used for purification of the enzyme and their chromatogram fig 13 and 14 does not show the anticipated pattern. The purity check of the sample using the SDS - page was inconclusive as the Purified fraction was so faint to examine. The crystal growth form plate b1 with crystallization condition 0.2 M Magnesium Acetate tetrahydrate, 0.1 MES and b2 with condition 0.2 M sodium acetate trihydrate were salt crystals because it dissolved in izit dye and very hard to break. The mass spectrometer and x-ray diffraction result gave no significant information on the protein.
In general, conclusion can be made that Aminox present in the starting sample is very little as seen from the results and the concentration of the purified sample is insufficient for further studies.
34
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