INOM
EXAMENSARBETE BIOTEKNIK, AVANCERAD NIVÅ, 30 HP
STOCKHOLM SVERIGE 2020,
Biosensor based on immobilized amine transaminase for detection of amphetamine
CLARA ÖH
KTH
SKOLAN FÖR KEMI, BIOTEKNOLOGI OCH HÄLSA
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Biosensor based on immobilized amine transaminase for detection of
amphetamine
Clara Öh
Degree project in biotechnology, 30 credits KTH Royal Institute of Technology
Stockholm, Sweden 2020
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Abstract
Amine transaminases (ATA) catalyse the transfer of an amino group from one molecule and replaces a ketone or aldehyde with the amino group, the amino group on the amino-donor is replaced with a ketone or aldehyde. This enzyme, ATA from Chromobacterium violaceum, has previously been used to catalyse the reaction involving amphetamine, therefore, it might be possible to use this enzyme to convert amphetamine and the product absorbs in the UV spectrum and can therefore be measured spectrophotometrically. The aim of the project was to explore the possibility of using ATA in a portable biosensor for the detection of amphetamine. A literature study of commercially available portable biosensors was performed, activity of the free enzyme was tested against two substrates, methylbenzylamine (MBA) and amphetamine. Research on immobilization techniques, materials, and surface functionalization was done to chose suitable methods for immobilizing ATA. Two immobilization methods were suggested and one of the methods, ionic immobilization through His- tag towards Ni2+ on the surface, was tested for enzyme activity toward MBA. The enzyme activity of the free enzyme in solution towards MBA was comparable to previously reported enzyme activity, however, no enzyme activity towards amphetamine was observed. No activity was observed for the immobilized enzyme, but it might be due to the experimental design, more experiments need to be performed to draw conclusions.
Keywords: amine transaminases (ATA), enzyme immobilization, biosensor, covalent immobilization, ionic immobilization, amphetamine, histidine-tagged enzyme, Poly(methyl methacrylate) (PMMA)
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Sammanfattning
Amintransaminaser (ATA) katalyserar överförandet av en amingrupp från en molekyl och ersätter en keton eller aldehyd med den amingruppen, amingruppen på amin-donatorn ersätts med en keton eller aldehyd. Det här enzymet, ATA från Chromobacterium violaceum (CvATA), har tidigare använts för att katalysera en reaktion som involverar amfetamin, därför skulle detta enzym kunna användas på amfetamin. Produkten av reaktionen absorberar i UV spektrumet och kan mätas med en spektrofotometer. Målet med projektet var att utforska möjligheten av att använda CvATA i en biosensor för att detektera amfetamin. En litteraturstudie på kommersiellt tillgängliga bärbara biosensorer genomfördes, aktiviteten av det fria enzymet testades mot två substrat, metylbenzylamin (MBA) och amfetamin. Information samlades om immobiliseringstekniker, material, och ytfunktionalisering gjordes för att välja ut lämpliga metoder för immobilisering av CvATA. Två immobiliseringsmetoder föreslogs och en av metoderna, immobilisering via enzymets His6-tagg och Ni2+ joner på ytan, testades för enzymaktivitet mot MBA. Enzymaktiviteten av det fria enzymet i lösning mot MBA var i samma storleksordning som tidigare rapporterad enzymaktivitet, men ingen enzymaktivitet mot amfetamin kunde observeras. Ingen aktivitet kunde observeras för det immobiliserade enzymet, men det kan vara på grund av designen på experimentet, fler experiment behöver göras för att kunna dra några fler slutsatser.
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Contents
Abstract ... i
Sammanfattning ... iii
1. Introduction ... 1
1.1 ATA enzyme ... 1
1.2 Immobilization ... 3
Background ... 4
Immobilization Methods ... 5
Support Materials ... 5
1.2 Immobilization of ATA ... 6
1.3 Aim of work ... 8
2. Materials and Methods ... 9
2.1. Enzyme Activity in Solution ... 9
Verification of activity using model substrate ... 9
Verification of activity using target substrate ... 10
2.2 Enzyme On a Surface ... 10
Enzyme Immobilisation ... 10
Enzyme Activity ... 11
3. Results and Discussion ... 11
3.1 Enzyme Activity In Solution ... 11
Verification of Activity Using Model Substrate ... 11
Verification of activity using target substrate ... 12
3.2 Enzyme on a Surface ... 13
Enzyme Immobilization ... 14
Enzyme Activity ... 15
4. Future Perspectives... 16
5. Acknowledgements ... 17
6. References ... 18
Appendix 1. Raw data ... 20
Appendix 2. Raw data ... 20
Appendix 3 ... 21
1
1. Introduction
A group at RISE is collaborating with the Swedish National Forensic Centre (NFC) to develop tools for analysis of blood aging and drug detection that can be used onsite at crime-scenes. Through the use of portable analytical tools, a pre-selection of relevant samples can be made so that fewer, and only samples of interest, need to be sent to lab for more thorough analysis. CvATA has been shown to catalyse the reaction with amphetamine in an enzymatic cascade in the production of the drug Vyvanse [1]. The working hypothesis is therefore to explore if CvATA could possibly be used in the chip under development at RISE for a portable tool that can be used onsite crime scenes for detection of amphetamine.
Devices like blood glucose test strips that use an enzyme, or other biological molecules like antibodies in pregnancy tests, to detect the presence of a compound by e.g. generating an electrical signal or a colour change are called biosensors. By immobilising the enzyme to a surface, the enzyme can be incorporated in a flow system biosensor of a similar concept as the blood glucose test strips [2].
Amine transaminases (ATA) are a subgroup of amino transferases with the ability to transfer an amino group from a molecule to a ketone or aldehyde with high regio- and stereoselectivity. What separates amine transaminases from other amino transferases is that the molecule “donating” an amino group (amino donor) does not need to have a carboxyl group [3], [4].
1.1 ATA enzyme
ATA from Chromobacterium violaceum (CvATA) is a homodimer where each monomer forms one active site towards the other monomer involving amino acid residues from both subunits. The homodimer has a molecular mass of approximately 100 kDa. [4] The enzyme is dependent on a co- factor called pyridoxal 5’-phosphate (PLP) for the transfer of an amino group, PLP binds covalently to a lysine in the active site of the enzyme. The mechanism of the transfer by ATA consists of two steps.
In the first step the amino group is transferred from the amino donor to PLP, through hydrolysis the amino donor is released from PMP (PLP with the amino group) as a ketone. In the second step, the amino acceptor binds to the PMP in the active site and the amino group is transferred to the carbonyl (see figure 1) [3], [5].
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Figure 1 Reaction catalysed by ATA. An illustration of the reaction ATA catalyses where it transfers an amino group to a ketone or aldehyde and the resulting products.
Figure 2 Structure of CvATA. Left: the structure of one monomer of CvATA. Right: the structure of the homodimer. Figure from
"Crystal structures of the Chromobacterium violaceum w-transaminase reveal major structural rearrangements upon binding of coenzyme PLP" by Berglund et al. [4]
ATA is of interest as a biocatalyst for its ability to use any primary amino group as the amino donor and transfer it to many different ketones, allowing for use of inexpensive amines as donors.
Additionally, its enantioselectivity makes it very useful in production of pharmaceuticals where enantiomeric purity is important, it is also important for intermediates in pharmaceutical, chemical and agrochemical industry [4].
ATA can act on amphetamine and use it as an amino donor, transferring the amino group to an amino acceptor, here sodium pyruvate (see figure 3). The product formed from amphetamine is phenylacetone, the formation of this can be measured over time by measuring the absorbance at a specific wavelength. Phenylacetone can be detected by spectrophotometry because it absorbs at around 255 nm and 283 nm but amphetamine also absorbs at 255 nm [6]. Before performing the tests with amphetamine, a model substrate methylbenzylamine (MBA) will be used, the product formed when ATA acts on this substrate will be acetophenone (see figure 3), and acetophenone absorbs at 245 nm [7].
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Figure 3 Reactions with model and target substrates. Top: illustration of the reaction with the model substrate MBA resulting in the product acetophenone. Bottom: illustration of the reaction with the target substrate amphetamine resulting in phenylacetone as the product.
There are different ways to determine enzyme activity, one method is to measure the formation of the product over time. The catalytic efficiency or the specific activity of the enzyme can be represented by moles of product formed per min per mass of enzyme. According to Lambert-Beer's law (equation 1) the concentration, and by extension, the amount of product formed can be followed over time by measuring how the absorbance changes over time.
By knowing the specific activity of the enzyme for a substrate-product the amount of enzyme in a reaction can be estimated from the measured total activity.
𝐴𝑏𝑠 = 𝑐 × 𝜀 × 𝑙 Equation 1 Lambert-Beer's Law 𝑐 =𝑛𝑣 Equation 2
𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = ∆𝑛
∆𝑡×𝑚𝑒𝑛𝑧𝑦𝑚𝑒 Equation 3
1.2 Immobilization
There are different strategies and support materials for immobilization of enzymes on a surface and no general methods working for all types of enzymes. Therefore the properties of the CvATA enzyme
4 and the intended use of the final product have to be considered and from them find a method that works for the enzyme and suites the requirements of the end product (or an intermediary of a possible end product).
Background
Today, there are a few products which use enzymes immobilized on a surface in a portable device to detect a certain target molecule that are commercially available: blood glucose test strips and pregnancy tests. Both are designed for use at home.
Glucose is transported from the intestine throughout the body by the bloodstream, insulin regulates uptake of glucose by cells. In diabetes the body produces too little insulin or none, or the cells are no longer responsive to insulin, which means it is important for diabetics to monitor their blood glucose level regularly to know when to administer insulin [8]. The purpose of the glucose sticks is to determine what concentration of glucose exists in the blood at a given time. Several different products from a few major pharmaceutical companies are available today for use. The most used enzymes on the test strips are glucose oxidase (GOx) and glucose dehydrogenase (GDH), which interact with the glucose molecules in the sample to generate a detectable signal. GOx is dependent on the co-factor FAD and GOx oxidises glucose in the presence of dioxygen (O2) or another mediator, yielding hydrogen peroxide (H2O2). The reaction can be divided into two steps where electrons are first transferred from glucose to GOx-FAD (the reduced state is GOx-FADH2), and GOx-FADH2 is then oxidized by dioxygen.
This can then be coupled to an electrode where hydrogen peroxide will be oxidized, and the electron flow will be proportional to the number of glucose molecules that have reacted [8].
Pregnancy tests measure the presence of the hormone human chorionic gonadotropin (hCG) which is present in the urine of pregnant women. The test is based on a method utilizing antibodies called enzyme-linked immunosorbent assay (ELISA). There are different versions of ELISA, the pregnancy tests use 3 antibodies and is a, so called, sandwich ELISA. One antibody that recognizes part of the hCG molecule, the antibody is therefore called “anti-hCG”, and this is loosely attached as a band at the bottom of the stick and the urine is wicked up to this band first. If there is hCG present in the urine it will be bound by the anti-hCG antibodies and travel up the test strip to where a secondary antibody is immobilized. This antibody recognizes hCG bound by anti-hCG and if there is no hCG present the lone anti-hCG antibodies will not bind to these secondary antibodies. Further up another secondary antibody is immobilized which recognizes the anti-hCG antibody that has not bound any hCG, this to confirm the test strip is viable and that the hCG has migrated up the test strip. Coupled to the secondary antibodies is a colour detection system which triggers when the secondary antibodies bind to their targets [9].
5 Immobilization Methods
The strategies for immobilisation can be divided into a few groups: adsorption, covalent bonding and cross-linking, and entrapment and encapsulation [2], [10], [11], []. Adsorption uses intermolecular forces between the enzyme and surface, for example, van der Waals forces, hydrophobic interaction, and hydrogen bonding []. Ionic interactions are also included in this group, though this interaction is stronger than the other interactions driving adsorption. Adsorption is the simplest of the immobilization methods, the enzyme 3D structure will not be affected by the interaction with the support material, and the enzyme can be released from the support by changing the conditions in the solution. A disadvantage with adsorption is that there is leaching of enzyme because of the weak interactions between the enzyme and the support [2], [10]. In covalent bonding of the enzyme to the support intramolecular bonds are formed, rather than intermolecular as in adsorption, between the enzyme and the surface and this interaction is much stronger than the forces driving absorption. The covalent bonds are formed with the side chains of amino acid residues at the surface of the enzyme, the most commonly used techniques form covalent bonds to lysine, cysteine, and glutamic and aspartic acid residues. The advantage of covalent bonding is that there is leaching of the enzyme because of the strong interaction, instead a disadvantage is that the bonds formed between enzyme and surface can change the 3D structure of the enzyme which may affect its catalytic activity [ ]. In entrapment, the enzyme is entrapped in a matrix which allows the passage of substrates and products but not the enzyme [] [10]. The enzyme is physically entrapped and not covalently bound to the matrix, this means that the structure of the enzyme is not affected as it may be in covalent bonding but instead there may be the problem of the enzyme leaching from the matrix.
Support Materials
There are many different materials that are used as supports for enzyme immobilization (for summary of materials see table 1), broadly they can be divided into organic materials and inorganic materials.
Important properties of the support are chemical inertness, to not disturb the enzyme-catalysed reaction, thermal, mechanical, and antimicrobial resistance, among others. Cost of the support material can also be a factor to consider, especially in the case of a disposable one-time-use product where there is no reuse of enzyme nor support.
Examples of inorganic support materials are glass, silica-based, alumina, and metal oxides, they generally have good microbial, chemical, and mechanical resistance, and low variance in pore size, however, some, e.g. metals, have low porosity and thus lower loading capacity [2].
6 Organic support materials can be further divided into natural and synthetic polymers. Natural polymers are polysaccharides like cellulose, collagen, chitin, chitosan, starch, agarose, etc. Properties making them suitable as supports for enzyme immobilization are that they are inexpensive, available in large quantities, they can form inert gels, and high thermal and mechanical resistance [2].
Table 1 Summary of different types of support materials [2], [10], [11].
Inorganic materials Zeolite, ceramics, celite, silica, glass, activated carbon, charcoal
Organic materials Natural polymers Alginate, Chitosan, chitin, collagen, carrageenan, gelatin, cellulose, starch, pectin, Sepharose
Synthetic polymers Polystyrene (PS), polyvinyl chloride (PVC), polyacrylate, polyamide, polypropylene, polymethacrylates (e.g. polymethyl methacrylate, PMMA), polydimethylsiloxane (PDMS), polyimide
1.2 Immobilization of ATA
From the literature, focus was given to two methods: covalent immobilisation by lysine residues on the surface of CvATA and coupled to an animated surface by a spacer molecule, this method has been used successfully previously by different groups [12] [13], and ionic immobilisation to nickel ions to the His6-tag on the enzyme used in the purification process.
Covalent immobilization of amine transaminases has been reported with an (S)-selective amine transaminase from Vibrio fluvalis on chitosan beads by Yi et al. [12], and two (R)-amine transaminases, from Gibberella zeae and Neosartorya fischeri, also onto a chitosan support by Mallin et al. [13]. Both groups first treated the chitosan support with glutaraldehyde (for chemical structure see figure 4) and then with the enzyme solution. This uses the amino group on the lysine residues as the reactive functionality which will bind to one of the aldehyde groups on glutaraldehyde. Glutaraldehyde is linked to the support in the same manner, chitosan has available amino groups which glutaraldehyde can react with. For illustration of linking of GA to amino groups on a surface and to ATA through lysine residues see figure 5.
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Figure 4 Chemical structure of glutaraldehyde.
Figure 5 The covalent linking chemistry through GA. [14]
Glutaraldehyde is reactive towards primary amine groups and can thus react with lysine residues on the surface of the enzyme, additionally, GA is bifunctional (see figure 4) because it has an aldehyde group on each end of its carbon chain and can thus react with two amino groups [15]. GA can then first be linked to amino groups on the support and then the enzyme can be linked to the other end of GA. This method with GA could then be applied to other support materials than chitosan for immobilization of the enzyme if the support has available amino groups, or can be treated to functionalize with amino groups. There are different ways of treating the different support materials to attain reactive amino groups depending on the support material.
Cerqueira et al. [16] describes a method for functionalization of a PMMA support (microchannels in a flow system) to yield amino groups and then linking to the enzyme with glutaraldehyde. The method they have used is inspired by Brown et al. [17], Brown et al. describes a quicker method to yield amino groups on PMMA than previously reported methods. With the use of ethylenediamine as NH2 source in dimethyl sulphoxide (DMSO) solvent they achieved a greater amount of amino groups on the surface by 2-fold when compared to other methods. This is of importance since more amino groups, or a higher density of amino groups, means more binding sites and thus the potential to immobilize more enzyme over the surface, i.e. increasing enzyme loading. Another group observed that polyethyleneimine (PEI) as source of NH2 resulted in the highest density of amino groups on the surface [18].
8 In the purification of CvATA the Bergland group uses IMAC, which separates the ATA enzyme by its His6-tag in the N-terminus from the rest of the molecules. The six histidine residues bind to metal ions, e.g. Ni2+, present in the column. [7]
This method was used to immobilise alanine racemase on silica with cobalt ions [19]. This concept can be applied to other support materials, for example PMMA. A chelator like AB-NTA can be coupled to glutaraldehyde and then nickel ions can be coupled to the chelator (see figure 6) [14]. Kulsharova et al. also describes a simplified method where the chelator is coupled directly to the PMMA support without the prior steps of amination and linking of glutaraldehyde, then the chelator, AB-NTA, is then coupled to methyl ester bonds that are available on the PMMA surface [14].
Figure 6 Linking chemistry for ionic immobilization. The first steps are the same as in the covalent linking chemistry, free amino groups on the surface that react with GA and then AB-NTA can bind to GA. Then nickel ions will be coordinated by AB- NTA and in turn immobilise the enzyme through the His-tag. [20]
1.3 Aim of work
Since the aim of the project was to explore the possibility of using the enzyme CvATA in a biosensor for detection of amphetamine, first the enzyme activity of free enzyme in solution was tested with two substrates, MBA and amphetamine. To determine possible ways of immobilizing the enzyme a literature survey was performed of immobilization techniques, support materials, functionalization of support materials, and cases where enzymes have been immobilized onto different surfaces using different methods. Both methods to immobilize ATA, Ni-His and covalently through GA, that has been discussed in the previous section seem promising with advantages and disadvantages. Bergland's group has used nickel coated plates with the enzyme before and therefore using these plates in a first test seemed like a good place to start when trying out the nickel-immobilization. Suitable support materials where chosen based on requirements for method of detection, simplicity of procedure for immobilization, and availability at RISE. The support material should be inexpensive, since the product
9 will be a disposable used only one time, it should also allow transmission of UV light to be able to measure the product spectrophotometrically at 245 nm. If the material is environmentally friendly is also a factor to be considered. PMMA is available at RISE, it is inexpensive, biocompatible, optical transparency at UV wavelengths, and there are many reported uses of PMMA for enzyme immobilization, some of which are microchannel flow systems [16], [14]. Lastly, a simplified version of one of two chosen immobilization methods was examined by use of ready-treated plates, amount of immobilized enzyme and enzyme activity with the immobilized enzyme was measured.
As a first test for proof-of-principle, when investigating the covalent immobilization method, would be to use microwell plates and treat the wells with poly-lysine, the treatment procedure only requires addition of poly-lysine to wells and incubation at room temperature for 1 hour. Coating microwell plates with poly-lysine would yield amino-groups from the lysine residues to use for covalent binding.
However, due to time constraints, only the Ni-His immobilization could be tested in this project.
2. Materials and Methods
The experiments performed can be divided into two parts: first verification of enzyme activity towards both model and target substrate with free enzyme, and second part performing the Ni-His immobilisation and measuring activity of the immobilised enzyme.
2.1. Enzyme Activity in Solution
To confirm the enzyme was active, and towards both substrates, the enzyme activity was measured for the free enzyme in solution. Further tests were performed with amphetamine as the substrate to investigate what wavelength phenylacetone had absorption maximum, and a test was done with both substrates present to investigate possible effect of amphetamine sulfate on the enzyme and the reaction.
Verification of activity using model substrate
Three reaction mixtures were prepared in UV cuvettes by addition of methylbenzylamine, MBA, sodium pyruvate, and PBS, the total volume after adding the enzyme was 700 μl. The reaction was started by adding the enzyme. Absorbance of acetophenone was then measured at 245 nm with a spectrophotometer, SpectraMax, at intervals of 30 s for 8 min from the start of the reaction. The reaction mixtures contained 5 mM MBA, 5 mM sodium pyruvate and 10 mg/ml ATA, the enzyme sample also contained PLP, in 50 mM PBS (pH 8.3, 50mM phosphate buffer, 13.5 mM KCl, 0.685 M NaCl). Reactants and enzyme were diluted with the PBS buffer. The reaction mixture before addition of enzyme was used as a reference.
10 Verification of Activity Using Target Substrate
The experiment to determine enzyme activity was carried out in the same way as above for MBA but using amphetamine instead.
To determine at what wavelength to measure absorbance of the product phenylacetone, at what wavelength there was the greatest difference in absorbance after the reaction compared to before the reaction, absorbance was measured over a broader wavelength spectrum. A reaction mixture was prepared in the same manner as above, the reaction was run and after about 10 min absorbance was measured over 230 nm – 400 nm. The reaction mixture before addition of enzyme was used as a reference.
To investigate whether the amphetamine sulfate was inhibiting or otherwise affecting the enzyme the reaction was run with both substrates present. The experiment was carried out the same way as previous experiments.
2.2 Enzyme on a Surface
First the enzyme was immobilized onto the nickel coated plates, then to estimate the amount of immobilized enzyme the enzyme solutions from the wells after immobilization was used to run reactions and activity determined. Then the reaction using MBA as substrate was run in the wells with immobilized enzyme and absorbance measured at three time points.
Enzyme Immobilisation
A dilution from the concentrated enzyme solution (37.9 mg/ml) to a concentration of 18 μg/ml ATA in 50 mM PBS buffer (0.05 M phosphate buffer, 0.135 M KCl, 0.685 M NaCl). Then 200 μl of this solution was added to each of 18 wells of the Pierce Nickel-coated 96-well plate (≈36 pmol enzyme per well).
After 1 hour, the wells were emptied and then rinsed two times with the 50 mM PBS buffer.
The enzyme solution from the wells were saved to measure enzyme activity for approximation of the amount of enzyme immobilized in wells.
To the 200 μl enzyme solution taken from a well, MBA, sodium pyruvate and buffer were added to a total volume of 700 μl and each with a final concentration of 5mM, performed the same way as previous measurements of enzyme activity.
11 Enzyme Activity
MBA, sodium pyruvate, and 50 mM PBS buffer were added to three of the enzyme-treated wells on the Ni-plate, a total volume of 200 μl per well. One well was emptied into a UV cuvette at 30 s from start of reaction and absorbance at 245 nm was measured. The next two wells were emptied, at intervals of 30 s, and absorbance was measured.
In one well the reaction mixture was left for 7-8 min and then absorbance measured and compared to the absorbance of the reaction mixture before it was added to the well.
3. Results and Discussion
The main purpose of this project was to explore the possibility of using the CvATA enzyme in a biosensor for the detection of amphetamine. During the project two promising methods were found and one of them was tested experimentally. From experimental testing of one of these methods, validation of the method failed the reason for this was likely due to the experimental design rather than failure of the actual method. The results do not prove that the method does not work, the conclusion from the results is that more experiments need to be performed.
3.1 Enzyme Activity in Solution
Specific activity for the CvATA free in solution towards MBA was found to be in the same range as reported by Bergland et al, however no activity could be observed towards amphetamine. Further experiments could not determine why no activity was observed for amphetamine.
Verification of Activity Using Model Substrate
The enzyme activity while free in solution was determined with MBA as the substrate by spectrophotometric measurements at 245 nm over time as the reaction progressed (the increase in concentration of acetophenone over time is illustrated in figure 6). From the absorbance values and time between measurements the rate of formation of the product acetophenone was calculated with Lambert-Beer's law (Equation 1) and Equation 2 (see table 2 for measured and calculated values). The measured values of the enzyme's specific activity, 3.8, 2.0, and 1.3 μmol min-1 mgCvATA-1, are comparable to the value reported by Bergland et al., which is 4.05 μmol min-1 mgCvATA-1 [21], and seemed reasonable.
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Figure 7 The change in concentration over time as calculated from the measured absorbances of the three reactions that were run.
Table 2 Calculated specific enzyme activities from measured absorbance. The values have been calculated from the measured absorbance over time with equation 1, 2, and 3 (from section 1.1).
ΔAbs Δn [µmol] Δt [min] mATA [µg] Specific activity [µmol min-1 mg-1]
0.24 13.4 0.5 7 µg 3.8
0.1 7 0.5 7 µg 2
0.09 4,67 0.5 7 µg 1.3
Verification of Activity Using Target Substrate
Since the structure of phenylacetone, the product when using amphetamine as substrate, differs slightly from acetophenone, product of MBA, the absorbance maximum can be slightly different from acetophenone. Therefore, absorbance of a reaction mixture 10 min after addition of ATA was measured over a spectrum of 230 nm to 400 nm. There was no difference in the absorbance over the spectrum for the reaction mixture before addition of ATA and after the reaction was run (see figure 8), implying there is no formation of the product phenylacetone.
When the reaction was run, no difference in absorbance over time was measured, at 255 nm or 245 nm. However, amphetamine also absorbs at 255 nm and since it is a 1:1 ratio of substrate and product this is probably why nothing is detected here. Phenylacetone also has absorption at 283 nm, which
-0,02 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14
0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480
Concentration [mM]
Time [s]
Formation of acetophenone over time
Run 1 Run 2 Run 3
13 would have been better to measure at then, unfortunately, this information was received after the experiments were performed. However, this still leaves for question why there was no difference in the absorbance spectrum before and after reaction, here it should have picked up a difference at 283 nm if there was any formation of phenylacetone. As we can see in the absorbance spectra (figure 8) there is no change at 283 nm either, there is little absorbance at all.
The first runs' failures were concluded to be because of loss of enzyme activity, after running reaction with MBA again and getting no change in absorbance over time, due to storage of enzyme over a longer period of time. However, when new enzyme was used, and tested with MBA to confirm activity, still no difference in absorbance was measured. The amphetamine used was in the form of a salt, amphetamine sulfate, C9H13N • ½ SO4. To test whether the sulfate is affecting the reaction, e.g.
through inhibition of the enzyme, the reaction was run with both reactants, MBA and amphetamine, in the mixture but the rection went as previously with MBA, ruling out the possibility of sulfate interfering with the enzyme. Running the reaction with double concentration of either amphetamine or the enzyme resulted in no change from the earlier experiments. However, this was measured at 255 nm and should have been measured at 283 nm to provide any information.
Figure 8 Absorbance spectra for the reaction run with amphetamine. The absorbance measured for the reaction mixtrure before and after running the reaction (by addition of ATA).
3.2 Enzyme on a Surface
The enzyme immobilisation in Ni-wells was performed, but no enzyme activity could be detected in the wells when they were tested with MBA. The result from the experiment to estimate the amount
0 0,5 1 1,5 2 2,5 3 3,5
230 240 250 260 270 280 300 320 340 360 400
Absorbance
Wavelength [nm]
Absorbance spectra
Absorbance before reaction
After reaction
14 of enzyme immobilised in the wells did not match either of the expected outcomes (positive or negative), the reason for which cannot be concluded from the experiments and currently available results.
Enzyme Immobilization
Wells on a nickel-coated plate were treated with enzyme solution to immobilize the enzyme on the surface of the wells. The enzyme solutions after immobilization were tested with the enzyme activity assay and compared to previous values of activity to estimate amount of enzyme left in the solution.
To the wells were added 3.6 μg CvATA (36 pmol), which is four times as much as the given capacity for one well, the capacity of the wells was given from the producer to be 9 pmol enzyme per well. When the enzyme solution from four wells were tested by using them in a reaction mixture and measuring the absorbance, two of the wells showed no difference in absorbance (fluctuations of ± 0.002) and two showed a slight but steady increase of only 0.01 points/min and over 10 min a total difference of 0.1 points (see figure 9). This suggested there might be some enzyme left in the solution, but the increase is so small that it could be random variation.
Calculations of the expected amount of enzyme left after immobilisation: The expected amount of enzyme left in the well if 9 pmol enzyme was immobilized of 36 pmol would be 27 pmol. 36 pmol and 27 pmol corresponds to 3.6 and 2.7 μg, respectively, of the ATA enzyme. 2.7 μg in 200 μl and then diluted 179 μl/700 μl (when added to reaction mixture) would mean 3.45 μg/ml, or 2.42 μg enzyme, expected in the measured reaction. If no enzyme immobilization occurred there would be 3.6 μg in 200 μl diluted 179 μl/700 μl, i.e. 4.60 μg/ml, or 3.22 μg enzyme, expected in the reaction.
Calculated amount of enzyme from the measured absorbance of the solution after immobilisation:
From the measured absorbance, the approximate amount of enzyme can be calculated from enzyme activity and the specific enzyme activity determined in previous experiments. The calculated amount of enzyme was 0.153-0.448 μg (see table 3), depending on which value of specific activity used. This is significantly lower than what was expected even with maximum enzyme immobilized in the wells.
However, it is not certain if this method of estimating the amount of immobilized enzyme would be accurate enough to discern differences of less than 1 μg enzyme in the reaction.
Table 3 The calculated amount of enzyme from the measured absorbance.
ΔAbs Δn [µmol]
Δt [min]
Activity [nmol min-1]
Specific activity (determined in 3.1.1)
mATA [µg]
(calculated)
mATA [µg]
left in well 0.01 0.833 1 0.583 3.8-1.3 μmol min-1 mg-1 0.153 – 0.448 -
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Figure 9 Absorbance over time measured using enzyme solution from wells. Absorbance of acetophenone (at 245 nm) over time when using enzyme solution from one well after immobilization to see how much enzyme is left in solution and how much have immobilized in well.
Enzyme Activity
The absorbance of three reaction mixtures from enzyme treated wells on the Ni-plate was measured at 245 nm at time points t1 = 30 s, t2 = 60 s, and t3 = 90 s (see figure 10). There was no difference in absorbance for the three reaction mixtures, nor for the reaction that was left for 7-8 min. The reaction volume was 200 μl for this experiment it would therefore be relevant to compare this to a reaction with free enzyme with a total reaction volume of 200 μl, if the reaction could be followed then.
Another possibility would be to pool three or more wells for each time point to achieve a greater volume to measure to compare to the previous experiment.
By immobilizing the enzyme through the His6-tag it is possible to somewhat control the orientation of the enzyme as it immobilizes to the support, which is not possible with covalent immobilization through lysine residues. The His6-tag is on the N-terminal of the chain, however, depending on the location of the N-terminal in the 3D structure of the enzyme, it might immobilize in an orientation that makes the active site unavailable or less available.
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Figure 10 Absorbance of acetophenone in reaction in well with immobilized enzyme.
4. Future Perspectives
During this project, the enzyme activity of CvATA towards the model substrate MBA was verified and the activity tested against the target substrate amphetamine. Two promising methods for immobilisation of CvATA were found and one of them was tested for activity of the immobilised enzyme. The immobilization methods described in this report should be performed, either on PMMA or first simplified methods for proof-of-concept. The enzyme activity for the different methods should be compared and compared to activity when free in solution. If there is great loss in activity when immobilized, mutant variants of the CvATA enzyme that have shown higher activity can be tested with the immobilization protocols. Storage stability, and under different conditions, should be examined since the final product might be stored in a car for longer periods, storage stability might also vary for different mutant variants of the enzyme and could possibly be examined if necessary. There was no activity of the enzyme towards amphetamine observed, and although further experiments as to why no activity could be detective the reason behind the lack of activity is still unclear. Since the reaction was successfully performed but in the opposite direction by Bergland et al. in the chemo-enzymatic cascade to produce the amphetamine-based drug Vyvanse, further experiments need to be performed with amphetamine as substrate to determine under what conditions this reaction will work.
Furthermore, if amphetamine should be measured by conversion to phenylacetone and spectrophotometrically or if further reaction steps to couple another detection method, e.g.
amperometric detection, should be considered. A biosensor based on CvATA for the detection of amphetamine is still promising but further experiments need to be performed to verify the theory that has been gathered and reported here.
0 0,05 0,1 0,15 0,2
30 60 90
Absorbance
17
5. Acknowledgements
First I would like to thank my supervisors Per Berglund at the Royal Institute of Technology (Sweden), main supervisor, and Ingemar Petermann at RISE, external supervisor, for making this project possible, for their support and encouragement. I would also like to thank Per Björk at RISE for help with theoretical and practical work. Thanks to Qin for a warm welcome, constant encouragement and for organizing fika meetings and opportunities for presenting project progress. Thanks to NFC and especially Simon Dunne at NFC for his participation in the project.
I would also like to thank RISE for the welcoming and supportive atmosphere.
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6. References
[1] L. Marx, "Chemo-enzymatic cascades for the synthesis of chiral high-value chemicals", KTH Royal Institute of Technology, 2019.
[2] V. L. Sirisha, A. Jain and A. Jain, "Enzyme Immobilization: An Overview on Methods, Support Material, and Applications of Immobilized Enzymes," Advances in food and nutrition research, vol. 79, pp. 179-211, 2016.
[3] F. Steffen-Munsberg, C. Vickers, H. Kohls, H. Land, H. Mallin, A. Nobili, L. Skalden, T. van den Bergh, H. J. Joosten, P. Berglund and e. al., "Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications," Biotechnol. Adv., vol. 33, no. 5, pp. 566-604, 2015.
[4] M. S. Humble, K. E. Cassimjee, M. Håkansson, Y. R. Kimbung, B. Walse, V. Abedi, H.-J. Federsel, P. Berglund* and D. T. Logan*, "Crystal structures of the Chromobacterium violaceum w- transaminase reveal major structural rearrangements upon binding of coenzyme PLP," pp. 779- 792, 2012.
[5] Y. C. Shin, H. Yun and H. H. Park, "Structural dynamics of the transaminase active site revealed by the crystal structure of a co-factor free omega-transaminase from Vibrio fluvialis JS17,"
Scientific reports, vol. 8, p. 11454, 2018.
[6] From correspondence with Simon Dunne (NFC), 2020.
[7] H. Land, F. Ruggieri, A. Szekrenyi, W. Fessner and P. Berglund, "Engineering the Active Site of an (S)-Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines," Adv.
Synth. Catal., vol. 362, no. 4, pp. 812-821, 2020.
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[11] S. Datta, L. Christena and Y. Rajaram, "Enzyme immobilization: an overview on techniques and support materials," 3 Biotech, vol. 3, no. 1, pp. 1-9, 2013.
[12] S. S. Yi, C. W. Lee, J. Kim, D. Kyung, B. G. Kim and Y. S. Lee, "Covalent immobilization of ω- transaminase from Vibrio fluvialis JS17 on chitosan beads," Proc. Biochem., vol. 42, no. 5, pp.
895-898, 2007.
[13] H. Mallin, U. Menyes, T. H. M. Vorhaben and U. T. Bornscheuer, "Immobilization of two (R)- Amine Transaminases on an Optimized Chitosan Support for the Enzymatic Synthesis of Optically Pure Amines," ChemCatChem, vol. 5, no. 2, pp. 588-593, 2013.
19 [14] G. Kulsharova, N. Dimov, M. P. C. Marques, N. Szita and F. Baganz, "Simplified immobilisation
method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices," New Biotechnol., vol. 24, pp. 31-38, 2018.
[15] F. López-Gallego, J. M. Guisán and L. Betancor, "Glutaraldehyde-Mediated Protein Immobilization," in Immobilization of Enzymes and Cells, Humana Press, 2013, pp. 33-41.
[16] M. R. F. Cerqueira, D. Grasseschi, R. C. Matos and L. Angnes, "A novel functionalisation process for glucose oxidase immobilisation in poly(methyl methacrylate) microchannels in a flow system for amperometric determinations," Talanta, vol. 126, pp. 20-26, 2014.
[17] L. Brown, T. Koerner, J. H. Horton and R. D. Oleschuk, "Fabrication and characterization of poly(methyl methacrylate) microfluidic devises bonded using surface modifications and solvents," Lab on a chip, vol. 6, no. 1, pp. 66-73, 2006.
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modification for enhancing antibody binding on polymer-based microfluidic device for enzyme- linked immunosorbent assay," Langmuir, vol. 22, no. 22, pp. 9458-9467, 2006.
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Alanine racemase in hydrophobic solvent," Biotechnol. Bioeng., vol. 99, no. 3, pp. 712-716, 2008.
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microquantification of L-phenylalanine," Anal. Biochem., vol. 359, no. 1, pp. 72-78, 2006.
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[ ] From correspondence with Simon Dunne, NFC, 2020.
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Appendix 1. Raw data - verification of activity towards model substrate
Table 4 Absorbances measured for the reaction with MBA with free enzyme in solution, the same reaction was run three times.
Time [s] Absorbance Run 1 Run 2 Run 3
0 0,06 -0,05 -0,08
30 0,3 0,05 0,01
60 0,53 0,17 0,09
90 0,73 0,27 0,17
120 0,9 0,37 0,25
150 1,06 0,46 0,31
180 1,17 0,55 0,39
210 1,26 0,63 0,46
240 1,32 0,7 0,51
270 1,37 0,78 0,6
300 1,4 0,84 0,67
330 1,44 0,9 0,71
360 1,45 0,96 0,79
390 1,47 1,01 0,84
420 1,48 1,06 0,89
450 1,49 1,1 0,95
480 1,49 1,14 0,99
Appendix 2. Raw data – absorbance spectra amphetamine reaction
Table 5 The measured absorbances over 230-400 nm of the reaction solution before and after the reaction was run (before/after addition of enzyme) transferred from the graph generated by the spectrophotometer.
Wavelength [nm] Before After
230 3 3,3
240 1,8 1,9
250 1,5 1,52
260 1 1,1
270 0,5 0,5
280 0,3 0,3
300 0,28 0,25
320 0,26 0,25
340 0,23 0,2
360 0,1 0,1
400 0,1 0,1
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Appendix 3
Table 6 Absorbance values measured from Ni-wells with reaction run with MBA.
Time [s] Absorbance
30 0,0083
60 0,0085
90 0,0083
