The information derived from the analysis (reported in Table 7) suggests that only laccase dopa-melanin can be identified as eumelanin (g = 2.0036) with the presence of carbon centered radicals.

69

used to study the melanin pigments and investigate composition of Sc- Ms 1 bacterial pigment.

Sc-Ms 1 bacterial melanin, dopa-melanin produced by tyrosinase and laccase were analysed using S and X-band EPR (Figure 25).

Figure 25 S (3.9 GHz) and X-band (9.8 GHz) EPR spectra of the (a) Sc-Ms1 natural melanin, (b) Sc-Ms1 tyrosinase dopa- melanin and (c) Tv laccase dopa-melanin samples. Spectra were recorded with 1.90 mW microwave power at S-band and 1.46 mW at X-band. Spline functions were used for the baseline correction of the S-band spectra.

The information derived from the analysis (reported in Table 7) suggests that only laccase dopa-melanin can be identified as eumelanin (g = 2.0036) with the presence of carbon centered radicals.

Sc-Ms1 tyrosinase dopa-melanin and Sc-Ms1 bacterial melanin were characterised by the g value= 2.0038 and 2.0047 respectively (Table 7), those data suggest the presence of more than one radical species.

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70 Table 7 Magnetic parameters for the natural and enzymic synthetic melanin samples

Sample A^Niso A^Nz 2A^Nz giso * gz gx= gy ΔBpp§

(mT) Sc-Mc1

melanin 2,0047 0,7

Sc-Ms1 Tyr.

Dopa melanin 2,0038 0,6

Tv Lac. Dopa

melnain 2,0036 0,5

Tv Lac.

Cysteinyldopa melanin

0.7 1,6 3,2 2,0050 2,0028 2,0060 3,2

*determined from the S-band (3.8 GHz) EPR spectra; ^§determined from the X-band (9.8 GHz) EPR spectra. Errors were estimated to g values ± 0.0002 and hyperfine splittings ± 0.05 mT.

The X- and Q-band were used to analyze the cysteinyldopa sample, reported in Figure 26 paired with the simulated spectra (red lines).

Considering that cysteinyldopa polymerizes into various benzothiazine derivatives [181], the spectrum at X-band has a broad signal amplitude (3.2 mT) and a high g value (2.0050) and resembles the EPR spectrum of an immobilised nitroxide [167,182]. At this frequency, the EPR spectrum is dominated by the z-component of the

N hyperfine splitting (Figure 26). The nitrogen coupling constant A

(1.6 mT) has been estimated from the Q-band cysteinyldopa spectrum as it is highlighted in Figure 26. The A

has been calculated as for nitroxides assuming an axial symmetry with A

= A

= 0.2A

and g

=g

. The best fit, at both frequencies, has been obtained using the same set of magnetic parameters reported in Table 2 changing only the frequency. The partly nitrogen centered free radicals in cysteinyldopa melanin remark the presence of the semiquinonimine radicals as was previously reported [142].

The Q-band spectra of Sc-Ms1 melanin and Sc-Ms1 tyrosinase melanin

(Figure 27a and 27b) show a complete different and more complex

lineshape compared to the single featureless line obtained at X-band

(Figure 25a and 25b).

71 Figure 26 X- (9.9 GHz) and Q-band (33.7 GHz) spectra of the cysteinyldopa melanin paired with their simulated spectra (red line).

Figure 27 Q-band (33.9 GHz) EPR spectra of the (a) Sc-Ms1 natural melanin, (b) Sc-Ms1 tyrosinase dopa-melanin, (c) Tv laccase dopa-melanin, (d) Tv laccase cysteinyldopa melanin samples. Spectra were recorded with 0.06 mW microwave power.

72

The Sc-Ms1 natural melanin shows a complex pattern of the EPR signal clearly addressing the presence of more than one radical species one of which was identified as the pheomelanin contribution. Pheomelanins and eumelanins are commonly produced in humans while only few reports address the presence of pheomelanins in bacteria and fungi [174,175,184,185]. In this context the results obtained represent one of the first reports detecting pheomelanins in bacteria. The natural, certainly more complex melanin sample, has been described on the basis of the enzymic synthesized eumelanin and cysteinyldopa melanin with the Q-band spectral simulation (Figure 27). The best fit was obtained taking into account the presence of the two different species, simulating the spectra with the magnetic parameters reported in Table 2. A contribution of 20% eumelanin and 80% pheomelanin was derived from the simulation. Notably, this is only a gross estimation of the two pigments contribution on the basis of the ones synthesized by the use of Tv laccase. Furthermore, as discussed in the introduction, cysteinyldopa in the cells is synthesized through dopa cysteinylation directly or by the mediation of glutathione depending by the presence of cysteine in the bacterial culture [186]. Again the Sc-Ms1 tyrosinase is also inactivated by an amount of cysteine greater than 0.01 mM [168]. The use of a cysteine-rich culture medium for Sc-Ms1 growth might justify the production of pheomelanin. The presence of the two different pigments, with the pheomelanin prevalence, is also confirmed by the high g value (2.0047). In literature, a calibration curve reporting the dependence of the amount of pheomelanin to g

-factor in human red hairs was built [187]. A g value of 2.0046 was reported for a pheomelanin/eumelanin ratio equal to 59% while the g value (2.0038) reported for the Sc-Ms1 tyrosinase dopa-melanin (Figure 27b), on this basis, accounts for a pheo/eu melanin ratio equal to 18%. This can be ascribed to the nature of tyrosinase which was purified from the culture broth. On the contrary, the laccase derived sample (Figure 27c) did not show any composite signal at high frequency, endorsing the assumption that laccase derived melanin is constituted of purely heterogeneous eumelanin units.

Nevertheless, the broad signal (DB

1.05 mT), recorded at Q-band, can

account for the presence of more than one radical species. Performing

hydration-controlled X-band analysis, it was seen that the water content

73

and pH can have strong influence on the solid-state EPR signal [178]. A model was proposed in which two coexisting free radical species are present in an eumelanin sample, the carbon centered (g = 2.0032) and semiquinone free radicals (g = 2.0045) whose intensity increases as the pH is increased. In the case here reported, the eumelanin sample (Figure 27c) with a g = 2.0036 fully agrees with the g value reported in literature for a powder sample at neutral pH supporting the hypothesis of a carbon center free radicals with a semiquinone free radical contribution whose formation is due to the comproportionation reaction [178]. Regardless, as Q-band frequency is not enough to solve the anisotropies of these two species, higher frequencies might be desirable to determine the different contributions.

3.4 Multifrequency CW EPR power saturation and Q-band pulse EPR relaxation studies on melanin pigments

The X-band CW-EPR analysis is the commonly technique used for melanin characterisation, however, the results do not give enough information. In this context, the preliminary combined pulse and multifrequency EPR analysis can give an important contribution for a more detailed characterisation of these pigments.

In the area of relaxation time measurements, melanin pigments characterisation was carried out in the pioneering work by Sarna and Hyde on 1978, whereas pulse EPR has been long underexploited for melanin radical characterisation, with the exception of the landmark X- band pulse EPR paper of Okazaki et al. dating back to 1985 [188].

Dopa-melanin and cysteinyldopa melanin synthetized using Laccase were analyzed and compared at increasing microwave power (max power value, M

=144.5 mW).

The X-band power saturation curves of dopa melanin

(g

=2.0036±0.0002) and cysteinyldopa melanin (g

=2.0050±0.0002)

samples are reported in Figure 28a and 28b. The dopa melanin free

radical signal (peak to peak signal amplitude, ∆B

= 0.5±0.1 mT, at

microwave power M=1.46 mW) identified the presence of carbon

centered (g~2.0032) and semiquinone (g~2.0045) free radical species,

whose respective contributions to the EPR signals are function of the

77

recovery of the equilibrium magnetization level was reached after approximately 4.5⋅10

s in the case of dopa melanin.

Table 8 Longitudinal relaxation times^a.

Dopa melanin Cysteinyldopa melanin T(K) T1f (µs)^b) T1s (µs) ^b) T1f (µs) ^b) T1s (µs) ^b)

20 1.15E+04 6.79E+04 - -

30 1.06E+04 4.90E+04 - -

40 5.42E+03 2.60E+04 6.07E+03 2.09E+04 50 3.93E+03 1.69E+04 4.91E+03 1.69E+04 60 2.95E+03 1.12E+04 2.74E+03 8.31E+03 70 2.09E+03 6.73E+03 1.94E+03 5.68E+03 100 1.53E+03 4.79E+03 7.16E+02 1.90E+03 110 9.70E+02 2.66E+03 4.14E+02 1.09E+03

aThe columns report the T1f and T1s values evaluated for the dopa melanin and cysteinyldopa melanin samples. ^bThe error on the reported T1f and T1s values obtained with the biexponential decay model, was estimated to ±3 µs

The presence of cysteinyldopa melanin is commonly detected by higher values of the electronic g-factor and by the more complex lineshape resolved by CW EPR. Due to the relatively high difference in terms of spin-lattice relaxation times for the two compounds at higher temperature (approaching 60% at 100 K), Q-band PFSR measurements can be proposed as a complementary tool to classic multifrequency CW EPR for the assessment of the nature of new melanin pigments of unknown composition, and as an insightful instrument in melanin radical characterisation.

Room temperature PFSR experiments were also performed to assess

the measurements of relaxation times as discriminant feature under

common melanins functional conditions (T = 298K) (Figure 29).

78 Figure 30 Q-band room temperature (298 K) PFSR curves recorded for dopa melanin (black) and cysteinyldopa melanin (orange). Dopa melanin n=33.843 GHz; cysteinyldopa melanin n=33.733 GHz.

Figure 30 and Table 9 show that cysteinyldopa faster longitudinal relaxation dynamics point out the feasibility of running T

measurement as discriminant feature for melanin characterisation even when EPR room temperature experiments are considered.

Table 9 Room temperature T1f and T1s values for the dopa and cysteinyldopa melanins.

Sample T1f (µs)^a) T1s (µs)^a)

Dopa melanin 61 216

Cysteinyldopa melanin 23 58

a) The error on the reported T1f and T1s values obtained with the biexponential decay model was estimated to ±3µs

The T

values obtained for the dopa melanin produced by laccase could be compared with those obtained by Okazaki et all., where values of T

~4 ms were recorded for dopa melanins (77 K), indicating consistency in T

values for dopa melanin pigments of different origin [193].

The use for melanin identification and the characterisation of the

relaxation properties of these natural pigments would certainly serve to

investigate the nature of their free radical centers, whose structure are

still object of intensive research.

79

3.5 Homogentisic Acid and Gentisic Acid biosynthesized pyomelanin mimics

Melanins are characterised by special physic-chemical properties, such as broad band visible light absorption, persistent paramagnetism related to their free radical properties, antioxidant activity and interesting conduction properties [194]. In the last years melanins have been used for different biotechnological applications in optoelectronics, biomaterials functionalization and biomedical applications [195–198].

Melanin was also proposed for optoacoustic applications due to its strong absorbance that extends into near infrared (NIR) conferring high contrast in tissues. However its poor solubility hampers a homogeneous tissue distribution. In this context, the researcher of new soluble compounds with similar physic-chemical properties has become important [199,200]. The soluble pigments obtained by polymerization of homogentisic acid have been proposed for photoacoustic imaging of macrophages [200]. This pigment is involved in Alkaptonuria, a rare disease characterised by high level of homogentisic acid which, even if excreted with urine, can originate brown pigment deposits in the cartilage of patients. The structure of this pigment has not yet been clearly identified, even if the autooxidation of HGA to benzoquinone acetic acid and self-polymerization to pyomelanin is the most accredited mechanism [201]. Hydroquinones are very reactive molecule and their enzymatic oxidation prompts to the formation of the semiquinone radical, which represents the reactive species towards the formation of the fully oxidized quinone and reduced quinol species. The persistent paramagnetism of this structures enables the use of the EPR as the election technique for their characterisation [172].

Pyomelanin from homogentisic acid and gentisic acid were synthetized

using 0.1 mM of Laccase Tv (0.92 U mg

) and 100 mM of substrate

(molar ratio 1:1000 lac:acid) in phosphate buffer (pH 7,100 mM). The

reaction mixture was leave to react for 48 hours in a shaker (150 rpm) at

room temperature in presence of oxygen. A brownish solution was

obtained and extensively dried under a nitrogen flux.

80

In Figure 31, the UV-vis spectra of the two samples of HGAm (black line) and GAm (red line) are reported and compared with the spectra of the HGA and GA monomers. The absence of the absorption peaks of the monomers in the HGAm and GAm spectra highlights that the oxidative enzymatic reaction is completed with the polymer formation.

The UV-vis spectra of HGAm and GAm are similar showing a broadband absorption typical of melanin pigments [96]. The UV-vis analysis shows that the HGA and the most common GA pyomelanin mimics pigments have a spectrum profile with absorption in the Vis region and high intense absorption close to the NIR region. The band at 390 nm is indicative of the presence of a quinonoid structure [202].

Figure 31 UV-vis spectra of HGAm (black line) and GAm (red line) paired with the corresponding UV-vis spectra of the monomers. The measurements were carried out at room temperature.

In Figure 32 the IR spectra of the two pyomelanin mimics are compared

with the IR spectra of the monomers. At a first analysis the FT-IR

spectra of the polymers are less resolved showing that all the typical,

well-defined peaks associated with HGA and GA are not present. In the

case of the polymers the region 3500-3000 cm-1 corresponding to the -

OH stretching band is decreased and shifted to higher frequencies

[203]. The sharp peak at 3489 cm

in the spectrum of HGA monomer,

82

The Dynamic Light Scattering experiments were performed with a Zetasizer NanoZS90 instrument (Malvern, Worcestershine, U.K. and Malvern Instruments Ltd., U.K.). HGAm and GAm were solubilised in distilled water with a concentration of 0.5 mg/ml. Three independent measurements were carried out and used in the analysis of the data.

Values of 13.1 kDa for HGAm and 11.5 kDa for GAm were determined.

These gross values, are in fair agreement with the data reported in literature for pyomelanin (10-14 kDa) [201]. The zeta potential values of -27.4 mV for HGAm and -8.5 mV for GAm, obtained in water, are reported in Fig. 34.

Figure 33 DLS of HGAm (a) and GAm (b). The spectra have been recorded in water.

Figure 34 Zeta Potential of HGAm (a) and GAm (b) in water

1

H NMR spectra of HGAm and GAm confirmed the disappearance of the corresponding monomers (Figure 35) [208].

H NMR spectra were recorded after solubilization of the sample (11.0 mg) in D

O (1.0 mL) and the spectra were recorded on a Bruker 400 MHz spectrometer.

P

a b

85 Table 10.Functional group distribution (mmol/g) in HGAm and GAm polymers^a

Sample Carboxylic acid

Aliphatic- OH

Condensed Phenolic

units

p-hydroxyphenyl units

HGAm 4.10 3.98 1.6 1.9

GAm 0.52 31.47 5.64 13.64

aQuantitative ³¹P NMR spectra of HGAm and GAm. The samples were recorded after derivatization with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP) using n- hydroxy-5-norbornene-2 3-dicarboximide (NHND) as internal standard in DMF/Pyridine.

The HGAm and GAm powder samples were characterised by CW X- band EPR at room temperature.

In Figure 37 (HGAm) the EPR spectra recorded at increasing

microwave powers (from bottom to top) is reported. The signal recorded

at lower microwave power (0.21 mW) is narrow without resolved

hyperfine coupling, similar to that recorded for other melanin samples. It

is relatively easy to saturate even at low microwave power. The X-band

saturation measurements show that, increasing microwave power, a

progressive saturation of the main signal with broadening and reduced

intensity is evident. Furthermore, when the microwave power is

progressively increased, new features appear in the spectrum. The

appearance and increasing intensity of a shoulder (indicated with the

red arrow) paired with other evident signals at a microwave power of

105.70 mW, are indicative of the presence of at least two different

radical species characterised by different relaxation times. The g-values

matrix for the two species are shown in Figure 37. Considering the g

values components, the two different radicals could be attributed to the

presence of carbon-centred (g

= 2.0055, g

= 2.0040, g

= 2.0025,

determined from Q-band spectrum) and oxygen-centred radical species

(g

= 2.0096 , g

= 2.0063, g

= 1.997). The g matrix values for the

oxygen centred radical are better separated in the spectrum due to the

much larger g

and g

values observed for phenoxyl radical (blue

values) exhibiting large spin density on the oxygen, which has a large

spin-orbit coupling, compared with radicals with spin density on carbon.

88 Table 11 Longitudinal relaxation times obtained for HGAm, Gam, and Dopa melanin samples, recorded at room temperature. A biexponential decay was used to interpret the saturation recovery results:

! = !_!!"# −_!^!

!! + !_!!"# −_!^!

!! + !. An experimental error of ±5 µs can be considered in the data reported.

T1 (µs) T1f (µs) Af/As

HGAm 254 59 1.51

GAm 170 44 1.13

Dopa melanin 216 61 1.50

As the results of a biexponential decay model for data analysis describe, the T1 values obtained from the higher-field spectral region of the GAm and HGAm were coherent with the T1 values obtained for dopa-melanin sample previously studied using the same experimental contour.[211]

Figure 39 Q-band saturation curves, Θ/Θ0 (Θ0 =1), obtained from the PFSR experiment performed at room temperature (298 K) on the a) HGAm, b) GAm, and c) Dopa melanin radical samples, for the determination of T1 relaxation times.

Natural melanins and related synthetic polymers show high antioxidant

activity and a correlation between the paramagnetic properties and

antioxidant activity of poly-phenolic polymers has been demonstrated

[212,213]. The antioxidant activities is mainly due to the capacity of this

class of compounds to donate electrons or protons with the consequent

89

extensive electron delocalization with the formation of π-conjugation and supramolecular structures [212].

In Table 12 the EC

value has been estimated for both the samples and compared with the EC

value for the Gallic Acid polymer. The EC

has been determined using the DPPH assay using the EPR and the UV-vis techniques. The DPPH assay is commonly used to measure the efficiency of electron transfer process. For EPR analysis, a stock solution of DPPH 0.4mM in ethanol with a fix volume of 100ml for each sample was used. The antioxidant was dissolved in H

O with a concentration of 0.127mg/ml for gallic acid, 1.77mg/ml for pyomelanin from gentisic acid, 0.26mg/ml for homogentisic acid and 0.59mg/ml for the gallic acid polymer. For each of these substances an increasing volume of solution ranging from 5 ml to 100 ml was added to DPPH solution to reach a final volume of 200 ml. The DPPH radical signal was monitored in all samples and the radical in the absence of antioxidant is the reference signal for the scavenger radical percentage determination.

After the spectra acquisition the double integral of each spectrum has been calculated and the scavenger effect percentage was determined using the following formula:

Scavenger effect % =

!

* 100

where A

represents the double integral of the DPPH radical without the addition of the antioxidant, A

is the double integral of the DPPH radical after the addition of antioxidant.

The H-transfer reaction from antioxidant to DPPH was monitored also by UV-Vis Spectrophotometer. The samples were prepared at room temperature and the reaction time was fixed at 15 minutes. The antioxidant activity was evaluated through the reduction of DPPH radical peak at 520 nm in presence of different concentrations of antioxidants.

The samples were prepared with 200 µl of DPPH (0,2 mM – 79 µg/ml)

and 200 µl of antioxidant at variable concentration. The antioxidant was

dissolved in H

O with a concentration of 0.0125mg/ml for gallic acid,

0.16mg/ml for melanin from gentisic acid, 0.1mg/ml for melanin from

homogentisic acid and 0.16mg/ml for the gallic acid polymer. For each

90

of these antioxidants an increasing volume of solution ranging from 20µl to 200µl was added to DPPH solution to reach a final volume of 400µl.

Plotting the DPPH scavenger percentage in function of the log of antioxidant concentration expressed in mg/ml, the EC

value was automatically calculated using GraphPad Prism 5.01. Log (inhibitor) vs.

normalized response (variable slope) was the statistical model used for data elaboration of DPPH

assay [214]. Spectra acquisition was run at a fixed time of 15 minutes after the addition of the antioxidant solution to the DPPH. All measurements were repeated in triplicate and the standard deviation error for each sample was calculated.

HGAm sample shows the lower value of the EC

demonstrating the higher antioxidant activity compared to GAm and the Gallic Acid polymer used as reference. The high antioxidant activity for HGAm hampered to determine its value by UV-vis technique as the absorbance value was not possible to be determined with precision due to the overlapping of the absorption curves.

Table 12 Antioxidant activity

Table 1. EC50 (µg/ml) values from DPPH assay

EPR UV-Visible

HGAm 2.7± 0.7 n.d.

GAm 27.2 ± 5.4 25.7± 2.6

Gallic acid

polymer 14.4 ± 3.3 12.7± 4.7

In Figure 40 the SEM image of the HGAm powder is shown. The

aggregates show different overlapping layers pointing out to an

extended π-conjugation system.

91 Figure 40 SEM of HGAm obtained at pH = 7.1 and 1:1000 Lac: acid molar ratio

These results show that the HGAm and GAm pigments have a common

radical composition where a carbon-centred radical species is paired

with the presence of an oxygen based radical species more probably

due to a semiquinone radical species involved in a π-conjugate

supramolecular assembly where the electron transfer is not only along

the polymer chain but also interchains [215]. This analysis shows also

that CW Multifrequency EPR with pulse EPR relaxation times

measurements could be employed as investigation tool to expand the

current knowledge on radicals structural and dynamical properties in

melanin and melanin-like pigments, where the solely X-band EPR

methodology is commonly employed for their characterisation. Open

questions regarding the actual structure of the radical species found in

melanin and melanin-like pigments, and their mesoscale structural

organization can be solved by this combined approach contributing to

link the molecular-level structural framework of melanin pigments with

their macroscopic physical and chemical properties.

93

and activity of 75%. The activity was observed for 10 consecutive cycles of reaction with a slight decrease in activity in the first reaction cycles.

The toxicity of MNPs-1 before and after laccase immobilisation was tested. The lower toxicity of nanoparticles after the immobilisation was obtained. This represents an important advantage for their applications.

In this thesis was studied also the use of enzymes to produce biomaterials as melanin pigments that found applications in several biotechnology fields. Melanins are heterogeneous macromolecules with persistent free radical signals. The EPR is the elective technique for the characterisation of their paramagnetism. The insoluble dopa-melanin (eumelanin) and cysteinyldopa melanin (pheomelanin) pigments were synthesized using Tv laccase. These pigments were used to identify and characterise the melanin produced by the Streptomyces cyaneofuscatus (Sc-Ms1) isolate coming from Algerian desert soil. The information derived from the analysis suggest that only laccase dopa- melanin can be identified as eumelanin (g = 2.0036) with the presence of carbon centered radicals. Sc-Ms1 tyrosinase dopa-melanin and Sc- Ms1 bacterial melanin were characterised by the g value= 2.0038 and 2.0047 respectively, those data suggest the presence of more than one radical species. The X- and Q- band were used to analyze the cysteinyldopa, the results remark the presence of the semiquinonimine radical. The composition of Sc-Ms1 natural melanin was estimated with a contribution of 20% eumelanin and 80% pheomelanin. Pulse Q-band EPR was used for longitudinal relaxation time (T

) determination with the aim of investigating the possible use of relaxation time as a novel observable to differentiate melanin samples. Cysteinyldopa melanin was characterised by a faster longitudinal relaxation time compared to dopa- melanin.

Soluble pyomelanin pigments from Homogentisic and Gentisic Acid

were produced (HGAm and GAm respectively) using the oxidative

action of Tv laccase. The X-band analysis indicated the presence of at

least two different radical species. The two different radicals could be

attributed to the presence of carbon-centred and oxygen-centred radical

species. The HGAm and GAm longitudinal relaxion times were coherent

with the T

values obtained for dopa-melanin. Furthermore, HGAm

94

shows a higher antioxidant activity compared to GAm and the Gallic

Acid polymer used as reference.

96

(GlcNAc)

Di-acetyl-glucosamine

(GlcNAc)

N,N’,N’’-tri- acetyl-glucosamine Glu Gluteraldehyde

Gnp Genipin

h Planck’s constant

HGA Homogentisic acid

HGAm Homogentisic acid melanin

HPAEC-PAD High performance anion exchange chromatography- pulsed amperometric detection

L-DOPA L-3-4 dihydroxyphenylalanine

Lac Laccase

M

Maximum of the microwave power

MALDI-TOF Matrix assisted laser desorption ionization-time of flight

MNPs Magnetic nanoparticles

m

Projection of the electron spin

MW Microwave

paCOS partially acetylated chitooligosaccaharides PBS Phosphate buffer

PFSR Picket fence saturation recovery

Sc-Ms1 Streptomyces cyaneofuscatus Ms1 strain SEM Scanning electron microscopy

T

Longitudinal relaxation time T

Transverse relaxation time

TEM Transmission electron microscopy

T

phase memory time

TRP-1 Tyrosinase-related protein-1 TRP-2 Tyrosinase-related protein-2

Tv Trametes versicolor

TYR Tyrosinase

Ua Activity of enzyme added

Ui Activity of enzyme immobilised UV-Vis Ultraviolet visible spectroscopy Uw Activity of enzyme in washing buffer

VA Violuric acid

β Bohr magneton

97

ΔB

Central peak to peak distance

ΔE Energy differences

µ Magnetic moment

ν Frequency of the radiation

99

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