Enzyme and Microbial Technology02c76e4f-7cf2-445d-a060-d734d62… · novel orange-coloured biodye...

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Enzyme and Microbial Technology

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Novel textile dye obtained through transformation of 2-amino-3-methoxybenzoic acid by free and immobilised laccase from a Pleurotusostreatus strain

Kamila Wlizłoa, Jolanta Polaka,⁎, Anna Jarosz-Wilkołazkaa, Rebecca Pognib, Elena Petriccib

a Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-031 Lublin, PolandbDepartment of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, 53100 Siena, Italy

A R T I C L E I N F O

Keywords:LaccaseDyesLaccase immobilisationBiotransformationFibre dyeing

A B S T R A C T

Transformation of 2-amino-3-methoxybenzoic acid into novel and eco-friendly orange dye (N15) was performedusing native and immobilised laccase (LAC) from Pleurotus ostreatus strain. A several parameters affecting lac-case-mediated transformation efficiency included the selection of type and pH value of buffer, reaction tem-perature, substrate and laccase concentration as well as the type of carrier and LAC storage conditions wereevaluated. The optimal conditions for N15 dye synthesis were 40mM sodium-tartrate buffer pH 5.5 containing3mM of the substrate, efficiently transformed by 2 U of free laccase per 1mmol of the substrate. Laccase wasimmobilised on porous Purolite® carriers, which had never been tested as a support for oxidoreductases.Immobilised laccase, characterised by a high immobilisation yield, was obtained by adsorption of laccase on aporous acrylic carrier with octadecyl groups (C18) incubated in optimum conditions of 40mM phosphate bufferpH 7.0 containing 1mg of laccase per 1 g of the carrier (wet mass). The immobilised LAC showed the higheststorage stability for 21 days and higher thermostability at 40℃ and 60℃ in comparison to its native form. TheN15 dye showed good dyeing properties towards natural fibres, and the dyed fibre demonstrated resistance todifferent physicochemical factors during use, which was confirmed by commercial quality tests. The N15 dye is aphenazine, i.e. a heterogenic compound containing amino-, methoxy-, and three carboxyl functional groups withthe molecular weight of approximately 449.37 U.

1. Introduction

Environmental pollution caused by harmful synthetic chemicals is awell-known worldwide problem, mainly due to the overproduction,excessive use, and low biodegradability thereof [1,2]. In the context ofsustainable development, the priority should be focused on the evolu-tion of a chemical synthesis method based on the white biotechnology,in particular on the oxidative enzymes used as catalysts. One of them islaccase, very useful enzyme with a broad range of potential applica-tions, mainly due to the low substrate specificity. Laccase was describedas an efficient biocatalyst in both the synthesis and degradation ofchemicals, e.g. the synthesis of a novel colourant as well as efficientdecolourisation of existing synthetic dyes [3–6].

An alternative to reduction of environmental pollution by colour-ants is the laccase-mediated synthesis of new, non-toxic, biodegradabledyes with a low environmental impact exerted by the enzymatic bio-transformation technology and the biodegradability of obtained

products. Due to its substrate promiscuity, the laccase is able to oxidisesimple phenolic and non-phenolic substrates, forming reactive radicals,which may undergo coupling reactions resulting in formation of col-ourful dimers, oligomers, or polymers, which are potentially useful astextile dyes [7]. Laccase was found to be able to transform benzene andnaphthalene derivatives to non-toxic orange-red dyes [8]. Some pro-ducts showed good dyeing properties and colour fastness combinedwith low ecotoxicity and cytotoxicity [9]. Laccase was also an efficientcatalyst in the synthesis of phenazine and phenoxazine compounds aswell as indigo dyes through heteromolecular oxidative cross-coupling ofaromatic amines and phenols [10]. Laccase-mediated transformation isan easy-to-handle and eco-friendly process occurring in mild conditionsof pH, temperature, and pressure. Unfortunately, from a commercialpoint of view, many limitations are observed in addition to the manyadvantages of the biotransformation process. One of them is the highcosts of the extracellular laccase production as well as the low stabilityof the enzyme, which makes it difficult to conduct the

https://doi.org/10.1016/j.enzmictec.2019.109398Received 5 July 2019; Accepted 8 August 2019

⁎ Corresponding author.E-mail address: jpolak@poczta.umcs.lublin.pl (J. Polak).

Enzyme and Microbial Technology 132 (2020) 109398

Available online 17 August 20190141-0229/ © 2019 Elsevier Inc. All rights reserved.

T

biotransformation process on an industrial scale. A possible way toovercome this problem is immobilisation of laccase on supports such asglass beads or polymers. Immobilisation of enzymes increases theirstability in adverse reaction conditions such as extreme pH, tempera-ture, and presence of organic solvents [11]. Moreover, immobilisedenzymes show higher stability during long-term storage and may bereused in several transformation processes. Improvement of the prop-erties and reusability of the biocatalyst reduce the costs of the bio-transformation and thus make it more accessible for industrial purposes[12]. Furthermore, given the different immobilisation techniques andthe different types and properties of carriers used, it is possible to takefull advantage of the catalytic properties of enzymes on an industrialscale [11,13].

The aim of the present work was to optimise the synthesis of thenovel orange-coloured biodye using two laccase-based reaction sys-tems: native and immobilised catalysts from a Pleurotus ostreatus strain.The product obtained was analysed in terms of dyeing properties,colour fastness, and structure. The analysis has shown that it can beconsidered as an interesting compound for textile industry.

2. Materials and methods

2.1. Microorganism and culture conditions

Extracellular laccase (LAC) from Pleurotus ostreatus (PO13) (FungalCollection of the Department of Biochemistry, Maria Curie-SkłodowskaUniversity in Lublin) was obtained after 18 days of cultivation onmodified Lindeberg-Holm medium [14]. Extracellular LAC was inducedby the presence of 250 μM of CuSO4 added on the 4th day of cultiva-tion. Culture liquid was decanted and concentrated by ultrafiltrationusing a Millipore polyethersulphate membrane (10 kDa) and purified byion exchange chromatography (HQ-Sepharose) using 1M (NH4)SO4 inTRIS-HCl (5 mM) with a linear grade from 0 to 50%. Purified laccasewas desalted, concentrated, and stored frozen at −18 °C until used forpreparing the working LAC with precise activity.

2.2. Enzyme assay

The activity of native or immobilised LAC was determined followingthe oxidation of 2.5mM 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sul-phonic acid) (ABTS) in 100mM Na-tartrate buffer at pH 3. The oxida-tion of ABTS was monitored spectrophotometrically at 414 nm(λ414= 34 450 M−1 cm−1). Laccase activity was expressed in U perlitre (UL−1). One unit of LAC (U) oxidised 1 μmol of ABTS per 1min at25 °C. In the case of immobilised LAC, samples of 5–10mg of LAC im-mobilised on the carrier were placed into a stirred vessel containing anappropriate amount of buffer and ABTS; the reaction was monitoredusing a Varian Carry 50 Bio spectrophotometer with a Peltier stirringmodule. The activity of immobilised LAC was expressed in Units (U) pergram of the carrier.

2.3. Chemicals, reagents, and carriers

2-amino-3-methoxybenzoic acid (N15 substrate), 2,2- azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), tris(hydroxymethyl)aminomethane (TRIS), glutaraldehyde, (3-aminopropyl)triethox-ysilane, and tartaric acid were purchased from MERCK-Sigma-AldrichCompany, whereas glucose, sodium hydroxide, and amonium sulphatewere purchased from Avantor Performance Materilas (Poland). Porouscarriers used for laccase immobilisation were purchased from Purolite®.

2.4. Optimisation of transformation of N15 substrate by native laccase

Optimisation of N15 substrate transformation by native LAC wascarried out in 3mL final volume under orbital shaking (140 rpm). In thefirst step of the study, the type of buffer (Na-tartaric, acetic, or McIlvain

buffer) and the value of buffer pH (3–8) for 1mM N15 transformationby LAC (with the final activity of 1 U per 1mmol of N15) were selected.Afterwards, the influence of LAC activity (0.5–5 U per 1mmol of N15)and the substrate N15 concentration (1–10mM) were tested. For theoptimal 3mM N15 concentration transformed by 2 U/mmol optimalLAC activity, the optimal sodium-tartrate buffer concentration was es-tablished (1–100mM). The last stage of the optimisation was the eva-luation of the optimal temperature for the N15 transformation, con-ducted in two variants differing in the final temperature (A and B). Invariant A, the N15 substrate transformation lasted 72 h at three dif-ferent temperatures: 23 °C, 28 °C, or 40 °C. In variant B, after 6 h ofincubation of the N15 substrate with LAC, only half of the transfor-mation mixture was incubated at 40 °C up to 72 h. Additionally, in thecase of both variants A and B, two different variants were performeddiffering in the presence of LAC. From half of the N15 transformationmixture incubated in each temperature after 6 h of the reaction, LACwas removed by ultrafiltration to assess the influence of the non-en-zymatic process on the formation of the major final product.

2.5. Optimisation of LAC immobilisation procedure and storage conditions

Purified LAC from Pleurotus ostreatus was immobilised covalentlyand through adsorption using the method developed by Bryjak [15] andRekuć [16], respectively. For covalent immobilisation, an acrylic sup-port with amino groups on the short spacer (Support A) was used,whereas two different supports were applied in the case of the ad-sorption, i.e. macroporous styrene (Support B) and acrylic beads withoctadecyl groups (Support C) (Table 1).

The concentration of LAC protein bound on each support (Pb) wascalculated based on the difference between the loaded concentration ofLAC protein on the support (Pl) and the concentration of protein presentin eluates obtained after washing out of the unbound laccase from thesupport (Pub), according to formula 1. The amount of protein bound tothe support (Pb) was expressed in mg of protein per g of wet mass of thesupport as well as in percent (P), based on formula 2.

⎜ ⎟⎛⎝

⎞⎠

= −Pbmg

gPl Pub

(1)

= ×P PbPl

(%) 100% (2)

The activity of immobilised LAC was measured in sodium-tartratebuffer 0.1 M pH 3, 25℃, using ABTS as a substrate. The activity wascalculated based on formula 3 and expressed in U per gram of wet massof the support (U/g), which is the amount of enzyme able to oxidize1 μmol of ABTS. In the formula, Amin is the absorbance per minute, Vt isthe total volume of sample (1mL), ε414 is the ABTS molar absorptioncoefficient, t is the time of reaction (1min), and m is the mass of thesupport used in the reaction (g).

Table 1Characteristics of supports used for immobilisation of LAC from Pleurotus os-treatus.

Parameter Support A Support B Support C

method ofimmobilisation

covalent adsorption adsorption

size of particle (μm) 150–300 300–700 150–300specific surface (m2/g) > 40 >750 nd*

pore diameter (Å) 450–850 900–1100 350–450pore volume (mL/g) 0.92 nd* 0.14functional groups amine, on the short

spacer (C2)none octadecyl (C18)

type of activator glutaraldehyde none none

* nd – no data.

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=Am A Vtε t m

** *

min

414 (3)

Selection of an optimal conditions for LAC immobilisation includedscreening the type of support and the immobilisation technique withthe use of the phosphate buffer concentration pH 7 (10–100mM) andthe LAC concentration (1–4mg of protein per 1 g of wet mass of thesupport). The main criteria for the selection was the highest im-mobilisation yield (Y) of LAC calculated based on formula 4, where Amwas the measured LAC activity on the support using ABTS as a substrate(U/g) and Al was the LAC activity loaded on the support in phosphatebuffer pH 7 (U/g).

= ×Y AmAl

100% (4)

The influence of the conditions of immobilised LAC storage wasassessed to find the optimal type of buffer (Na-tartaric, McIlvain,acetate), pH value (4.5–6.0), and concentration of Na-tartaric buffer(1–100mM) during 21-day storage at 4 °C. The activity of both im-mobilised and free LAC was measured every 7 day and expressed as apercentage of the initial LAC activity (time 0).

2.6. Operational stability and activity of immobilised LAC

The immobilised LAC was used for N15 substrate transformation.The reaction was conducted in 5mL of a transformation mixture inconditions analogous to those optimised for native LAC, i.e. 100mMNa-tartaric buffer, pH 5.5, and 3mM N15 concentration. The reactionwas catalysed by 25mg, 50mg, and 100mg of support with im-mobilised LAC, which was equal to 0.084 U, 0.167 U, and 0.334 U ofLAC activity, respectively. After calculation per 1mmol of the N15substrate, the activity of LAC amounted to 5.6 U, 11.1 U, and 22.3 Urespectively. The transformation of the N15 substrate by immobilisedLAC lasted 48 h (at 28 °C and 140 rpm); afterwards, the coloured re-action mixture containing the synthesised product was separated fromthe catalyst by decantation and the reaction was continued at a tem-perature of 40 °C for the next 72 h. The concentration of the substrateand the amount of the product obtained were monitored using capillaryelectrophoresis. The immobilised LAC activity was determined after thetransformation using ABTS as a substrate and compared with the ac-tivity of control immobilised LAC incubated for 48 h in reaction bufferwithout the addition of the N15 substrate.

2.7. Analytical assays

2.7.1. Micellar electrokinetic chromatography (MECK)Analysis of products and precursors was performed in a capillary

filled with borate-SDS buffer (both in 100mM concentration) using7100 Capillary Electrophoresis System (Agilent Technologies). The ca-pillary length was 50 cm and the diameter was 50 μm. The appliedvoltage was 24 KV (positive polarity) and the temperature was 20 °C.The analysis was started with preconditioning the capillary with 30-second flushing under pressure of 50mbar. The same value of pressurewas used for injection of the sample for 5 s. During sample separation,signals at a wavelength of 210 nm and 440 nm were recorded.

2.7.2. Dyeing of natural fibersFive natural fibers of animal and plant origin such as wool, cotton,

silk, flax, and viscose were dyed using N15 lyophilised product powder.The N15 dye solution at a concentration of 0.2% and 0.5% was adjustedto pH 4 using 10% acetic acid and incubated with 0.3 g of apriopriatefibre for 30min at 100 °C. Afterwards, unbound dye was washed outwith distilled water and the fibres were dried at room temperature. Theintensity of dyeing was evalauted visually and by absorbance mesaur-ments, which compared the absorbance of the dye solution before andafter the fibre dyeing process.

2.7.3. Colour fastnessWool fibres dyed with the N15 dye at a concentration of 0.5%, 1%,

and 3% were analysed for their colour fastness by TKANLAB Laboratoryin Łódź, according to ISO standards. The tested parameters includedartificial light, distilled water, washing at 40 °C, alkaline and acidicsweat, and dry and wet rubbing. The results of colour fastness in arti-ficial light were presented according to an 8-point index blue scale,where “1” means great change of colour and “8” means no change ofcolour. The other factors were expressed in a 5-point index grey scale,where “1” means great change of colour and “5” means no change ofcolour.

2.8. Molecular characterisation of products

Mass spectroscopy analysis was recorded using an LC/MSD chro-matography system (1100 Agilent) connected to a UV detector. Theanalysis was performed in a mixture of MeOH/H2O 95:5 at 0.4mLmin−1 by direct injection in Electrospray Ionization (N2 = flow 9 Lmin−1, T =350 °C, solubilisation P=40 PSI, potential difference =70 eV).

3. Results and discussion

3.1. Laccase-mediated transformation of 2-amino-3-methoxybenzoic acidinto orange dye

Synthetic dye production is one of the largest and most profitablebranches of industry. About 0.7 million tons of dyes are consumedannually by textile industry, and 10%–40% of them are introduced tothe environment, exerting a highly toxic effect on many living organ-isms [17,18]. Moreover, the reaction conditions themselves can nega-tively affect the natural environment, e.g. harmful and toxic couplers,extreme temperatures, and concentrated acids are used in the synthesisof azo dyes [19].

To solve the environmental issue caused by the classical chemicalsynthesis of dyes, their eco-friendly alternative processes can be con-ducted, e.g. laccase-based enzymatic catalysis. The application of bio-catalysts gives a possibility to obtain non-toxic biodyes without usingharsh reaction conditions. Laccase is a natural biocatalyst requiringonly the presence of oxygen during substrate oxidation and mild con-ditions of the catalysed reaction. Laccase-mediated dye synthesis hasbeen studied intensively and successfully. This made it possible to ob-tain dyes with diverse chemical structures and colours, classified asphenolic, phenoxazine, or indigo dyes [7,20]. Among coloured productsobtained using laccase, special attention was focused on phenazine andphenoxazine compounds with mainly yellow-orange colouration ob-tained from action of fungal LAC on different aromatic hydroxyaminessuch as 3-amino-4-hydroxybenzenosulfonic acid [8,21] or the differentstructures of phenazines and phenoxazines obtained via the action ofbacterial CotA laccase on aromatic diamines and hydroxyamines[20,22]. Some of the products are characterised by low ecotoxicity andlow cytotoxicity with simultaneously good dyeing properties [9]. De-spite the high application potential, implementation of enzymaticsynthesis on an industrial scale is limited due to the high cost of laccaseproduction, enzyme stability, and process efficiency. Therefore, alongwith optimisation of process conditions, studies should be concentratedon better utilisation of laccase through immobilisation thereof and,consequently, its possible reusability.

In our paper, the LAC-mediated synthesis of the novel orange dyewas demonstrated with an additional step of optimisation of severalparameters such as the composition and pH value of the reaction buffer,substrate concentration, LAC activity, and temperature.

3.1.1. Influence of buffer type and pH valueIn the first step of the study, the effect of the type and pH value of

buffer on both the synthesis of the N15 dye and free laccase activity was

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determined. The N15 substrate possesses amino and methoxy groups,i.e. very important substituents involved in LAC-mediated couplingreactions due to their potential of benzene ring activation duringelectrophilic substitution. Even though it does not belong to phenolics,substrate N15 exhibited a bell-shape pH profile, typical for phenolicsubstrates, with the optimum 5.5, regardless of the type of buffer used(Fig. 1.A) [23].

It was found that LAC incubated with the presence of the testedbuffers exhibited slightly different pH stability (measured with ABTS asa substrate), depending on the type of the buffer used (Fig. 1.B). In thecase of the Na-tartrate buffer, the highest LAC activity was determinedat pH in the range between 5 and 5.5, while in the case of McIlvainbuffer, the highest laccase activity was found at pH 7. Nevertheless, thehighest LAC activity was detected in both types of buffers with themaximum values maintained at a comparable level of 91 ± 1% of theinitial LAC activity.

The second step of the optimisation studies involved selection of theoptimal concentration of the N15 substrate and LAC activity for 48 hN15 dye synthesis.

For the substrate concentrations up to 5mM, the application of LACwith the activity of 0.5 U per mmol of the substrate was sufficient forN15 dye synthesis efficiency. In the case of the highest concentration ofthe N15 substrate (10mM), the maximum absorbance was noted usingLAC with the activity of 3 U/mmol (Fig. 2) with concomitant lack ofautooxidation in control samples.

Since the 3mM concentration of the N15 substrate was the max-imum concentration for which at least 90% of product N15 was ob-tained after 6 h of transformation (data not shown), the 3mM con-centration of N15 was selected for detailed research under optimal LACactivity in the range from 0.5 U to 5 U per mmol of the substrate. Themaximal absorbance of the N15 dye was obtained after 48 -h transfor-mation of the N15 substrate by LAC with the activity of 4 U per mmol,

whereas the minimal LAC activity required for efficient (at least 90%)N15 substrate transformation was 2 U per mmol of the substrate andsuch values was selected as optimal (data not shown).

3.1.2. Concentration of bufferTo ensure both the best efficiency of the 3mM of N15 substrate

biotransformation and the minimal buffer concentration, the optimi-sation of the Na-tartrate buffer concentration (pH 5.5) in the range from5 mM to 100mM was performed, using LAC with the activity of 2 U permmol of N15. During the transformation, the absorbance of the N15product was monitored spectrophotometrically, while the substrate andproduct concentrations were determined by capillary electrophoresis.The results showed the highest absorbance of the dye in the case of100mM sodium-tartare buffer used as the reaction environment withsimultaneous 100% efficiency of the N15 substrate transformation afterthe 48 h reaction (Fig. 3). Nearly 100% efficiency of N15 transforma-tion was also noted in samples containing 40mM and 80mM buffer.Lower values of dye absorbance were noted for such samples; however,they were not lower than 85% of maximal absorbance.

Together with the decreasing concentration of the buffer, the rate ofsubstrate transformation and the maximal absorbance of the productdecreased to the values of 58% and 7.51 a.u., respectively, which wasnoted when the 5mM tartrate buffer was used. This resulted from thelower buffering capabilities of the less concentrated buffers observedduring the reaction. The pH value was 6.94 for the 5mM buffer and5.68 pH for the 100mM buffer. Moreover, there was also no change inthe colour of the control solution of the substrate at any of the con-centrations of the sodium-tartrate buffer because of lack of autoxida-tion.

After 48 h of transformation in 40mM buffer, the N15 substrate was

Fig. 1. Influence of the type of buffer on the absorbance of the N15 dye (A) andLAC stability (B) during 24 -h incubation in Na-tartrate buffer (solid line) orMcIlvain buffer (dotted line); 100% is the activity of LAC at the beginning of theexperiment.

Fig. 2. Influence of LAC activity and N15 substrate concentration on themaximal absorbance of the N15 product obtained during 48 h LAC-mediatedtransformation.

Fig. 3. Influence of the sodium-tartrate buffer concentration (5mM–100mM)on the yield of N15 substrate transformation and the maximal absorbance of theN15 dye noted during 48 -h incubation with LAC.

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completely consumed, and such a buffer was selected for further ex-periments.

3.1.3. Homogeneity of the N15 dyeThrough LAC-mediated synthesis, novel compounds can be obtained

due to enzymatic as well as non-enzymatic coupling reactions betweenradicals formed from oxidised substrates after the action of LAC[24,25]. The formation of end products during LAC-mediated trans-formation is in some cases unpredictable because they are a result ofspontaneous interactions between very active different radicals witheach other or with other aromatic compounds. These coupling reactionsof radicals are dependent on the substituents increasing or decreasingthe electron density of aromatic compounds, which is directed towardsthe ortho or meta position during electron substitution [7,22,26].Therefore, products obtained by the action of LAC are very often nothomogenous. In the case of the N15 dye synthesis, four different or-ange-coloured products were detected in different quantities, changingduring the process (Fig. 4).

The analysis of the transformation mixture (run at 28 °C) showedthe total consumption of the N15 substrate during the first 6 h of thereaction and the formation of four products with different retentiontime and peak areas, indicating their different structures and con-centrations (Fig. 4, peaks A–D). After 6 h of transformation, the highestpeak area was noticed for compound A. During the next hours oftransformation, the amounts of products A, B, and D decreased, with asimultaneously increasing amount of compound C, which was indicatedas the main component of the N15 dye.

Non enzymatic coupling reaction: influence of the reaction tem-perature and the presence of LAC. Interestingly, the analysis of thechromatograms and UV-spectrum of the products differed when thetransformation of the N15 substrate occurred at the higher temperature(40 °C). During the last step of N15 dye synthesis optimisation usingpurified free LAC, the temperature effect on the rate of substratetransformation was assessed. The rate of the transformation proceedingat 23 °C and 28 °C was relatively slow; product A dominated in thereaction mixture, both in the presence of the enzyme and after removalthereof. Raising the reaction temperature to 40 °C accelerated theprocess of product A transformation into product C almost twice, re-sulting in a drop of the concentration of products A and B after 72 h ofreaction by 66% and 92%, respectively, with a simultaneous 84% in-crease in the amount of product C. In the case of product D, there was a24% increase in the amount after 48 h, followed by a 50% decrease inits amount on the next day (data not shown). Moreover, it is possible toobserve that the removal of LAC after complete transformation ofsubstrate N15 (after 6 h) did not have a significant influence on theamount of the main reaction product C (Fig. 5.). This indicates that therole of LAC in the synthesis of the main product C is limited to acti-vation of the substrate, e.g. a radical formation during the first 6 h, andthe formation of the end product C occurs non-enzymatically and thisprocess might be accelerated by elevation of the temperature up to

40 °C (Fig. 5).

3.2. N15 dye synthesis using immobilised LAC

3.2.1. Optimal conditions for LAC immobilisation and storageTo achieve a high yield of transformation with a simultaneous low

environmental impact of the process, immobilised LAC should be re-cognised as a potential biocatalyst for industry. To optimise the

Fig. 4. Chromatograms of the transformation mixture during72 h of N15 dye synthesis using LAC in optimal conditions of0.1M sodium-tartrate buffer pH 5.5, 3mM of the N15 sub-strate, and temperature of 28 ℃ (in the first 6 h) and 40 ℃ (forthe next 64 h); the intermediate products of the synthesis aremarked with letters A, B, and D, whereas the main product ismarked with letter C.

Fig. 5. Amount of the N15 C product (expressed as peak area) synthesised fromthe 6th to 72nd hour at different values of reaction temperature and in thepresence (solid line, LAC+) or absence (dotted line, LAC-) of LAC as a catalyst;during the first 6 h, all samples were incubated with LAC at a proper tem-perature of 23℃, 28℃, or 40℃.

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conditions of the laccase immobilisation procedure and storage, threetypes of porous carriers used for LAC immobilisation were tested instarting conditions - an acrylic carrier functionalised by an aminefunctional group used in covalent bonding of laccase (carrier A; NH2), amacroporous polystyrene carrier without any functional groups (carrierB; S0), and a porous acrylic carrier functionalised by octadecyl groups(carrier C; C18). These supports had never been tested before for laccaseimmobilisation. However, modified acrylic resins are well-known lac-case carriers, for example EUPERGIT C 250L© or Sepabeads EC-EP3,and they have been used in decolourisation of synthetic dyes [27,28].

As can be noticed, the presence of octadecyl groups in the surface ofthe porous carrier (carrier C18) had a positive effect on both the yield ofprotein and the yield of LAC activity immobilisation using the 40mMphosphate buffer and 1mg of LAC per 1 g of the support (Table 2). Thedifferences between the activity of covalently immobilised laccases andlaccase adsorbed on the carrier are caused mainly by structural changesin the enzyme itself. In the case of adsorbed laccase, the weak bindingforces between laccase and the carrier cause slight structural changes,preserving laccase activity, while stabilisation of the quaternary struc-ture of immobilised laccase through covalent bonds has an impact onthe laccase active centre and, therefore, loss of activity [11,29]. How-ever, the structure of the support has an impact on the immobilisationyield as well. As can be observed in the case of the S0 carrier used forlaccase adsorption, the activity of laccase was very low, although theamount of bounded protein was comparable to that on the C18 carrier.One of the possible reasons may be the blocking of the laccase activesite by the matrix, which is one of the reported disadvantages of ad-sorption [11]. Another reason might be the adsorption of ABTS duringactivity measurements, which was observed as intensive colourisationof the S0 carrier with the green ABTS+ %radical obtained through lac-case oxidation. Another important factor influencing the yield of im-mobilised laccase is the protein concentration. Although the percent ofprotein bound to the C18 carrier was high for the range of concentrationfrom 1mg to 4mg per 1 g of the carrier, it did not correspond with theincrease in the activity of immobilised laccase and was the highest for1mg/g. This phenomenon was caused by intermolecular space inhibi-tion of laccase particles bound on the surface of the carrier, which limitsthe dispersion of the substrate and the product. The limitation of im-mobilised laccase activity caused by its concentration has been fre-quently reported, e.g. after adsorption of Trametes versicolor laccase on

bentonite-based mesoporous materials in the protein concentrationrange from 0.5 to 4mg/mL, the activity of the enzyme increased onlyup to the concentration of 2mg/mL [30]. A similar correlation formagnetic silica nanoparticles was observed, for which adsorption oflaccase was efficient only up to an initial concentration of 0.1mg/ml or1mg/ml in the case of carbon-based magnetic composites; exceedingthis concentration resulted in a decrease in immobilised laccase activity[31,32].

One of the main goals of laccase immobilisation is to maintain LACactivity during long-term storage and thus extend its half-life.Therefore, in the second step, the conditions for carrier storage werechecked, including the type, pH value, and concentration of the bufferapplied for both free and immobilised LAC storage at 4 °C for 21 days(Table 3). Among the three tested buffers, the highest activity wasobserved in the case of immobilised and free LAC incubated in the Na-tartrate buffer, pH 5.5. The immobilisation of LAC on the C18 supportsignificantly increased its stability, for which 40% higher activity wasnoted after 21 days of storage in comparison with the activity of freeLAC.

This correlation was consistent with reported results of laccaseimmobilised on bimodal micro-mesoporous Zr-metal organic frame-work or biochar, which clearly indicated the positive effect of laccaseimmobilisation on its activity during storage in comparison to the ac-tivity of the free form [33,34]. Moreover, our preparation showedhigher stability in comparison to those reported in the other articlesmentioned above. The stability of Pleurotus ostreatus laccase adsorbedon the C18 carrier was comparable to the results obtained for storage ofP. ostreatus laccase immobilised covalently on perlite [35]. However, itshould be emphasised that covalent bonds are stronger than forces ofphysical adsorption and yet, due to the storage conditions used, themaintenance of the activity of laccase adsorbed on the C18 carrier wasas efficient as that when covalent immobilisation was used.

3.2.2. Thermostability of immobilised laccaseThermostability of both native and immobilised LAC was evaluated

at a temperature of 40 °C and 60 °C in optimal storage conditions. Asshown in Fig. 6, the immobilisation of LAC on the C18 support has apositive effect on its thermostability. After 20min of incubation, theimmobilised LAC exhibited ca. 15–20% higher activity in comparison tofree LAC in the case of both tested temperatures. After one hour of

Table 2Influence of immobilisation conditions on the parameters of immobilised LAC from Pleurotus ostreatus.

Immobilisation conditions Parameters of immobilised LAC

Type of carrier Concentration of buffer[mM]

Concentration of laccase[mg/g]

Protein bounded [mg/g]

Yield of protein[%]

Activity of immobilisedlaccase [U/g]

Yield of activity [%]

Starting conditionsA; NH2 100 1.5 0.43 ± 0.1 28 ± 0.1 1.27 ± 0.1 1.17 ± 0.1B; S0 100 1.5 0.13 ± 0 9 ± 0 0.1 ± 0 0.052 ± 0C; C18 100 1.5 0.15 ± 0.1 10 ± 0.1 2.10 ± 0.2 1.92 ± 0.1Optimal conditionsC; C18 40 1 0.82 ± 0.2 68 ± 4.9 2.66 ± 0.2 3.17 ± 0.3

Table 3Immobilised (on C18) and free LAC stability in different storage conditions.

Buffer parameters Remaining activity of immobilised LAC [%] Remaining activity of free LAC [%]

type pH concentration [mM] 7 days 14 days 21 days 7 days 14 days 21 days

Starting conditionsphosphate-citrate 4.5 100 85 ± 3 85 ± 1 81 ± 11 62 ± 1 41 ± 2 36 ± 1acetate 4.5 100 90 ± 5 94 ± 1 75 ± 4 57 ± 3 32 ± 1 27 ± 3Na-tartrate 4.5 100 89 ± 7 90 ± 0 86 ± 8 68 ± 1 43 ± 1 46 ± 1Optimal conditionsNa-tartrate 5.5 100 97 ± 2 99 ± 4 94 ± 0 91 ± 2 77 ± 2 74 ± 2

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storage, free LAC incubated at 60 °C lost almost 100% of initial activity,whereas immobilised LAC exhibited around 23% of initial activity(Fig. 6). This result confirmed the well-known fact that laccase im-mobilisation improves its properties, including thermostability, viaprevention of loss of the three-dimension structure and, therefore, de-naturation of laccase. For example, Myceliophtora thermophila laccaseadsorbed on Sephabeads after 2 h of incubation showed about 15% and10% higher stability at 60 °C and 70 °C, respectively, in comparison toits free form [27].

3.2.3. Transformation of the N15 substrate by immobilised LACSince the reusability of an immobilised biocatalyst is essential for its

continuous bioreactor usefulness, subsequent N15 dye synthesis cycleswere carried out. Three different activities of LAC immobilised in op-timal conditions on the C18 support were used as biocatalysts of N15substrate transformation into the coloured N15 dye. Over the first 48 h,the reaction proceeded in the presence of immobilised LAC. Afterwards,the transformation mixture containing the synthesised dye was dec-anted from the carrier and the reaction was continued. The N15 sub-strate was totally transformed by immobilised LAC during the first two

Fig. 6. Thermostability of immobilised (solid lines) and free (dotted lines) LACduring incubation at a temperature of 40 °C (black lines and b) and 60 °C (greylines c and d).

Fig. 7. Dyeing properties of the N15 dye against natural fibres of animal and plant origin dyed with the N15 dye at concentration of 0.2% and 0.5%.

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48 h long transformation cycles in every activity tested (data notshown). In the third cycle, the presence of a small amount of the sub-strate was observed in the reaction mixture after transformation of theN15 substrate by 5.6 U/mmol of immobilised LAC per mmol. After thefourth cycle, 100% substrate transformation was noted only in the caseof the highest activity of immobilised LAC (22.3 U/mmol) (data notshown).

The activity of immobilised laccase detected after four transforma-tion cycles retained 63% of initial activity, which indicates its highstability and possible suitability for the synthesis of the dye in a con-tinuous system. This result is consistent with the activity of adsorbedlaccase, which retained 65.5% of its initial activity after five cycles ofABTS transformation [33]. Operational stability is a frequently eval-uated parameter of immobilised laccase as a biocatalyst; for example,synthetic dyes such as malachite green, for which six transformationcycles were performed with efficiency from 80% in the first cycle to20% in the sixth cycle of decolourization, retained 41% of its initialactivity [27]. There are also reports on highly reusable immobilisedPleurotus ostreatus laccase adsorbed on an agarose-based carrier pro-viding 15 times use of the same biocatalyst in bisphenol A removal,maintaining removal efficiency>90% [36]. Simultaneously, the ac-tivity of immobilised laccase used as control samples incubated in the

sodium-tartrate buffer without the N15 substrate exhibited 76% of itsinitial activity. The most probable cause of those differences was theN15 dye adsorption on the surface of the C18 carrier. Adsorption of asubstrate and/or product is a common problem during work with im-mobilised laccase, limiting or even completely inhibiting the enzymeactivity, as in the case of highly hydrophobic anthraquinone and azodye decolourization [37,38]. There are also some reports on the ad-vantage of covalent immobilisation over adsorption in terms of betterreusability. This is mainly related to the stronger attachment andmultipoint bonds between the LAC and the carrier, preventing laccaseleaching from the carrier pores [33]. However, measurements of theimmobilised laccase control samples showed that the laccase leachingfrom the tested C18 carrier was negligible (data not shown).

3.3. Dyeing properties of the N15 compound

The ability of the N15 product to dye natural fibres was tested to-wards wool, cotton, silk, viscose and flax. The fibres were dyed using aconcentration of 0.2% and 0.5% of the N15 dye solution, which re-presented a concentration of 4.6% and 11.6% per fibre mass. Thedyeing properties of the dyes resulted from the interactions between thedye and fibre as well as the chemical structure, presence of functionalgroups, and polarity of both. In 95%, cotton is made from cellulose andit therefore possesses hydroxy functional groups on its surface, whichare essential for hydrogen as well as covalent bond formation betweenfibres and direct dyes [39]. In the case of wool fibre, which as a proteinpossesses free amino groups, ionic bonds can be formed between theamino groups of the fibre and the carboxyl or sulphonic groups of aciddyes [39,40]. The N15 product exhibited good dyeing propertiesagainst animal fibres (wool and silk), which indicated the acid nature ofthe N15 dye correlated with the substrate structure (2-amino-3-methoxy carboxylic acid) (Fig. 7). This feature is in agreement withliterature data on good dyeing properties of other acid dyes synthesisedusing LAC as a biocatalyst for colourisation of wool [8]. The dyeingactivity toward plant fibres was weaker, which was caused mainly bythe lack of interactions with hydroxyls at the cellulose surface.

Dyed wool fibres were tested for their resistance to physicochemicalfactors such as artificial light, distilled water, washing at 40 °C, alkalineand acidic sweat, and dry and wet rubbing (Table 4) by the TKANLABLaboratory. In terms of most of the tested parameters, the N15 dyeshowed good dyeing quality. Reduced resistance of the N15 dye wasonly observed in the case of alkaline sweat treatment, since the N15 dyeis well soluble in alkaline solvents. Nevertheless, the results indicatedthe high dyeing potential of the tested compound as fabric dye.

3.3.1. Chemical structureThe mass spectra of the main N15 C product as showed relatively

intense peaks at m/z449.37 U. Based on the literature data, the phe-nazine structure of the N15 product was proposed (Fig. 8). The goodwater solubility of the dye and the good dyeing properties against woolindicated the presence of a carboxy substituent in the chemical struc-ture. The amino groups present in the structure of the substrate areinvolved in the phenazine formation, which is in agreement with theliterature data [26,41]. The phenazine N15 dye contains imine,methoxy, hydroxy, and carboxy groups and its proposed name is 7-(2-carboxy-6-metoxyphenyl)amine)-4,9-dicarboxy-6-hydroxyphenazine.

4. Conclusions

The use of enzymes in textile processes has many advantages as faras the environmentally friendlier processes are concerned and theseinclude (1) water and energy savings, (2) the use of lower amounts ofchemicals, and (3) milder process conditions. Laccase, i.e. a fungaloxidoreductase with a broad substrate spectrum, belongs to a largegroup of enzymes with commercial potential. This enzyme is able totransform simple aromatic precursors into dyes with very effective

Table 4Colour fastness of wool fabric dyed with the N15 dye at a concentration of0.5%, 1%, and 3%.

Tested parameters ofcolour fastness

Dye concentration [%] ISO standard

0.5 1 3

1 Artificial light1) a) 5-6 5-6 5 PN-EN ISO 105-B02:2014-11

2 Distilledwater2)

a) 4 4-5 4 PN-EN ISO 105-E01:2013-06b) 4-5 4 4-5

c) 4-5 4 43 Washing 40 °C2) a) 3 3 3-4 PN-EN ISO 105-

C06:2010 accordingto A1S

b) 4 3-4 4c) 4 4 4

4 Alkalinesweat2)

a) 4 4 4 PN-EN ISO 105-E04:2013-06b) 3 2-3 2-3

c) 3-4 3-4 2-35 Acidic sweat2) a) 4 4 4 PN-EN ISO 105-

E01:2013-06b) 4 4-5 3c) 4-5 4-5 3

6 Dry rubbing2) b) 4 4 4 PN-EN ISO 105-X12:2005

7 Wet rubbing2) b) 4 3-4 3-4 PN-EN ISO 105-X12:2005

Colour fastness according to a blue1) or grey2) scale, in which index “8” or “5”,respectively, means no change of colour and “1” means great change of colour;according to PN-EN20105-A02:1996 and PN-EN 20105-A03:1996 standards; a)change in the colour of the tested sample; b) soiled whiteness of the accom-panying fabric – cotton; c) soiled whiteness of the accompanying fabric – wool.

Fig. 8. Proposed structure of the orange N15 dye obtained through homo-molecular transformation of 2-amino-3-methoxybenzoic acid mediated by LACfrom P. ostreatus.

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properties and can be used in free and immobilized forms. Immobilisedbiocatalysts offer many advantages: they are simple to prepare and canbe re-used in continuous systems. An advantage of the laccase-mediatedtransformation into potential new colour molecules is the very rapidreaction resulting in synthesis of generally stable products with colourslasting for a long time without specific stabilisation. Other advantagesof this type of synthesis include simplicity and the use of oxygen as the“clean oxidant”. Considering the small quantities of the enzyme em-ployed and the possible use of the immobilised form of laccase, thelaccase-mediated transformation of simple compounds as potential dyeprecursors turns out to be effective. The use of enzymes for bio-transformation processes ensures a minimal number of steps required toobtain new valuable products. An appropriately designed bioprocessyielding products with specific parameters, such as the structure,purity, and activity, can be carried out using highly selective enzymaticsystems.

Funding

This work was partially supported by the National Science Centre,Poland (2015/17/N/NZ9/03647; 2016/21/D/NZ9/02460) and theMinistry of Science and Higher Education Iuventus Plus Program(0433/IP1/2011/71).

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