Supplementary material

Enhanced production, one-step affinity purification, and characterization of

laccase from solid-state culture of Lentinus tigrinus and delignification of pistachio

shell by free and immobilized enzyme

Salar Sadeghian-Abadia, Shahla Rezaeia, Mahsa Yousefi-Mokria, and Mohammad Ali

Faramarzi*,a

a Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and

Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box

14155–6451, Tehran 1417614411, Iran

-------------------------------

*Corresponding author: Tel: +98-21-66954712; Fax: +98-21-66954712; E-mail:

faramarz@tums.ac.ir (M.A. Faramarzi)

1

S1. Additional experiments

S1.1. Isolation and screening

To isolate laccase-producing fungi, wood-decay fungi containing samples were

collected from live decaying trees in urban environments, i.e. street trees, gardens, and

parks in different parts of Iran. The samples were dispersed in a sterile saline solution

(0.9% NaCl, pH 5.0), filtered to remove particulates, and spread on Sabouraud dextrose

agar (SDA) plates containing a laccase indicator at an appropriate concentration [0.1%

(w/v) guaiacol (GUA), 0.05% (w/v) tannic acid (TA), or 0.1% (w/v) 2,6-

dimethoxyphenol (2,6-DMP)] and 0.01% chloramphenicol to prevent bacterial

contamination. Plates were then incubated at 30 °C until colored zones appeared

(reddish-brown, dark-brown, and orange-brown in the presence of GUA, TA, and 2,6-

DMP, respectively). Several laccase-producing strains were isolated and sub-cultured in

SDA with and without indicators to confirm laccase activity. Colored halo size (HS)

around the colonies to colony size (CS) ratios were determined to select suitable isolates

with highest laccase activity. Colonies with a color zone to colony size ratio of more

than 2 were selected for quantitative enzyme activity measurement. Colonies (three

plaques (1×1 cm2) from actively growing 7 days old culture on SDA plates) were

transferred to Sabouraud dextrose broth (SDB) medium (50 ml) in 250-ml Erlenmeyer

flasks. After incubation at 30 °C and 150 rpm for 2 weeks, cultures were collected at

regular 24-h intervals and centrifuged at 8000×g for 15 min to remove mycelia and

medium debris and the cell-free supernatant was used as a crude enzyme solution to

measure laccase activity. Enzyme assay was performed using 2,2'-azino-bis(3-

ethylbenzothiazoline-6-sulphonic acid) (ABTS) according to the method described in

2

this paper. Isolate with the highest laccase production was selected for further

investigations.

S1.2. Identification

Macroscopic morphological studies using different solid media, such as SDA,

potato dextrose agar (PDA), and malt extract agar (MEA), as well as microscopic

characterization after slide culturing on SDA, were performed using the methods

described by Damm et al. (2008) following instructions of the literature. The

sequencing of 18S and 5.8S rDNA genes was carried out to identify the selected strain.

Genomic DNA was extracted from fungal cells using a modified phenol-chloroform

extraction method (Sambrook and Russell, 2001) to use as a template for PCR

amplification. The ratio of A260/A280 was determined to confirm the purity of template

DNA. NS1 and NS4/NS7 primers were used for 18S rDNA amplification (Borneman

and Hartin, 2000; Damm et al., 2008), and 5.8S was amplified using ITS1 and ITS4

primers (Damm et al., 2008). The PCR mixture (12.5 μL) contained 50 mM Tris (pH

8.3), 250 μM dNTP mixture, 400 nM of each forward and reverse primers, 40–60 ng

template DNA, 2.5 mM MgCl2, and 0.25 U Taq DNA polymerase. The reagents were

combined in 200 μL micro-tubes and placed in a Primus 96 advanced thermal cycler

(PEQLAB, Erlangen, Germany) programmed as 35 cycles, 94 °C for 1 min

denaturation, 56 °C for 90 s primer annealing, and 72 °C for 60 s extension. The

amplified DNA was separated on 1% agarose gel in TEA buffer. Sequence comparisons

were performed using BLAST (NCBI, USA). The 1015 bp (18S rDNA) and 602 bp

(5.8S rDNA) sequences were submitted to GenBank under accession numbers of

KY563123 and KY563124 (http//www.ncbi.nlm.nih.org/ available May 08, 2019),

3

respectively. Phylogenetic and molecular evolutionary analyses were conducted using

the molecular evolutionary genetics analysis (MEGA) software version 6.06. Distances

and clustering were calculated using the neighbor-joining method. Bootstrap analysis

was used to evaluate tree topology of neighbor joining data by performing 1000 re-

samplings (Samaei-Nouroozi et al., 2015). The fungus was maintained on SDA culture

at 4 °C for long storage and sub-culturing was done every month.

S1.3. Enzyme production using solid-state fermentation (SSF)

To evaluate the effect of various factors on laccase production, the basal

medium was supplemented with 0.1‒2 mM concentrations of laccase inducers including

caffeine, catechol, gallic acid (GA), guaiacol (GUA), hydroquinone, pyrogallol, Tannic

acid (TA), tyrosine, vanillin, or veratryl alcohol or 1‒10% (v/v) concentrations of

ethanol, 0.5‒10 mM concentrations of trace elements including Al3+, Ba2+, Ca2+, Co2+,

Fe2+, Mg2+, Mn2+, Ni2+, and Zn2+, and 0.1‒2% (w/v) concentrations of glucose. In order

to study the effects of process parameters, the pH (3.0‒8.0), temperature (20‒45 °C),

initial moisture (30‒80% w/w), inoculum size (plaque sizes of 1×1, 1×2, and 1×3 cm),

and pistachio shell particle size (<500 to >2000 μm) used for SSF experiments were

altered in the given ranges.

S1.4. Laccase activity assay

To start the reaction, 100 µL enzyme solution was mixed with 900 µL of 50 mM

citrate buffer pH 4.5 containing 2 mM ABTS and incubated at 40 °C for 10 min. The

reaction was stopped by adding 100 µL of 1 mM sodium azide, and resulting products

were measured by reading the absorbance at 420 nm. Readings were compared to a

4

standard curve made using a dilution series of product from 100 to 1000 µmol mL−1. To

prepare products, a stock solution of substrate was mixed with 0.5 mM CuSO4 and

allowed to stand in the dark at room temperature for 6 h to complete the reaction before

the precipitation of products. The reaction mixture contained product was used to create

diluted solutions using a serial dilution method.

S1.5. Enzyme purification, SDS-PAGE, FPLC, and zymography

For synthesize of large pore SBA-15 with magnetic nanoparticles (m-SBA), 2 g

of triblock copolymer Pluronic P123 and 0.014 g of NH4F were dissolved completely in

70 mL of 2 M HCl while stirring for 2 h at room temperature. Then, 1.5 g FeCl2 was

added to the solution slowly and stirred for about 1 h. Twenty mL of hexane was mixed

with 8.5 mL of tetraethyl orthosilicate (TEOS), added dropwise into the mixture, and

vigorously stirred (1500 rpm) at 15 °C for 24 h. The product was then transferred to a

Teflon-sealed autoclave at 130 °C for 24 h. The resultant precipitate was filtered,

washed with distilled water and ethanol, and air-dried overnight at room temperature.

The calcination of the solid was performed at 550 °C for 6 h. The final product was then

filtered, washed with distilled water and ethanol, and air-dried over night at 50 °C.

The enzyme was purified using the method described by Rezaei et al. (2017). Briefly,

enzyme production was performed according to the method described in this paper. The

clear supernatant was precipitated using PEG 4000 at a final concentration of 15%. The

precipitate was then recovered using centrifugation at 18,000×g for 15 min, re-

suspended in a minimum volume of citrate buffer, and dialyzed overnight against

repeated changes of the same buffer followed using centrifugation at 8,000×g for 10

min.

5

After loading enzyme-containing concentrate, the column was washed with 50

mL of phosphate buffer and eluted with a linear gradient of pH, urea, (NH4)2SO4, and

CuSO4 (Rezaei et al., 2017). Fractions were concentrated, dialyzed against 50 mM

citrate buffer (pH 4.5), analyzed for laccase activity and protein concentration, and

stored at −20 °C for subsequent experiments. SDS-PAGE was carried out using 10%

gels and the Page Ruler Plus Prestained Protein Ladder (Fermentas, Lithuania) as

weight marker (Adrangi et al., 2010). Samples were loaded into the wells and run at a

constant current of 30 mA. For denatured gels, laccase samples were mixed with

loading buffer (containing β-mercaptoethanol and SDS) and heated in a water bath at

100 °C for 10 min. For FPLC-anion exchange chromatography, an active fraction was

subjected to a 10/100 GL Mono Q FPLC column (Pharmacia, Uppsala, Sweden) with an

HPLC apparatus (Knauer, Germany). The column was eluted with buffer in a gradient

of increasing concentrations from 0 to 1 M NaCl at a flow rate of 0.5 mL min−1 and the

spectra was scanned for 10 min (Moshfegh et al., 2013).

S1.6. Enzymatic characterization

S1.6.1. Redox potential determination

The redox potential (E°) of the T1 copper was measured using the cyclic

voltammetry approach. Cyclic voltammetry of a solution of 100 U enzyme in 50 mM

citrate buffer (pH 4.5) was recorded with planar ‘BAS’ gold electrode in a 1-mL volume

electrochemical cell. In these measurements, an Ag|AgCl|3 M NaCl reference electrode

(BAS) and a platinum counter electrode were used and potential was referred to the

normal hydrogen electrode (NHE). Electrode surface of the working gold electrode was

polished on DP-Suspension and on alumina FF slurry (0.25 µm and 0.1 µm; Stuers,

6

Copenhagen, Denmark), rinsed with Millipore water, and sonicated between polishings

and after final polishing for 10 min. Then, the electrode was kept in concentrated H2SO4

with 10% H2O2 for 1 h, subjected to 30 cycles in 0.5 M H2SO4, and rinsed with

Millipore water (Shleev et al., 2005; Macellaro et al., 2014).

S1.6.2. Enzyme optimal activity

Optimal pH and temperatures were determined by measuring free and

immobilized enzyme activity in the pH range 3.5‒6.0 at different temperatures from 20

to 45 °C. The activity of each sample was measured as previously described and the

maximal enzyme activity was subjected as the control (100%). The effect of incubation

time (0.5‒20 min) on laccase activity from L. tigrinus was also checked. The obtained

optimal conditions were subjected to subsequent experiments.

S1.6.3. Enzyme stability

Temperature and pH stabilities were studied simultaneously by incubating free

or immobilize enzyme at different temperatures (25‒55 °C) and pH values (2.0‒11.0)

for 6 h and determining the residual enzyme activity under standard assay conditions.

These results were expressed as the enzyme residual activity (%) related to laccase

activity measured in start time of the study. The following buffer systems were used at

50 mM: citrate for pH 2.0–6.0, phosphate for pH 6.5–8.0, and Tris-HCl for pH 8.5–

11.0. Stability against freeze and thaw treatment was evaluated by subjecting the soluble

and free enzyme to repeated cycles of freezing at −80 °C and thawing at room

temperature. Enzyme activity was considered to be 100% in initial cycle. Activity in

each cycle was defined as the residual enzyme activity in reaction mixture. For testing

7

storage stability of both forms of enzyme, laccase in 50 mM citrate buffer (pH 4.5) was

stored at 25 °C for several days. The remaining activity of enzyme at desired times was

measured under standard conditions. The inhibitory effect of metal ions (Al3+, AS5+, B3+,

Ba2+, Bi3+, Br-, Ca2+, Co2+, Cu2+, F−, Fe2+, Fe3+, I−, K+, Li+, Mg2+, Mn2+, Mo+, Na+, Ni2+,

and Zn2+), common enzyme inhibitors (cysteine, dithiothreitol (DTT),

ethylenediaminetetraacetic acid (EDTA), H2O2, kojic acid, 2-mercaptoethanol (2-ME),

NaN3, oxalic acid, phenylmethylsulfonyl fluoride (PMSF), and thiourea), and

surfactants (cetyl trimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS),

Triton X-100, and Tween 80) was examined by adding the desired substance at a final

concentration of 1-10 mM and incubating the free and immobilized enzyme at 25 °C for

6 h before measuring activity. Activity in the absence of any agents was the control

(100%). Stability against organic solvents (dimethyl sulfoxide (DMSO), 1,4-dioxane,

methanol, acetonitrile, ethanol, acetone, isopropanol, ethyl acetate, butanol, and toluene)

and the hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate

([Bmim][PF6]) was assessed by adding the desired compound in a final concentration of

10−90% to the enzyme, incubating the resulting mixture at 25 °C and 200 rpm for 6 h,

and determining the residual laccase specific activity in the aqueous fraction. All assays

were performed in triplicate. The operational stability of the immobilized laccase was

determined by repeated oxidation reaction of ABTS in citrate buffer over several

consecutive cycles. At the end of each oxidation cycle, the immobilized laccase was

separated by a magnet, washed three times with the same buffer, and the procedure was

repeated with a fresh aliquot of substrate. The activity of immobilized enzyme was

considered to be 100% in the initial cycle. Activity in each cycle was defined as the

residual activity of enzyme in the reaction mixture.

8

S1.6.4. Substrate specificity

Substrate specificity of free laccase was qualitatively determined against a panel

of 38 structurally different potential substrates of both phenolic and non-phenolic types

under optimal assay conditions. The assays were performed under optimal assay

conditions (incubation of the reaction mixture containing 1 mM substrate and 10 U

mL−1 purified laccase in 50 mM citrate buffer (pH 4.5−6.5) for 10 min at 40 °C). A

UV/Vis scan between 200‒800 nm was performed before and after the reaction and

changes in absorption spectra of enzyme-treated substrates were recorded. To exclude

the possibility of non-enzymatic oxidation, blanks lacking laccase were considered in

parallel.

S1.6.5. Kinetic parameters

To determine kinetic constants, Michaelis-Menten constant (Km) and maximal

reaction velocity (Vmax) values were estimated using Lineweaver-Burk plots. The value

of Vmax and the concentration of the purified enzyme ([E]) were used to calculate the

catalytic constant (Kcat) using the equation of Kcat = Vmax [E] (Rezaei et al. 2017). The

ratio of Kcat/Km was used to determine the specificity constant of enzyme with each

substrate. Substrate concentrations ranging from 0.1 to 2 mM were used to calculate the

initial rate of the reaction with 10 substrates. Standard assays were performed in sodium

citrate and sodium phosphate buffers (pH 4.0−8.0) at 40 °C. The amount of product was

quantified colorimetrically at the related wavelengths.

S1.7. Delignification study

9

S1.7.1. Total phenol and reducing sugar contents

Delignification was performed in a reaction mixture containing purified free and

immobilized laccase (about 50 U) and pistachio shells (1 g) in 1 mL of 50 mM citrate

buffer (pH 4.5) and incubated at 35 °C and 40 °C for free and immobilized state,

respectively and 150 rpm shaking for 12 h. After this period, immobilized enzyme and

then the mixture were separated using a magnet and filtration, respectively. The filtrate

was used to measure reducing sugars and total polyphenol contents employing the

methods of dinitrosalicylic acid and Folin-Ciocalteu, respectively. To measure reducing

sugars released from the bio-waste, the supernatant was mixed with dinitrosalicylic acid

(DNS) reagent at a 1:1 ratio, placed in boiling water for 10 min, and read against its

blank at 540 nm after cooling. Total polyphenol content was also measured with Folin-

Ciocalteu. Briefly, 4 mL of 10% Folin-Ciocalteu reagent (FCR) was added to 1 mL of

supernatant and equilibrated at room temperature for 5 min. After adding 10 µL of 7.5%

NaHCO3, the mixture was diluted to 8.5 mL with distilled water, incubated at 45 °C,

and shaked at 150 rpm for 15 min. Finally, the mixture was left in a dark place at room

temperature for 2 h and the absorbance was read at 765 nm against a proper blank.

Control samples without enzyme were processed in parallel with test samples.

S.1.7.2. Physicochemical characterizations of laccase treated pistachio shells

For chemical characterization of laccase-treated pistachio shell, sample was

prepared by incubating 5 g bio-waste and 375 U laccase in 50 mL citrate buffer (50

mM, pH 8.0) with or without 10 mM mediator (2,6-DMP, gallic acid, HBT, TEMPO, or

vanillin) at 40 °C, 150 rpm agitation, and 90% aeration for 1‒8 h. After desired

incubation time, the mixture was centrifuged at 8000×g for 10 min and solid fraction

10

was used for chemical analyzes. For scanning electron microscope (SEM) imaging, the

sample was prepared using incubation of 1 g pistachio shell with 75 U laccase at 40 °C,

150 rpm, and 90% aeration. The solid fraction was separated for SEM after 1 h

incubation. To take the SEM images (Bruker-XFlash 6|10 Detector), raw and pretreated

solid residues were dried in a vacuum dryer oven at 45 °C for 24 h followed by

gradually dehydrating. Dehydrating was performed using acetone-water mixtures with

increasing the concentration of acetone up to 100%. Finally, dehydrated samples were

mounted on the metal stubs using double-faced tap and images were taken using an

accelerating voltage of 10 kV. Before imaging, samples were coated with gold-

palladium in a sputter coater.

S.1.7.3. Gas chromatography-mass spectroscopy (GC-MS) analysis

Gas chromatography-mass spectrometry (GC-MS) analysis was performed as

follows. Samples containing 5 g pistachio shell and 375 U laccase in 50 mL citrate

buffer (50 mM, pH 8.0) were incubated at 40 °C, 150 rpm, and 90% aeration. After 1.5

h, the mixtures were centrifuged at 8000×g for 10 min and the supernatant was

extracted with ethyl acetate (1:1 ratio) and concentrated to a volume of less than 1 mL.

GC-MS spectra were obtained using an Agilent GC 7890A/5975C Inert MSD

instrument equipped with a Triple-Axis Detector (EI at 70 eV) employing a 30 m×0.25

mm Rtx-5MS column (film thickness 25 μm). Approximately 1 μL of sample were

deposited on a ferromagnetic wire and inserted into the glass liner. The oven was

programmed from 40 °C (1 min) to 230 °C at a rate of 6 °C min−1. Final temperature

was held for 20 min and the carrier gas (He) was set at 1 mL min−1.

11

S2. Additional results

S2.1. Isolation and identification

A total number of 250 fungal colonies with the ability to produce activity zones

on SDA screening plates were obtained. Among these, 6 colonies were selected for

quantitative evaluation of laccase activity based on their higher color zone to colony

size ratios. Since laccase activity cannot be quantitatively measured on agar plates, these

isolates were transferred to liquid culture media. One of these isolates (designated

isolate SS-100) showed higher levels of laccase activity when cultured in the liquid

medium and, therefore, was chosen for further studies. Identification studies based on

18S and 5.8S rDNA sequencing revealed that this isolate most likely belongs to the

genus Lentinus. The 1014 bp 18S rDNA sequence showed 100% identity over its entire

length to L. tigrinus and Polyporus arcularius 18S rDNA while the 606 pb 5.8S rDNA

sequence was 99% identical to several L. tigrinus entries and only 92% to P. arcularius

sequences.

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Fig. S1. Phylogenic tree showing the inter-relationships of L. tigrinus and its closest

relatives based on the 18S rDNA sequences.

S2.2. Enzyme production and effective factors on laccase production

Laccase production was found to be dependent on the presence of an adequate

mass of active cells. The strain identified as L. tigrinus was selected after screening 250

fungal isolates for laccase activity. This strain was able to produce high quantity of

laccase (i.e. 20.9 U mg−1 extracellular protein after 7 days incubation). As shown in Fig

S2, pistachio shell resulted in the highest laccase production compared to other bio-

wastes tested. Rice straw, wheat straw, and SDB led to production levels of 17.3, 15.6,

and 2.8 U mg−1 laccase by the strain.

13

The extracellular laccase production was also studied in the presence of five

concentrations of laccase inducers. The presence of inducers in culture medium

increased the laccase activity level, but the highest induction (6.7-fold) was obtained

with 0.5 mM 2,5-xylidine followed by 1 mM TA (5.6-fold) (Fig. 1b). In addition to 2,5-

xylidine and TA, GUA, catechol, hydroquinone, caffeine, and vanillin also increased

laccase production to more than 2-fold compared to the control with no inducer.

Inductive effect of different inducers on laccase production was also reported in other

studies, such as laccase production by Pleurotus sajor-caju in the work of Murugesan et

al. (2006), in which 1 mM 2,5-xylidine enhanced its maximal laccase production to 2.5-

fold. Nandal et al. (2013) also reported that 2,5-xylidine at a concentration 0.5 mM

induced laccase production by Coriolopsis caperata. The promoter region of fungal

laccases may contain xenobiotic response elements (Piscitelli et al., 2011; Janusz et al.,

2013). These regions control the expression of laccase genes in response to the presence

of aromatic compounds. The increased expression of laccases in this case is believed to

protect the host from the lethal effect of oxygen radicals that may be produced by

chemical reactions involving such compounds (Piscitelli et al., 2011).

In addition, the determination of the effective variables on SSF process showed

an enhanced production around pH 6.0 and temperature 30 °C (approximately 28 U

mg−1) (Fig. 1c). Enzyme production was more than 25 U mg−1 at a pH of 6.0 and a

temperature range of 25‒35 °C. In addition, laccase activity at all pH values tested (3.0‒

8.0) and 25‒35 °C was greater than 20 U mg−1. The lowest enzyme activity (2.1 U mg−1)

was obtained at pH 8.0 and 45 °C. Nandal et al. (2013) obtained similar results with

Coriolopsis caperata in which its maximum laccase production occurred at pH 5.0 and

30 °C. In contrast, Bertrand et al. (2015) reported that a pH of 6.0 and a temperature of

14

50 °C were optimal for Trametes versicolor laccase production. Chhaya and Gupte’s

optimization studies for laccase production from Fusarium incarnatum (Chhaya and

Gupte, 2013) revealed that laccase yield was maximal at a pH of 5.0 and a temperature

of 28 °C. Wu and Nian (2014) and Irshad et al. (2011) also reported different results on

optimal pH and temperature for laccase production under SSF by Fusarium solani (pH

6.5 and 20 °C) and Schyzophulum commune (pH 4.5 and 35 °C), respectively.

Laccase expression is influenced by pH through the action of the pH-dependent

activation factor PacC (Cañero and Roncero, 2008). Not all laccase genes, however,

respond identically to PacC. While some laccase genes are up-regulated, others appear

to be down-regulated by this transcription factor (Cañero and Roncero, 2008). It should

be noted that increase of enzyme production in some pH values and temperatures may

have resulted, at least partly, from altered fungal metabolism and does not necessarily

reflect an equivalent increase at laccase expression level.

Among all trace elements tested, 10 mM Cu2+ and Mn2+ resulted in a high

laccase yield of about 72.2 and 71.9 U mg−1, respectively, more than 3-fold greater than

the medium with no trace element (Fig. 1d). Unlike Cu2+ and Mn2+, Co2+, Fe2+, and Mg2+

were negative signals for laccase production by the strain. CuSO4 has shown to

stimulate laccase production in other strains such as Cladosporium cladosporioides

(Halaburgi et al., 2011), Pleurotus florida (Sathishkumar et al., 2010), Trametes trogii

(Zouari-Mechichi et al., 2006), Schyzophylum commune (Irshad et al., 2011), Polyporus

sp. (Guo et al., 2011), and Pleurotus ostreatus (El-Batal et al., 2015). It is known that

the promoter region of some laccase genes contains a metal responsive element (MRE)

that is responsible for the metal-induced increase in laccase expression observed in

some fungal species (Janusz et al., 2013). Laccase production by many fungal species is

15

affected by Cu2+ in a similar manner. However, this cannot be considered a general rule

as some strains appear to be unaffected by Cu2+ ions (Piscitelli et al., 2011; Yang et al.,

2016). Some researchers suggest that copper might increase laccase production through

mechanisms other than MRE-mediated induction (Collins and Dobson, 1997). There are

also some reports describing increased laccase expression by fungal strains in the

presences of Mn2+ ions (Piscitelli et al., 2011). Again, this is not a universal rule and

Mn2+ ions do not necessarily increase laccase production by all fungal strains (Yang et

al., 2016).

Fig. S2. Comparative study on laccase production by L. tigrinus using SDB and three

solid cultures. Error bars represent standard deviations of the means (n=3). Asterisks

denote the most appropriate point with the statistically significant difference from the

control (at a minimum of p<0.05).

16

Culture medium

Sabouraud dextrose broth

Wheat straw

Rice straw

Pistachio shells

Laccase productiom

(U m

g-1

protein)

0

5

10

15

20

25 *

Enzyme production was also affected by inoculum size, incubation time, glucose

concentration, moisture content, and solid substrate particle size (Fig. S3).

Obtained results showed that inoculum size can influence the growth and enzyme

production and maximum enzyme production (25 U mg−1) at the shorter fermentation

time was obtained after 5 days fermentation and inoculation of 3 plaques with a size of

3×1 cm2 (Fig. S3a). Vikineswary et al. (2006) and Irshad et al. (2011) presented similar

results in inoculum size and incubation time effects on laccase production by

Pycnoporus sanguineus and Schyzophylum commune, respectively.

From different tested concentrations of glucose, lower concentrations of 0.1%

and 0.2% glucose enhanced laccase production (Fig. S3b). Palvannan and Sathishkumar

(2010), and Halaburgi et al. (2011) also reported the same or slightly higher range of

optimum glucose concentrations for laccase production. The effect of glucose is more

complicated than other factors tested. Laccase production by fungi is subject to carbon

catabolite repression (Piscitelli et al., 2011; Janusz et al., 2013). Higher glucose

concentrations are thus expected to reduce or even completely abolish laccase

expression. However, increasing glucose concentration can also boosts fungal

metabolism and, as a result, indirectly improve laccase production (Irshad et al., 2011).

So there must be an optimum glucose concentration at which these contradicting effects

balance each other. Reduction in particle size resulted in an enhanced enzyme

production due to increase in the substrate available to the fungus.

Furthermore, laccase production by L. tigrinus was affected by particle size of

the solid substrate. According to the results reported in Fig. S3c, laccase production by

L. tigrinus was maximal (39.9 U mg−1) by applying a particle size of <500 μm. Enzyme

production was enhanced by decreasing particle size and a particle size of <500 μm had

17

a clear positive effect on enzyme production (39.9 U mg−1). It has been widely reported

that initial moisture of culture medium strongly influences enzyme production under

SSF conditions due to substrate utilization by the microorganism (Chhaya and Gupte,

2013). In this study, maximal laccase production (23 U mg−1) occurred at initial

moisture 50%, and reduced in the presence of higher moisture contents (Fig. S3d).

Similar results in effectiveness of initial moisture on laccase production were observed

using cultures of Schyzophylum commune (Irshad et al., 2011) and Fusarium

incarnatum (Chhaya and Gupte, 2013). The initial moisture content of the solid

substrate can have a major impact on enzyme production in SSF systems as well. Too

high or too low moisture levels may decrease enzyme production by limiting oxygen

and nutrient accessibility, respectively (Pandey, 2003). Moisture level in SSF varies

between 30% and 85%, and the optimal moisture content for growth of the most fungi

and substrate utilization is between 40% and 70%. However, enzyme production at

higher moisture levels is believed to reduce the porosity of the substrate and to limit the

transfer of oxygen and growth within whole substrate (Chhaya and Gupte, 2013).

Overall, a strong increase in enzyme production was observed at 7 days after incubation

by applying the conditions obtained above, i.e. pH 6.0, temperature 30 °C, 3×1 cm2

inoculum size, 50% initial moisture, <500 μm particle size of pistachio shell, 0.5 mM

2,5-xylidine, 10 mM CuSO4, and 0.2% glucose. As the industrial standpoint, high

expression level reached in the relatively optimized conditions shows the scalability of

enzyme production.

18

Fig. S3. The effect of (a) inoculum size, (b) glucose concentration, (c) pistachio shell

particle size and (d) initial moisture on laccase production by L. tigrinus. Error bars

represent standard deviations of the means (n=3). Asterisks denote the most appropriate

point with the statistically significant difference from the control (at a minimum of

p<0.05).

S2.3. Enzyme affinity purification

The purification results are given in Table S1. These data are comparable to

those obtained from a few studies on laccase purification using affinity strategies. For

example do Rosário Freixo et al. (2012) applied an affinity chromatographic matrix

19

Time (day)

0 2 3 4 5 7 9 10 12 13 14 16

Laccase productiom (U

mg

-1 protein)

0

5

10

15

20

251×1 cm2 plaque2×1 cm2 plaque3×1 cm2 plaque

Initial mouisture (% w/w)

30 40 50 60 70 80

Laccase productiom (U

mg

-1 protein)

0

5

10

15

20

25

Glucose concentration (% w/v)

0.1 0.2 0.5 1 2

Laccase productiom (U

mg

-1 protein)

0

5

10

15

20

25

Particle size (um)<500 um

500-1000 um

1000-1500 um

1500-2000 um

>2000 um

Laccase productiom (U

mg

-1 protein)

0

10

20

30

40

*

*

*

*

d)c)

b)a)

containing urea as an affinity ligand for affinity purification of laccase and obtained

final enzyme recovery of approximately 60%, specific activity of 18.0 U mg−1 protein,

and a purification factor of 46.0.

Laccase from L. tigrinus was a monomeric protein like the majority of fungal

laccases with almost the same molecular mass (about 65 kDa), such as laccases from

Pleurotus ostreatus (Patel et al., 2014), Schyzophylum commune (Irshad et al., 2014),

Pleurotus ferulae (Ding et al., 2014), Aspergillus ochraceus (Telke et al., 2010),

Trametes pubescens (Si et al., 2013), Trametes trogii (Zouari-Mechichi et al., 2006),

Hericium coralloides (Zou et al., 2012), Clitocybe maxima (Zhang et al., 2010), and

Coltricia perennis (Kalyani et al., 2012).

S2.4. Enzyme immobilization

Optimum conditions for laccase immobilization can be seen in Fig. S4.

20

Fig. S4. The effect of (a) enzyme quantity, (b) attachment time, and (c) pH-temperature

on the yield of laccase immobilization. Error bars represent standard deviations of the

means (n=3). Asterisks denote the most appropriate point with the statistically

significant difference from the control (at a minimum of p<0.05).

21

Table S1. Purification summary of laccase produced by L. tigrinus.

Purification step Volum

e (mL)

Total

activity

(U)

Total

protein

(mg)

Specific

activity

(U mg−1)

Yiel

d

(%)

Purificatio

n (fold)

Crude enzyme 15 1428.1 62.9 22.7 100.

0

1.0

Dialysed

precipitate

1 699.6 14.5 48.2 49.0 2.12

Affinity

chromatography

0.5 598.2 1.1 543.6 41.9 23.9

S2.5. Free and immobilized enzyme characterization

S2.5.1. Redox potential

Progressive reduction of the fully oxidized T1 copper was followed

spectroscopically using disappearance of the absorption at 608 nm. Under experimental

conditions, the E° value determined for enzyme was 0.72 V vs. Ag|AgCl electrode (Fig.

S5). As low, medium, and high potential laccases have the E° values <0.43 V, 0.47‒0.71

V, and >0.71 V, respectively, laccase from L. tigrinus can classify as a high redox

potential laccase (Singh et al., 2015). This value is consistent with those previously

reported for fungal laccases.

22

E (V)

-0.5 0.0 0.5 1.0 1.5

-10

0

10

20

Figure S5. Cyclic voltammetry of the purified laccase from L. tigrinus.

S2.5.2. Optimal conditions for free and immobilized enzyme activity

Optimal conditions for laccase activity with ABTS were obtained at pH 4.5, 35‒

40 °C, and 10 min reaction time for free enzyme and pH 4.5, 40‒45 °C, and 15 min

reaction time for immobilized enzyme (Fig. S6). The free enzyme showed more than

75% of its optimal activity at the temperature range 25 °C to 45 °C, when pH was

adjusted at 4.5 (Fig. S6a). The lowest activity of free enzyme was obtained at pH 6.0

and 45 °C with only 0.13% of original activity and at pH 6.0 and 20 °C with more than

50% of original activity for immobilized enzyme (Fig. S6b). The pH-temperature profile

of free laccase was similar to that observed for laccase from Pleurotus ostreatus (El-

Batal et al., 2015) that showed high activity toward ABTS occurring at a pH of 5.0 and

40 °C. However, contradictory results were published by Wu et al. (2010), in which

Fusarium solani produced a laccase showing maximal activity at 70 °C and a pH of 3.0.

Laccase from Pleurotus sajor-caju also showed its maximal activity at pH 5.0 and 40

°C. Maximal activity of laccase from Trametes pubescens occurred at 50 °C and a pH of

23

5.0 (Si et al., 2013). In another study, a laccase from Trametes versicolor reported by

Bertrand et al. (2015) demonstrated an optimal pH and temperature of 6.0 and 50 °C,

respectively. Zhang et al. (2010) also reported a fungal laccase from Clitocybe maxima

with an optimal activity at pH 3.0 and 60 °C and a thermostable laccase from Polyporus

sp. reported by Guo et al. (2011) showed its maximal activity at 75 °C and pH 4.0. By

assessing influence of time on oxidation rate of substrate, the maximal laccase activity

against 1 mM ABTS occurred after 10 and 15 min incubation in with 5 U free and

immobilized laccase, respectively (Fig. S6c).

Fig. S6. (a, b) Optimal pH-temperature and (c) reaction time for L. tigrinus free and

immobilized laccase activity. Error bars represent standard deviations of the means

(n=3). Asterisks denote the most appropriate point with the statistically significant

difference from the control (at a minimum of p<0.05).

24

S2.5.3. pH- and thermal- stability

As shown in Fig. 3a, b, both free and immobilized laccases from L. tigrinus

were highly stable over broad ranges of temperature (25–55 °C) and pH (2.0–11.0),

with an optimum at 25 °C and pH 5.0 for both forms. The thermal stability profile

indicated that free enzyme retained more than 70% of its initial activity at 25–55 °C (pH

5.0) after 6 h of incubation. Free enzyme retained about 50% of its activity at 55 °C

when the pH was at the range 4.0–7.0. In addition, the pure laccase displayed high

stability in the pH range 2.0–11.0 when the temperature was 25‒35 °C, although

enzyme activity was gradually reduced to 30% after treatment at temperatures above 45

°C. The pH-temperature stability of immobilized laccase was improved compared with

that of free enzyme at all the tested pH and temperature values. Similar results were also

observed for other fungal laccases from Fusarium solani (Wu et al., 2010), Trametes

torgii (Zouari-Mechichi et al., 2006), and Pycnoporus sanguineus (Ramírez-Cavazos et

al., 2014), Trametes torgii (Grassi et al., 2011), Cerrena sp. (Yang et al., 2016), and

Cladosporium cladosporioides (Halaburgi et al., 2011). In addition, 10 cycles of freeze-

thaw stability testing showed that free and immobilized enzyme retained more than 70%

and 90% of their initial activity, respectively (Fig. S7a). Incubations performed at room

temperature and a pH of 4.5 demonstrated that free and immobilized laccase did not

show more than 27% and 11% reduction in their activity for at least 45 days,

respectively (Fig. S7a). Overall, pH- and thermal-stabilities measured for this laccase in

both free and immobilized forms were higher than those reported for most other

mesophilic fungal laccases.

25

Fig. S7. (a) Efftect of storage and freeze/thaw cycles, (b) repeated use, and (c, d)

inhibitors on the stability of free and immobilized enzyme. Error bars represent standard

deviations of the means (n=3).

S2.5.4. Stability with organic solvents and other additives

The stability of free and immobilized enzyme in different organic solvents is

shown in Table S2. From ten solvents and one IL tested in this study, significant

deactivation of free enzyme was observed with 90% acetone and isopropanol (less than

20% of initial activity retained after 6-h incubation), which was probably due to the

stripping-off of the crucial bound-water monolayer essential for enzyme activity from

26

the macromolecule (Samaei-Nouroozi et al., 2015). Interestingly, ethyl acetate, butanol,

and toluene at 90% concentration enhanced free and immobilized enzyme activity up to

167%. At low concentration of 10%, all the solvents tested had no significant effect on

the stability of free enzyme. [Bmim][PF6] at a concentration of 10% had a strengthening

effect on stability and activity of both forms. Similar behavior was observed for other

laccases that were stable or unstable in organic solvents. Wu et al. (2010) have reported

a laccase from Fusarium solani with an organic solvent stability that showed enhanced

activity with 5% methanol, ethanol, and dimethyl sulfoxide (DMSO). A fungal

thermostable laccase characterized from Pycnoporus sanguineus has also shown high

stability in ethanol, acetonitrile, and acetone (Ramírez-Cavazos et al., 2014). Cerrena

sp. has also been reported to produce a stable laccase with stability against different

solvents of methanol, ethanol, acetonitrile, DMSO, and dimethylformamide (DMF)

(Yang et al., 2016). Another study by Yang et al. (2013) showed a laccase produced by

Shiraia sp. was stable against methanol, ethanol, isopropanol, acetone, heptane, hexane,

and t-butanol.

27

Table S2. Organic solvent stability of soluble and immobilized laccase from L. tigrinus

after 6 h incubation at 25°C. Asterisks show the significance level of the difference

between the means of the treatments and the controls as defined in the text.

Organic solvent Log P Relative residual activity (%)

10% (v/v) 50% (v/v) 90% (v/v)

FE IE FE IE FE IE

Control - 100 100 100 100 100 100

[Bmim][PF6] −2.39 120.1*** 109.

4**

85.3*** 100ns 71.4*** 96.7ns

DMSO −1.30 88.9* 98.7ns 51.0*** 91.2* 4.3*** 76.9***

1,4-Dioxane −1.10 83.7*** 95.4ns 78.0*** 90.5* 13.4*** 77.5***

Methanol −0.76 89.1* 98.8ns 27.5*** 88.2** 6.6*** 64.3***

Acetonitrile −0.33 81.4*** 90.6** 38.2*** 81.8*** 6.8*** 60.7***

Ethanol −0.24 87.2** 98.1ns 27.2*** 92.5ns 6.5*** 70.6***

Acetone −0.23 85.4** 96.5ns 14.1*** 90.7* 2.4*** 53.3***

Isopropanol 0.05 86.9** 97.5ns 17.5*** 88.8** 5.9*** 60.8***

Ethyl acetate 0.68 113.6** 106.5ns 128.5*** 109.9* 167.5*** 122.6***

Butanol 0.80 103.8ns 100ns 126.5*** 106.

6ns

137.2*** 119.6***

Toluene 2.5 101.5ns 100ns 109.5*** 108.7* 136.2*** 120.5***

FE= Free enzyme; IE= Immobilized enzyme

28

The effect of ions on laccase activity is shown in Table S3. For concentrations

up to 10 mM, only Fe2+ and Fe3+ showed significant inhibitory effects on free laccase,

and Li+ and Mn2+ at a concentration of 10 mM caused less than 50% inhibition on the

activity of free enzyme. Cu2+, Ca+2, Co+2, As5+, Ni+2, Bi3+, Zn+2, Mo+2, and I− produced an

increase in the activity of free laccase from 135% to 195% compared to that attained

with no ions. Other ions tested had no significant effect on enzyme activity and stability.

Free enzyme showed a particular sensitivity to Ca+2, Co+2, Ni+2, Zn2+ and Mo+2. The

enzyme from Clitocybe maxima had enhanced activity with Fe3+ (Zhang 1t al. 2010).

Mo2+, Ni2+, and Mn2+ also had an activation effect on a laccase from Trametes trogii

(Zouari-Mechichi et al. 2006). Forootanfar et al. (2011) reported a fungal laccase with

increased stability with Cu2+. Daâssi et al. (2013) reported a laccase from Trametes sp.

that was activated with Cu2+ and slightly inhibited with Fe2+, Zn2+, Cd2+, and Mn2+. In

another studies, thermostable laccases from Cladosporium cladosporioides (Halaburgi

et al., 2011) and Trametes versicolor (Zhu et al., 2011) were stable with the tested

metals.

29

Table S3. Stability of free and immobilized laccase from L. tigrinus against three

concentrations of different ions after 6 h incubation at 25 °C. Asterisks show the

significance level of the difference between the means of the treatments and the controls

as defined in the text.

Ions

Concentration

1 mM 5 mM 10 mM

FE IE FE IE FE IE

Control 100 100 100 100 100 100

Al3+ 106.8*** 101.7ns 106.4** 100.0ns 106.1** 100.0ns

As5+ 126.0*** 111.6*** 130.6*** 119.4*** 157.0*** 148.5***

B3+ 101.1ns 100.0ns 84.4*** 99.8ns 78.4*** 98.5ns

Ba2+ 99.9 ns 100.0ns 79.8*** 98.4ns 67.3*** 97.7ns

Bi3+ 119.3*** 110.3*** 130.9*** 118.2*** 153.8*** 139.1***

Br− 150.0*** 137.5*** 121.5*** 135.5*** 110.1*** 136.0***

Ca2+ 135.9*** 124.5*** 151.5*** 145.4*** 188.9*** 167.4***

Co2+ 130.8*** 127.6*** 135.8*** 130.5*** 163.8*** 150.5***

Cu2+ 143.4*** 131.7*** 195.3*** 157.7*** 119.7*** 164.3***

F− 92.3*** 112.3*** 83.0*** 111.4*** 67.9*** 111.6***

Fe2+ 57.9*** 82.1*** 24.5*** 81.9*** 0.9*** 75.5***

Fe3+ 49.8*** 86.8*** 27.2*** 86.0*** 11.0*** 83.2***

I− 126.2*** 118.7*** 134.9*** 125.6*** 105.7** 126.1***

K+ 85.7*** 98.8ns 81.2*** 91.2*** 56.5*** 90.1***

Li+ 93.6** 100.0ns 55.7*** 99.1ns 55.0*** 93.3**

Mg2+ 100.7ns 109.5*** 106.4** 115.3*** 115.2*** 120.1***

Mn2+ 97.2ns 108.4*** 67.1*** 107.1** 58.2*** 107.0**

30

Mo+ 119.8*** 112.5*** 143.5*** 132.2*** 115.8*** 140.1***

Na+ 114.5*** 109.5*** 94.2* 109.1*** 91.2*** 108.0**

Ni2+ 115.3*** 110.0*** 117.2*** 121.5*** 153.8*** 140.5***

Zn2+ 108.5*** 113.5*** 123.5*** 128.3*** 148.5*** 135.4***

FE= Free enzyme; IE= Immobilized enzyme

Amongst ions tested, only Fe2+ and Fe3+ had a reducing effect on the free laccase

activity (Table S3), while immobilized enzyme was nearly complete stable with all ions

and lost less than 25% of its initial activity with Fe2+. Particular sensitivity of laccase

from L. tigrinus to ions might be related to favorable or unfavorable chainging the

conformation of enzyme. The activation of the enzyme by Cu+2 may be happened due to

filling the type-2 copper binding sites with copper ions.

After incubating the free and immobilized enzyme with various inhibitors, the

activity of free enzyme was found to be inhibited with all inhibitors tested, while

immobilized enzyme maintained fully stable and active (Figs. S7c and S7d). A

significant activity loss was observed when free enzyme was incubated with 1,4-DTT,

NaN3, H2O2, 2-ME, and L-cysteine at all concentrations tested (Fig. S7c). These

findings are in consonance with Asgher et al. (2017) which showed the alginate-

chitosan immobilized laccase was more tolerant against the activity inhibition caused by

EDTA, cysteine, and several metal ions than the free enzyme in solution. Free laccases

from Pleurotus sajor-caju (Murugesan et al. 2006), and Fusarium solani (Wu et al.

2010) also showed similar behaviors. Similar behavior was also observed for purified

laccases from Pleurotus ostreatus (Patel et al., 2014), Aspergillus ochraceus (Telke et

al., 2010), Paraconiothyrium variable (Forootanfar et al., 2011), and Cladosporium

31

cladosporioides (Halaburgi et al., 2011). Reducing agents of 2-mercaptoethanol (2-

ME), 1,4-dithiothreitol (1,4-DTT), oxalic acid, and H2O2 inhibit the enzyme by affecting

the important sulfhydryl groups required for enzyme activity. Iinhibition activity of

kojic acid is by reducing Cu II to Cu I. The thiol group of cysteine as a strong

nucleophile can disrupt enzyme action. Ethylenediaminetetraacetic acid (EDTA) as a

chelating agent and phenylmethylsulfonyl fluoride (PMSF) as a serine modifier can

result in activity loss of enzymes. NaN3 as a common metalloenzyme inhibitor binds to

Cu ions in laccase and deactivates the enzyme. Thiourea is also able to reduce Cu ions

interfering laccase activity (Rezaei et al. 2017).

The effect of Tween 80, Triton X-100, CTAB, and SDS on enzyme stability was

also investigated. All surfactants tested showed enhancement effects on both free and

immobilized laccases stability to a maximum of 223% with 5 mM CTAB as an ionic

surfactant (Figs. 3c and 3d). In concordance with these results, the addition of SDS to

the enzyme mixture in the investigation of Wu et al. (2010) led to enhanced activity of

laccase. In contrast to the results on surfactant stability of laccase from L. tigrinus,

Halaburgi et al. (2011), Zhu et al. (2011), and Yang et al. (2013) reported decreased

enzyme activity for three laccases from Cladosporium cladosporioides, Trametes

versicolor, and Shiraia sp. with SDS.

S2.5.5. Substrate specificity

In this study, after qualitatively substrate screening of laccase from L. tigrinus,

purified laccase showed activity toward 30 out of 38 substrates tested (Table S4). No

color change was detected for non-phenolic substrates of biphenyl and 1,4-

dichlorobenzene. For 4-nitrophenol with -NO2 substitution, no activity was also

32

observed. 5-bromosalicyl aldehyde with a halogen substitution was not oxidized. These

results are consistent with the data obtained by Reiss et al. (2011) and Rezaei et al.

(2017). Important factors in the rate of laccase-catalyzed reactions are the redox

potential difference between substrate and enzyme, pH-temperature, enzyme-substrate

ratio, size and structure of substrate, and substitution pattern of the substrate (Rezaei et

al., 2017).These results indicated that the purified laccase from L. tigrinus has a broad

substrate spectrum.

33

Table S4. Substrate specificity of laccase produced by L. tigrinus.

Substrate No. Substrate Enzyme assay

1 ABTS +

2 2-Aminobiphenyl +

3 p-Aminoanisole +

4 Biphenyl −

5 5-Bromosalicyl aldehyde −

6 Catechol +

7 p-Cresol +

8 1,4-Dicholoro benzene −

9 2,3-Dicholorophenyl +

10 2,4-Dicholoro phenyl +

11 2,6-DMP +

12 L-DOPA −

13 Gallic acid +

14 Guaiacol +

15 1-Hydroxybenzotriazole (HOBT) +

16 Hydroquinone +

17 4-Hydroxybenzoic acid +

18 2-Methyl-1,4-naphthoquinone +

19 3-Methoxysalicyl aldehyde +

20 4-Methoxy salicyl aldehyde +

21 α-Naphtol +

22 4-Nitrophenol −

23 5-Nitrosalicyl aldehyde +

34

24 Phenol +

25 Phenylenediamine +

26 Pyrogallol +

27 Resorcinol +

28 Salicyl aldehyde +

29 Syringaldazine +

30 Tannic acid +

31 TEMPO +

32 o-Toluidine +

33 p-Toluidine −

34 3,4,6-Tricholoro phenol +

35 Tyrosine +

36 Vanillin +

37 Veratryl alcohol −

38 2,5-Xylidin +

S2.5.6. Kinetics

Laccase from L. tigrinus followed a classical Michaelis-Menten kinetics for 10

substrates used, with Km, Vmax, Kcat, and Kcat/Km values. As shown in Table S5, the values

for Km and Vmax obtained for enzyme showed that laccase from L. tigrinus had a greater

affinity toward ABTS and pyrogallol with a Km of 2.1 and 10.5 μM, respectively. This

enzyme also presented high efficiency with Kcat values of 492, 480, 472, 448, and 432

sec−1 toward pyrogallol, gallic acid, ABTS, hyroquinone, and 2,6-DMP, respectively.

Indeed, the most efficient catalysis resulted for substrates of ABTS, pyrogallol, 2,6-

35

DMP, and SGZ. These kinetic parameters values were in the range of those found for

other laccases of fungi (Baldrian, 2006).

Table S5. Kinetic parameters of laccase produced by L. tigrinus.

Substrates Wavelength

(nm)

Kinetics

Km

(μM)

Vmax (μkatal

mg−1)

Kcat

(Sec−1)

Specificity

constant (Sec−1

μM−1)

ABTS 420 2.1 11.8 472.0 224.8

Catechol 450 57.2 10.7 428.0 7.5

2,6-DMP 470 24.7 11.3 452.0 18.3

Gallic acid 250 51.4 12.0 480.0 9.3

Guaiacol 470 36.2 7.6 304.0 8.4

Hydroquinone 525 61.8 11.2 448.0 7.2

Pyrogallol 450 10.5 12.3 492.0 46.9

SGZ 530 21.6 5.4 216.0 10.0

Tannic acid 250 95.8 2.1 84.0 0.9

2,5-Xylidin 505 71.9 3.7 148.0 2.1

S2.6. Delignification results

S2.6.1. Effect of enzyme dosage and reaction time

Incubation time and enzyme dosage can play a substantial role in maximizing

the yield of a reaction. In this study, the observed delignification rate quite depended on

enzyme dosage and specially reaction time.

36

As shown in Table 1, 50, 75, and 100 U mL−1 of enzyme resulted in 25%, 55%,

and 75% delignification yield for free form and 13%, 43%, and 44% for immobilized

form, respectively after 1 h incubation. The lignin was removed up to 50%, 75%, and

85% after only 2 h of incubation when the concentrations of free enzyme were 50, 75,

and 100 U mL−1, respectively and these results for immobilized laccase were 30%, 63%,

and 67%, respectively with equal quantities of enzyme. By increasing incubation time

up to 6 h, delignification efficiency increased to 85%, 95%, and 100% for soluble form

and 67%, 85%, and 90% for immobilized form, respectively. These results imply that

time is a more effective factor than enzyme dosage. However, enzyme dosage of 75 U

mL−1 and 1 h reaction time were selected to apply in subsequent experiments.

S2.6.2. Effect of pH, temperature, and aeration

As shown in Fig. 4b, laccase from L. tigrinus could delignificate the bio-waste

after 2 h incubation at 20–45 °C and pH 3.0–6.0 with a maximal yield (80%) at 35 °C

and pH 4.5 for soluble enzyme and a maximal yield (74%) at 40 °C and pH 4.5 for

immobilized enzyme related to optimal conditions of enzymes activity. Delignification

of pistachio shell varied with pH and temperature similarly to that observed for enzyme

activity. In addition, the maximal delignification activity (56% and 43% for free and

immobilized enzyme, respectively) was occurred at an aeration of 80%‒90% (v/v)

(Table 2).

S2.6.3. Effect of different additives

37

Specific features of enzyme, such as a great stability with several surfactants,

organic solvents, and [Bmim][PF6] as a hydrophobic ionic liquid were used to design of

experiments for testing the effect of several additives on the yield of delignification. All

tested surfactants, organic solvents, and [Bmim][PF6] had a positive effect on the

efficiency of process, but the most important effect (73% delignification) was observed

when the reaction was conducted with free enzyme and 5 mM CTAB (69%

delignification) followed by SDS at the same concentration (Table 3). As it shown in

Table 3, among all solvents tested, the treatment of the bio-waste with laccase and 10%

[Bmim][PF6] resulted in a maximal improvement in delignification yield (65% and 54%

for free and immobilized state, respectively). Delignification efficiency was around 6%

after 1 h from the incubation of lignocellulose with [Bmim][PF6] alone. Rezaei et al.

(2017) obtained a notable delignification yield of 78.4% when enzymatic delignification

was conducted in 50% [Bmim][PF6] by a stable laccase from a halophilic bacterium. In

study of Rezaie et al. (2017) except for CTAB and Triton X-100, other additives

including solvents, surfactants, mediators, and [Bmim][PF6] increased the efficiency of

delignification reaction.

Among all mediators investigated, a more remarkable effect on delignification

yield was observed in with 10 mM TEMPO by a delignification efficiency of 69%

followed by 68%, 63%, and 62% yield obtained using 5 mM TEMPO, 10 mM gallic

acid, and 1 mM TEMPO, respectively (Table 4). Best yields of delignification with

immobilized enzyme obtained with 10 mM TEMPO (65%), 5 mM TEMPO (64%), 10

mM GA (63%), 1 mM TEMPO (62%), and 10 mM HOBT (60%). Other mediators

applied had also an incremental effect on process efficiency.

38

S2.6.4. Physicochemical analysis of treated pistachio shell

SEM images of pistachio shells before and after enzymatic treatment are shown

in Fig. 5. The images show that surface structure in the control is rigid and highly

ordered and the structure is distorted after pretreatment. The changes are due to the

degradation of lignin after enzymatic pretreatment, increasing the surface area of

cellulose, and making it more accessible to use. Starting composition of pistachio shell

and the changes in composition after enzymatic treatment are shown in Fig. 6. Some

differences, such as lower lignin content and higher insoluble ash, hemicellulose, and

cellulose are resulted from a successive delignification treatment. Among different shell

fractions, cellulose and lignin showed the largest proportionate changes.

Table S6. GC-mass analysis for products of pistachio shells delignification with free

laccase.

No. Compound Retention time (min)

1 Methylene chloride 1.6

2 2-Butanone 1.8

3 Ethyl propanoate 2.4

4 Unidentified compound 2.8

5 1-Methylpropylacetate 2.9

6 N-(2-Aminoethyl)acetamide 3.8

7 Unidentified compound 4.6

8 Syringol 13.0

9 Unidentified compound 13.2

10 Vanillin 14.1

39

11 Acetovanillone 15.2

12 4-Ethylsyringol 17.5

13 Syringaldehyde 18.4

14 Acetosyringone 20.5

15 Synapaldehyde 20.6

16 Unidentified compound 24.6

The identification of the main products of decomposition based on mass spectral

libraries, literature data, and their intensity are listed in Table S6 and Fig. S8. As shown

in Table S6 and Fig. S8, both carbohydrate and lignin products derivatives are produced

after enzymatic treatment of bio-waste. Although the main decomposition product of

cellulose, i.e. 1,6-anhydro-β-D-glucopyranose, was not detected, other carbohydrate

products derivatives, are present. Significant amount of smaller molecular products,

mostly aldehydes, ketones, and acetic acid derivatives are also released from

polysaccharide molecules. Degradation of lignin produces mainly aromatic products

that can be guaiacol or syringol derivatives. In this study, lignin degradation produced a

high yield of acetosyringone, syringol, 4-ethylsyringol, vanillin, syringaldehyde,

acetovanillone, and synapaldehyde which are valued products from enzymatic

delignification.

40

Fig. S8. GC-MS spectrum of delignification products from laccase-treated

pistachio shells.

Additional references

Adrangi, S., Faramarzi, M.A., Shahverdi, A.R., Sepehrizadeh, Z., 2010. Purification and

characterization of two extracellular endochitinases from Massilia timonae.

Carbohydr. Res. 345, 402–407.

Asgher, M., Wahab, A., Bilal,M., Iqbal, H.M., 2017. Delignification of Lignocellulose

Biomasses by Alginate-Chitosan Immobilized Laccase Produced from Trametes

versicolor IBL-04. Waste Biomass Valor., 1‒9.

Baldrian, P., 2006. Fungal laccases-occurrence and properties. FEMS Microbiol. Rev.

30, 215‒242.

Bertrand, B., Martínez-Morales, F., Tinoco-Valencia, R., Rojas, S., Acosta-Urdapilleta,

L., Trejo-Hernández, M.R., 2015. Biochemical and molecular characterization of

laccase isoforms produced by the white-rot fungus Trametes versicolor under

submerged culture conditions. J. Mol. Catal. B: Enzym. 122, 339–347.

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