Skip to content

Advertisement

  • Original article
  • Open Access

Lyoprotection and stabilization of laccase extract from Coriolus hirsutus, using selected additives

AMB Express20188:152

https://doi.org/10.1186/s13568-018-0683-3

  • Received: 23 March 2018
  • Accepted: 24 September 2018
  • Published:

Abstract

The development of stable lyophilized laccase, obtained from Coriolus hirsutus, using a wide range of temperature treatments and storage conditions, was investigated. Using selected lyoprotectants, including, dextran 6 kDa, sucrose and a mixture of sodium benzoate, potassium sorbate and sorbitol (BKSS) (1.5:1.0:98.5; w/w/w) resulted by 2.4, 1.4 and 1.8-fold increase in laccase activity after lyophilization as compared to the fresh enzyme, respectively, whereas the addition of mannitol preserved 98.2% of its activity. Using 2.5% (w/v) dextran (15–25 kDa) or mannitol appeared to be the most appropriate lyoprotectants for the laccase activity. The laccase stability of the lyophilized enzymatic extract was greatly enhanced with the presence of mannitol, with 96.2, 38.9 and 24.7% of residual activity after 4 weeks of storage at − 80, 4 and 25 °C, respectively. The inactivation constant (kinactivation) value and the amount required to decrease 50% of the laccase activity (C1/2) showed that Carbowax® polyethylene glycol (PEG)-8000 was the most appropriate additive for laccase activity, followed by glycerol and CuSO4. When the enzymatic extract was incubated at 50 °C in the presence of either CuSO4, PEG-8000 or glycerol, the time required to decrease 50% of the laccase initial activity (t50), were 52.9, 54.6, 50.2 h, respectively, as compared to that of the control trial of 38.9 h.

Keywords

  • Laccase
  • Coriolus hirsutus
  • Stabilization
  • Lyoprotectants
  • Additives

Introduction

Laccases (EC 1.10.3.2) are o- and p- diphenol: dioxygen-oxidoreductase (Dalfard et al. 2006). Laccases belong to the polyphenol oxidases that catalyze the oxidation of a large number of phenolic and non-phenolic compounds, with a parallel reduction of the molecular oxygen as the electron acceptor (Tarasi et al. 2018).

Laccases are widespread in nature and they are produced by plants, fungi, bacteria, insects and crustacean. Most laccases have been isolated from white rot basidiomycetes, since mostly fungal laccases were found as extra-cellular enzymes (Strong and Claus 2011).

The broad substrate specificity, the high catalytic rate and the ability of laccases to use the environmental oxygen as a co-factor, made them highly useful as biocatalysts in versatile biotechnological applications, including lignin biodegradation, biobleaching, detoxification, organic synthesis, biosensing and biografting (Freire et al. 2001; Gianfreda and Bollag 2002; Jeon and Chang 2013; Thakur et al. 2016; Tarasi et al. 2018; Yesilada et al. 2018; Agrawal et al. 2019; Rocha-Martín et al. 2018). As green catalysts, laccases have also been increasingly employed in various industrial, environmental and medical applications leading to rapid growth in the demand for these enzymes (Liu et al. 2016). Gap between production and demand was reported owing to their high production and purification cost as well as to their poor stability (Nunes and Kunamneni 2018).

Lyophilization, thermal treatments, reaction process, protease as well as laccase-catalyzed polymerization, depolymerization and detoxification reaction products could destabilize the enzyme (Chansanroj and Thanawattanawanich 2016). Various chemicals were employed to stabilize laccase. The results have shown that the enzyme activity and stability was enhanced in presence of polyethylene glycol (PEG) (Kim and Nicell 2006a, b; Modaressi et al. 2005; Ghosh et al. 2008), CuSO4 (Baldrian and Gabriel 2002; Papinutti et al. 2008) mannitol and glycerol (Papinutti et al. 2008). The use of polysaccharides, including guar gum, starch, agarose and agar, for the prevention of the laccase denaturation and retention of the enzyme activity for long period in solution state, was also reported (Poonkuzhali and Palvannan 2011; Poonkuzhali et al. 2011). Several additives were also employed to enhance the stability of the vacuum dried laccase including polyvinyl alcohol, dextran, lactitol and polyacrylic (Stepanova et al. 2003).

Although the literature reported on the stability of laccase in relation to the vacuum drying (Stepanova et al. 2003), spray drying (Liu et al. 2016), thermal treatment (Papinutti et al. 2008), storage and biocatalysis (Kim and Nicell, 2006a, b; Papinutti et al. 2008); there is a wide range of discrepancy in the reported experimental findings especially due to the source, the nature of the enzyme and its application. Moreover, to our knowledge there is lack of information regarding the production of stable lyophilized laccase with high activity and extended shelf life.

The present work is part of ongoing research in our laboratory aimed at the biotechnological applications of laccase in non-conventional media. The aim of the present study was to prepare stable lyophilized powdered laccase with long-term conservation of its activity and high operating stability. The stabilization of laccase activity of the enzymatic extract, obtained from Coriolus hirsutus, after lyophilization using selected additives, was investigated. It was aimed to use the enzymatic extract prior purification to avoid further increase of the cost of process. The efficiency of selected lyoprotectants on laccase thermal and storage stability as well as the effect of selected additives on laccase activity and stability in the reaction media were also evaluated.

Materials and methods

Materials

Coriolus hirsutus (MYA-828) was obtained from the American Type Culture Collection (ATCC; Manassas, VA). The additives include KCl and mannitol (BDH Inc.; Toronto, ON), glycerol (MP Biomedicals; Solon, OH), sucrose and CuSO4 (ACP Chemical Inc.; Montreal, QC) as well as bovine serum albumin (BSA), dextran, sodium benzoate, sorbitol, potassium sorbate and the substrate syringaldazine (SG) (Sigma Chemical Co.; St. Louis, MO). The anhydrous ethanol was obtained from Commercial Alcohols Inc. (Branchville, QC). Other chemicals used for the preparation of reagents and buffers were purchased from Fisher Scientific (Fair Lawn, N.J.) and prepared in deionized water, using Milli Q plus (Millipore; Milford, MA).

Preparation of the enzymatic extract

As indicated by the ATTC protocol, C. hirsutus was maintained through periodic transfer onto malt agar media plates and incubated at 20 °C. For laccase production, a basal medium was prepared using the method described by Shin and Lee (2000). The fermentation of C. hirsutus as well as the recovery and enrichment of the enzymatic extract were carried out according to Taqi (2012). The production of laccase was induced with the addition of 50 mL ethanol/L of culture medium. After 14 days of incubation, the mycelium pellets were removed with cheese cloth and the extracellular enzyme recovered from the culture media was ultra-filtered through a Prep/Scale TFF Cartridge (2.5 ft2) (Millipore) of polyethersulfone low protein-binding membranes of 10 kDa cut-off filter and a pressure of 10 psi. All steps were performed at 4 °C unless otherwise stated.

Protein determination

The protein content of the enzymatic extracts was determined according to a modification of Lowry method (Hartree 1972), using BSA (Sigma Chemical Co.) as a standard for the calibration curve.

Enzyme assay

Laccase activity was performed following the optimized procedure described by Taqi (2012). Lyophilized laccase extract was suspended in sodium acetate buffer (0.1 M, pH 5.0). A 0.2 mL aliquot of enzyme suspension or the fresh enzyme extract (0.2–0.5 mg protein/mL) was added to a total 0.7 mL of the acetate buffer, containing 15 µL of 4.0 mM SG solution in ethanol. Laccase activity was assayed by measuring the initial oxidation rate of SG monitored at 525 nm (ε525, 65,000 cm−1·M−1), using DU 650 spectrophotometer (Beckman Instruments Inc.; San Raman, CA). One unit of laccase activity was defined as 1 mol of product per mL of enzyme per min. The enzymatic reaction was performed at 55 °C, otherwise mentioned. All laccase assays were performed in triplicate and run in tandem with control trials containing 15 µL of 4.0 mM SG, but without an enzyme extract, in a total volume of 0.7 mL.

Effect of selected lyoprotectants on laccase stability

Prior to lyophilization, selected lyoprotectants (w/v), including 0.5% BSA, 5% mannitol, 5% sucrose, 1% dextran 6 kDa, 70% (w/w) KCl or a 5% of the mixture of sodium benzoate, potassium sorbate and sorbitol (BKSS) (1.5:1.0:98.5; w/w/w), were added to the fresh ultra-filtered concentrated exo-laccase. The effect of dextran, with a molecular weight of 1.5, 6, 15–25, 40 and 70 kDa, as well as mannitol with a wide range of concentration (0–10%, w/v) on the stability of laccase was investigated and compared to the lyophilized enzyme without additive (control). After lyophilization, the enzymatic preparation was reconstituted in the acetate buffer and the laccase activity was assayed (Hall et al. 2008).

Effect of selected lyoprotectants on laccase thermostability

The effect of temperature on the laccase stability, prepared with 2.5% (w/v) dextran or mannitol, was investigated. The lyophilized laccase preparations were reconstituted in the acetate buffer and pre-incubated for 2 h in a wide range of temperatures, 4, 20, 30, 40, 45, 50, 55, 60 and 70 °C. In addition, the different laccase preparations were reconstituted in the acetate buffer and incubated in water-bath for 0–24 h at 4, 25 and 50 °C. The treated enzymatic extracts were immediately cooled down with the use of an ice-bath before measuring the laccase activity, using the standard assay.

Effect of selected lyoprotectants on laccase storage stability

The lyophilized enzymatic preparations, including that prepared with 2.5% (w/v) mannitol as well as with dextran 15–25 kDa, were stored at 4, 25 and − 80 °C for a period of 0–4 weeks. The laccase activity was determined each week, using the standard assay.

Effect of selected additives on laccase activity

The effect of selected additives, including CuSO4, Carbowax® PEG-8000 and glycerol, on laccase activity of the enzyme prepared with 2.5% (w/v) mannitol was investigated, by varying their concentrations in the reaction mixture from 0 to 10% (w/v), according to the procedure described by Hall et al. (2008). The laccase relative specific activity was calculated and defined as the concentration of oxidized syringaldazine (nmol product/mg protein/min) in the reaction trial containing the optimum additive concentration relative to that without additive. All trials were performed in triplicate and run in tandem with the control without additive. Relative percentage standard deviation was defined as the standard deviation of laccase triplicate trial divided by their respective means, multiplied by 100.

The inactivation rate of laccase activity was estimated by calculating the first-order inactivation constant (kinactivation) on semi logarithm plots. The amount (%, w/v) of the additive, required to decrease half of the initial laccase activity (C1/2), was calculated by using the linear equation:
$$\ln \;(\text{A}/\text{A}_{0} ) = - k_{\text{inactivation}} \text{C}$$
(1)
where, C is the concentration of the chemical additive; A and A0 are the laccase activities at C concentration of the chemical additive and without it, respectively.

The rate constant of inactivation (kinactivation), expressed as 1/% (w/v) of the additive per reaction volume, was estimated by plotting the ln percentage residual laccase activity versus the percentage of additive in the reaction trial for laccase. The C1/2, expressed in % (w/v) of the additive per reaction volume, defined as the additive concentration at which 50% of the initial activity was reached.

Effect of selected additives on laccase thermostability

The effect of selected additives, including CuSO4, Carbowax® PEG-8000 and glycerol, on laccase thermostability at 4, 25, 37 and 50 °C, was evaluated by pre-incubating the reconstituted lyophilized enzymatic extract. Samples were withdrawn after 7 days of incubation. The residual laccase activity was measured, using the standard assay. Residual laccase activity (%) was calculated by dividing the specific activity (nmol product/mg protein/min) of a given sample after 168 h of incubation at a defined temperature to that at time 0, multiplied by 100.

The thermal deactivation rate of laccase, at 50 °C, in presence of the selected chemical additives, was estimated by calculating the first-order deactivation constant (kt) on semi logarithm plots. Samples were withdrawn at different incubation times. The residual laccase activity was measured, using the standard assay. The thermal stability time (h) required decreasing 50% of the initial laccase activity (t50) was calculated using the equation:
$$t_{50} = \ln \;(2)/k_{\text{t}}$$
(2)

Statistical analysis

Data were expressed as means of triplicate trials and their respective standard deviation (SD). The percent relative standard deviation (RSD) was calculated as the SD divided by the mean multiplied by 100. Correlation analyses were performed, using the SigmaPlot-Systat Software V. 11 (Systat Software Inc., Chicago, IL). Data were analyzed, using PROC ANOVA analysis, performed with the same statistical program. A post hoc comparison was made, using Tukey’s test. Values of P < 0.05 were considered to be significant.

Results

Effects of selected lyoprotectants on laccase stability

The effects of selected lyoprotectants, including BSA, mannitol, sucrose, dextran 6 kDa, KCl and the mixture BKSS, were investigated in terms of their efficiency to maintain the laccase activity during the lyophilization of the enzymatic extract and to enhance its solubility. Although Table 1 shows that the residual laccase activity of the lyophilized enzymatic extract of the control trial was 92.1%, the solid protein was not easily reconstituted or manipulated. The addition of 70% (w/w) of KCl inhibited completely the laccase activity, whereas the presence of 0.5% (w/v) BSA resulted by 17.3% of the residual enzyme activity. The results also show that the addition of 5% (w/v) mannitol maintained 98.2% of the original laccase activity. In addition, the laccase activity of the lyophilized enzymatic extract was determined to be 2.4, 1.4 and 1.8-fold higher than that of the fresh one, in the presence of 1% dextran 6 kDa, 5% sucrose and 5% of the mixture BKSS, respectively. The addition of BSA, mannitol, dextran 6 kDa and KCl resulted by a higher solubility of the enzymatic extract, whereas the use of sucrose and the mixture BKSS resulted in a collapsed crystalline formulation of poor solubility.
Table 1

Effect of different lyoprotectants on laccase stability, nature of the extract and its solubility

Extract

Residual laccase activity (%)a

Nature of extractb

Solubilityc

Fresh without additive

100.0

Liquid

n.d.

Lyophilized without additive

92.1 (6.2)e

Sticky

+

Lyophilized with 0.5% (w/v) BSA

17.3 (11.6)

Powder

+++

Lyophilized with 5% (w/v) mannitol

98.2 (2.3)

Powder

+++

Lyophilized with 1% (w/v) dextran 6 kDa

241.0 (5.4)

Powder

+++

Lyophilized with 5% (w/v) sucrose

141.2 (14.0)

Collapsed

Lyophilized with 5% (w/v) mixture (BSKS)d

184.3 (2.5)

Collapsed

++

Lyophilized with 70% (w/w) KCl

n.d.

Powder

+++

n.d. Not detected

aResidual laccase activity (%) was defined as the specific activity (nmol product/mg protein/min) of the lyophilized trial in comparison to that of the fresh ultrafiltrated enzymatic extract before lyophilization without additive

bNature of the extract was defined as the final texture of the lyophilized enzymatic

cDegree of solubility of the enzyme preparation in sodium acetate buffer (0.1 M, pH 5.0), after lyophilization, was expressed with qualitative evaluation +++, ++, + and − from the highest solubility to the lowest one, respectively, taking into consideration the required time for solubilization

dMixture was composed of sodium benzoate/potassium sorbate/sorbitol (BKSS) (1.5:1.0:98.5, w/w/w)

eRelative percentage standard deviation was defined as the standard deviation of laccase triplicate trial divided by their respective means, multiplied by 100

Effect of dextran molecular weight on laccase stability

The effect of dextran molecular weight (1.5, 6, 15–25, 40 and 70 kDa) on the laccase stability of the lyophilized enzymatic extract was investigated. The results (Fig. 1) show that the use of dextrans 15–25 or 6 kDa resulted in a significant increase in laccase activity by 2.6 and twofold, respectively, as compared to the lyophilized extract trial without additive. However, using dextrans 1.5, 40 and 70 kDa resulted in a residual laccase activity of 47.0, 50.1 and 73.1%, respectively.
Fig. 1
Fig. 1

Effect of dextran molecular weight on the residual laccase activity after lyophilization, using syringaldazine as substrate. The residual laccase activity was defined as the specific activity of lyophilized enzyme to that of the lyophilized enzyme trial that is lacking the additive (Control)

Effects of dextran and mannitol concentrations on laccase stability

The effects of dextran (15–25 kDa) and mannitol concentrations were investigated in term of their efficiency to stabilize the laccase activity during lyophilization. Figure 2 indicates that the addition of 2.5% (w/v) dextran (15–25 kDa) resulted by a 2.1-fold activation of laccase activity; whereas it was increased from 80 to 102%, with the increase in mannitol concentration, from 1 to 10%.
Fig. 2
Fig. 2

Effect of the lyoprotectant concentration (%, w/v) of dextran 5 to 25 kDa (black circle) and mannitol (white circle) on the residual laccase activity. The residual laccase activity was defined as the specific activity of lyophilized enzyme to that of the lyophilized enzyme trial that is lacking the additive

Thermostability of laccase

Effect of temperature on laccase stability

The effect of lyoprotectants on laccase thermostability was investigated. The results (Fig. 3) show that the different laccase preparations, including the control trial and those treated with dextran and with mannitol demonstrated similar thermostability profiles, where the enzyme retained its full activity after 2 h of incubation up to 50 °C.
Fig. 3
Fig. 3

Thermostability profile of the lyophilized laccase activity, after 2 h of incubation at different temperatures, assayed with syringaldazine as substrate. The reaction mixture contains the enzymatic preparations including the control being the lyophilized enzyme without additive (black circle) with 2.5% (w/v) mannitol (black down-pointing triangle) and 2.5% (w/v) dextran 15 to 25 kDa (black square) as additive

The energy required for the maximum activation of laccase of the enzymatic preparations, without any additive, as well as those with dextran or with mannitol, were of 26.9, 31.2 and 21.5 kJ/mol, with a coefficient Q10 of 1.4, 1.4 and 1.5, respectively (Data Not Shown). These findings suggest that the activity of laccase preparations using mannitol as lyoprotectant was the most prone to variation with the increase of temperature as compared to that with dextran and the control.

Effect of incubation time on laccase stability

The thermostability of laccase activity of the enzyme preparations, including the control trial and those treated with dextran and with mannitol, was investigated by incubating the reconstituted enzyme preparation in the acetate buffer at different temperatures and incubation times. The thermostability profiles (Fig. 4) show that the residual laccase activity, after 24 h of incubation at 4, 25 and 50 °C, for the control trial was 271, 277 and 98%, respectively; it was also 302, 240 and 78% with 2.5% (w/v) dextran (15 to 25 kDa) and 276, 256 and 89% with 2.5% (w/v) mannitol, respectively. The results also showed high thermostability of the laccase prepared with mannitol and the control when incubated at 25 and 50 °C for 1–4 h as compared to shorter incubation time.
Fig. 4
Fig. 4

The thermostability profiles of the residual laccase activity in the lyophilized enzymatic extract from Coriolus hirsutus, after incubation at a 4, b 25 and c 50 °C. The reaction mixture contains the enzymatic preparations including the control being the lyophilized enzyme without additive (black circle) with 2.5% (w/v) mannitol (black down-pointing triangle) and 2.5% (w/v) dextran 15 to 25 kDa (black square) as additive. The residual laccase activity (%) was defined as the specific activity of each treated enzyme extract at a defined temperature and time in comparison to that at time zero of each incubation temperature

Laccase storage stability

The stability of the lyophilized laccase extracts, prepared in the presence of 2.5% (w/v) dextran or mannitol and stored for different periods and temperatures, was investigated. The results (Table 2) indicate that mannitol was the most effective stabilizer, with 96.2, 38.9 and 24.7% of residual laccase activity after 4 weeks of storage at − 80, 4 and 25 °C, respectively; whereas in the absence of any additive, it decreased steadily to 56.6, 8.5 and 17.8% at − 80, 4 and 25 °C, respectively. In the presence of dextran, it decreased to 57.1, 19.8 and 0% after 1 week of storage at − 80, 4 and 25 °C, respectively.
Table 2

Long-term stability of laccase activity of the lyophilized enzymatic extract from Coriolus hirsutus, prepared with 2.5% (w/v) mannitol or dextran 15–25 kDa

Storage (week)

− 80 °C

4 °C

25 °C

Controlb

Mannitol

Dextran

Controlb

Mannitol

Dextran

Controlb

Mannitol

Dextran

Relative residual laccase activity (%)a

 0

100.0

100.0

100.0

100.0

100.0

100.0

100.0

100.0

100.0

 1

85.0

96.9

57.1

48.1

95.3

19.8

46.3

49.7

0.0

 2

78.6

95.8

50.6

28.2

28.3

21.0

24.4

25.6

0.0

 3

78.5

101.9

47.0

8.8

33.9

9.0

16.4

26.0

0.0

 4

56.6

96.2

38.5

8.5

38.9

4.4

17.8

24.7

0.0

aRelative residual laccase activity (%) was calculated by dividing the specific activity (nmol product/mg protein/min) of a given sample at defined storage time and temperature to that at time 0, multiplied by 100. The enzyme activity was assayed with syringaldazine as substrate

bControl was defined as the lyophilized enzyme without additive

Effects of concentrations of selected additive on laccase activity

Table 3 summarizes the experimental findings for the effects of selected additives, including CuSO4, PEG-8000 and glycerol, on laccase activity. The addition of 0.02% (w/v) PEG-8000 or 0.4% (w/v) glycerol to the reaction medium showed an increase of 10% in the laccase activity, whereas the addition of 0.002% (w/v) CuSO4 resulted by a 13% decrease of the enzyme activity.
Table 3

The effect of selected additives used in the reaction mixture on the laccase specific activity of an enzymatic extract from Coriolus hirsutus

Additive

Relative concentration (%)a

Relative specific activity (%)b

None

100

CuSO4

0.002

87.7 (7.0)c

PEG-8000

0.02

117.2 (6.9)

Glycerol

0.4

118.2 (9.4)

aRelative concentration of the additive was expressed in percent (w/v), was the one at which the laccase specific activity was at its optimum

bRelative specific activity was calculated and defined as the concentration of oxidized syringaldazine (nmol product/mg protein/min) in the reaction trial containing the optimum additive concentration relative to that without additive

cRelative percentage standard deviation was defined as the standard deviation of laccase triplicate trial divided by their respective means, multiplied by 100

The effect of additive concentrations on the laccase activation rate was also investigated. The results (Table 4) show that PEG-8000 was the most appropriate additive for laccase activity, followed by glycerol, with an inactivation rate constant (kinactivation) of 0.088 and 0.103, respectively. C1/2, which is the concentration (%, w/v) of PEG-8000, glycerol and CuSO4 required to decrease 50% of the initial laccase activity, were 7.8, 6.7 and 1.6% (w/v), respectively.
Table 4

The effect of selected additives used in the reaction mixture on laccase activity of an enzymatic extract, obtained from Coriolus hirsutus in aqueous medium composed of acetate buffer (0.1 M, pH 5.0), using syringaldazine as substrate

Additive inactivation parameter

Additive

CuSO4

PEG-8000

Glycerol

k inactivation a

0.441

0.088

0.103

C 1/2 b

1.6

7.8

6.7

aThe rate constant of inactivation (kinactivation), expressed as 1/% (w/v) of the additive per reaction volume, was estimated by plotting the ln percentage residual laccase activity versus the percentage of additive in the reaction trial for laccase

bThe C1/2, expressed in % (w/v) of the additive per reaction volume, defined as the additive concentration at which 50% of the initial activity was reached

Effects of selected additives on laccase thermostability

The effects of selected additives, including CuSO4, PEG-8000 and glycerol, on laccase activity at different incubation temperatures were investigated. Table 5 shows that the residual laccase activity of the control trial without additives was 81.0, 65.7, 49.0 and 4.1% after 168 h of incubation at 4, 25, 37 and 50 °C, respectively. A significant stabilization was obtained with the addition of CuSO4, where 116.4, 88.0, 62.8 and 12.6% of the laccase activity was retained after 168 h of incubation at 4, 25, 37 and 50 °C, respectively. The addition of 0.02% (w/v) PEG-8000 to the reaction mixture, resulted by 112.5, 96.2, 99.3 and 11.8% residual laccase activity after 168 h of incubation at 4, 25, 37 and 50 °C, respectively. In the presence of 0.4% (w/v) glycerol, the laccase activity was 114.1, 94.0, 79.2 and 9.8% of its initial activity, after 168 h of incubation at 4, 25, 37 and 50 °C, respectively.
Table 5

Effect of additives used in the reaction mixture on laccase thermal stability in aqueous medium composed of acetate buffer (0.1 M, pH 5.0), using syringaldazine as substrate

Additive

Relative concentrationa

Residual laccase activity (%)b

4 °C

25 °C

37 °C

50 °C

None

 

81.0 (3.0)c

65.7 (4.2)

49.0 (7.3)

4.1 (0.7)

CuSO4

0.002

116.4 (1.6)

88.0 (5.1)

62.8 (9.6)

12.6 (4.6)

PEG-8000

0.02

112.5 (4.0)

96.2 (1.3)

99.3 (7.6)

11.8 (0.3)

Glycerol

0.4

114.1 (10.1)

94.0 (10.8)

79.2 (1.3)

9.8 (0.4)

aRelative concentration of the additive, expressed in percent (w/v), was the one at which the laccase specific activity was at its optimum

bResidual laccase activity (%) was calculated by dividing the specific activity (nmol product/mg protein/min) of a given sample after 168 h of incubation at a defined temperature to that at time 0, multiplied by 100. The enzyme activity was assayed with syringaldazine as substrate

cRelative percentage standard deviation was defined as the standard deviation of laccase triplicate trial divided by their respective means, multiplied by 100

The laccase thermal stability at 50 °C, was also investigated over 7 days period of incubation. The semi-logarithmic plots of the inactivation kinetics, fitted in linear regression curves and with high correlation coefficient values, indicate (Table 6) that the thermal inactivation of laccase at 50 °C followed the first order kinetic behavior. The results also show the rate constant of deactivation (kt) and half-life time (t50), estimated from the semi-logarithmic plots. The t50 for the laccase control trial at 50 °C was 38.9 h, whereas that in the presence of CuSO4, PEG-8000 or glycerol was of 52.9, 54.6 and 50.2 h, respectively.
Table 6

Effect of additives used in the reaction mixture on laccase thermal stability in aqueous medium composed of acetate buffer (0.1 M, pH 5.0), using syringaldazine as substrate

Thermal inactivation parameter

Additive

None

CuSO4 0.002% (w/v)

PEG-8000 0.02% (w/v)

Glycerol 0.4% (w/v)

kt (h−1)a

0.0178

0.0131

0.0127

0.0138

T50 (h)b

38.9

52.9

54.6

50.2

aConstant of inactivation was determined from the first order kinetics behavior of the inactivation effect of increasing incubation time at a specific temperature

bT50 is defined as the incubation time at the specific temperature in acetate buffer (0.1 M, pH 5.0) required to report a 50% decrease in the initial activity

Discussion

The experimental findings suggest that the addition of mannitol, dextran, sucrose and the mixture BKSS enhanced the laccase activity of the enzymatic extract from C. hirsutus during freeze-drying as compared to the control without additives. Moreover, mannitol and dextran were the most appropriate lyoprotectants for the preparation of highly soluble powder freeze-dried laccase. Stepanova et al. (2003) showed that the addition of 1% (w/v) dextran 17 kDa resulted in 95 and 88% of residual laccase activity after a vacuum-drying of the enzymatic extract, obtained from C. hirsutus and Coriolus zonatus, respectively. Urena et al. (2016) reported that the use of the lyophilization agent maltodextrin in an aqueous suspension of the enzyme laccase from Trametes versicolor was an optimum mixture for the bio-functionalization of carbon surfaces. Hall et al. (2008) indicated that the residual activity of lipoxygenase (LOX) and hydroperoxide lyase (HPL) of the enzyme extract, obtained from Penicillium camemberti, was 55.0 and 29.7%, respectively, when 60% (w/w) dextran 72.2 kDa was added to the enzymatic extract before its lyophilization; these authors also reported that the addition of 86% (w/w) of KCl maintained 92.9% of the LOX residual activity and resulted by 2.25 times of enhancement of the HPL activity. In contrast, Capolongo et al. (2003) findings indicated that the addition of mannitol and dextran to the lignin peroxidase extract was not suitable to protect the enzymatic extract during its lyophilization, since their interactions with the protein destabilize it by decreasing its unfolding temperature; these authors also indicated that although the sucrose had a stabilizing effect on the lignin peroxidase, the sucrose containing-solutions were difficult to be lyophilized since the glass transition was at − 33 °C, which resulted in a rubbery product after their lyophilization. The stabilization of enzyme activity, obtained by the addition of sugar and poly-alcohol, could be related to their ability to replace the water molecule by involving the hydrogen bonding with polar groups on the protein (Crowe et al. 1993; Allison et al. 1998). Tanaka et al. (1991) demonstrated that sugars and poly-alcohols could protect the catalase by helping their direct interaction with the protein. The beneficial lyoprotectant effects of dextran and mannitol may be also attributed to the alteration of the glass transition temperature (Tg) of the enzymatic preparation (Carpenter et al. 1997). Wang (2000) suggested that the Tg of the stabilized formulation is reached, the greater the degree of structural preservation is obtained with less protein aggregation.

The results reported in this study also suggested that the laccase stability is dependent on the dextran molecular weight. Similarly, Gloger et al. (2003) indicated that the addition of 4% (w/v) dextran 10 kDa provided the highest relative residual activity of 104% for the carbohydrate-binding activity of aviscumine, whereas the addition of the same concentration of dextran 75 and 1 kDa resulted by 83 and 92% of its residual activity, respectively. These authors also reported a 20% decrease in the protein-binding activity of aviscumine when the molecular weight of dextran was increased from 1–75 kDa. The inactivation effect obtained with the use of large dextran polymer (40 and 70 kDa), may be attributed to the ability of the dextran to change the glass transition temperature (Tg) of a protein formulation (Wang 2000). High molecular weight dextrans show more intramolecular hydrogen bonds which lead to a loss in intermolecular bonding capacity resulting in decreased protein stability (Tanaka et al. 1991). The failure of dextran with very low or very high molecular weight to stabilize laccase activity may be due also to the low or high content in residual water in the lyophilized enzyme preparation resulting with under- or over-drying of the protein (Carpenter et al. 1997; Gloger et al. 2003). The reasons of the instability of laccase in presence 40 kDa dextran are not clear. This could be related to proteins impurities or to the presence of two laccase isozymes in the enzymatic extract obtained from C. hirsutus as characterized by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) of the concentrated protein extract, the partially purified and purified enzymatic fraction, with estimated molecular weights of 31 and 56 kDa (Taqi 2012).

Moreover, the experimental findings suggested that the stabilization effect, obtained with the use of dextran or mannitol, was concentration dependent. Similarly, Hall et al. (2008) indicated that the increase in dextran concentration was associated with a decrease in LOX and HPL activities. On the other hand, Stepanova et al. (2003) reported that the increase in dextran concentration, from 0.5 to 1%, resulted by an increase in the residual laccase activity, from 67 to 95%, after a vacuum-drying of the enzyme extract; these authors also reported that the increase in the polyvinyl alcohol concentration, from 3 to 5%, resulted in a decrease from 75 to 69% of the enzyme activity, obtained from C. hirsutus, and an increase, from 48 to 85%, in that obtained from C. zonatus. It was demonstrated that the maximum protection of a protein by carbohydrates is achieved at the concentration that allow forming a monomolecular layer on the protein surface (Tanaka et al. 1991). It is also suggested that the optimal protection effect is obtained by amorphous mannitol, but the stabilizing effect decreases with an increase in its crystallinity (Izutsu et al. 1994).

Similar to the findings reported in this study, the increase in laccase activity of the enzymatic extract after a pre-incubation in the acetate buffer at different temperature and incubation time was also reported for other fungal laccases, including those from Fomes sclerodermeus (Papinutti et al. 2008), Chaetomium thermophilum (Chefetz et al. 1998) and C. zonatus (Koroleva et al. 2001). The activation of laccase after the thermal treatment could be due to a change either in the ratio of monomeric and aggregated enzyme molecules in solution or in the conformation of its active site (Stepanova et al. 2009).

In addition, the activation rate, obtained during the pre-incubation of the different laccase preparations at different temperatures, was relatively higher than that for the fresh enzyme preparation reported previously by Taqi (2012). Similar findings were reported by Stepanova et al. (2009) where the activity of the native laccase preparation, cooled to 4 °C, did not vary during the subsequent incubation for 4 h at room temperature, whereas that of the frozen one at − 18 °C did not exhibit any enzyme activity immediately after it was thawed. Yaropolov et al. (1994) suggested that the activation of laccase, obtained after thermal pre-incubation, could be attributed to the effect of such treatment on the transition of the enzyme state from the so-called dormant form to the active one. Stepanova et al. (2009) reported that the ratio of monomeric and aggregated laccase molecule was unchanged before and after incubation at room temperature, whereas the structure of all the three copper centers (Type-I ion copper, Type-II and Type-III) was rearranged during the incubation. On the other hand, Koroleva et al. (2001) reported that the decrease in laccase activity at the temperature range of 55–65 °C was caused by the release of Type-II ion copper which was completely absent at 70 °C; in addition, the Type-I and Type-III sites were completely disintegrated at temperatures higher than 70 °C, while the overall protein conformation was still maintained intact.

Moreover, the overall results show that the reconstituted laccase was stable at different thermal treatment, while the use of dextran and mannitol did not confer any significant stabilization of laccase as compared to the control trial. Papinutti et al. (2008) indicated that, with or without mannitol, the residual laccase activity of F. sclerodermeus after 24 h of incubation at pH 4.5 and at 40 °C was 50%. Moreover, Stepanova et al. (2009) reported that dextran did not provide an appropriate stabilization effect on the laccase activity of C. hirsutus and C. zonatus, after 196 h of incubation at 40 °C, with a residual enzymatic activity of 16.1 and 23.5%, respectively. Hall et al. (2008) reported that the addition of 5% (w/v) mannitol to the reaction mixture resulted by an increase in the thermostability of LOX from P. camemberti.

The experimental findings (Fig. 4) also suggest that the laccase preparation with mannitol and the control without lyoprotectant showed higher thermostability when it was stored for 1–4 h at 25 and 50 °C as compared to shorter incubation time. Similar findings were also reported by Saparrat et al. (2008) in which the relative laccase activity of the crude enzyme, obtained from Grammothele subargentea, increased after 4 h of its incubation at 40, 50 and 60 °C. The results are also in agreement with those of Chefetz et al. (1998) in which a preincubation of the purified laccase, obtained from C. thermophilum, at 40 to 60 °C and up to 1 h increase the enzyme activity. This increase could be due to conformational changes of the enzyme, which may increase its flexibility and therefore its catalytic activity. Saparrat et al. (2008) suggested that conformational changes in metalloproteins, such as laccase, can lead to more efficient electron transfer rates in the reaction. Coll et al. (1993) have reported that fungal laccases may be thermally activated by their pre-incubation at elevated temperatures up to 60 °C. In addition, it is suggested that mannitol did not have significant effect on the conformational changes of the enzyme in solution during thermal treatment.

The assessment of the storage stability of the different laccase preparation for 4 weeks at − 80, 4 and 25 °C showed that with mannitol was the most stable as compared to that with dextran and the control lacking any additive. Wang (2000) reported that the presence of additives in an enzyme preparation may have adverse effects, which could be due to the crystallization of the additives during storage; the destabilization can be also attributed to the failure of the dextran to interact effectively, by hydrogen bonding, with the protein molecules. The stabilization mechanism of the additives could be attributed mainly to their ability to inhibit the aggregation of the protein by physical or chemical interactions, the chemical degradations, the deamination, the oxidation of side chains or the hydrolysis of the lyophilized protein formulations.

The effect of additives and their concentrations on the laccase activity and its activation rate were also investigated. The results show that PEG-8000 was the most appropriate additive for laccase activity, followed by glycerol, while the addition of CuSO4 resulted by a decrease of the enzyme activity. Stepanova et al. (2003) reported that the addition of various salts (10−3–10−1 M) to laccase suspensions showed a concomitant decrease in the laccase activity with the increase in salt concentrations; these authors also reported that the addition of Cu2+ to the enzyme suspension resulted by a 25% decrease in its activity. Kim and Nicell (2006a) reported also that the laccase suspension, treated with 1 mM cyanide, Cu2+ and Fe3+, showed a significant decrease in the conversion of triclosan by 55.8, 28.0 and 6.2%, respectively; since this decrease may be due to the fact that these ions may interrupt the electron transport systems of the enzyme activity decreasing hence its activity. Modaressi et al. (2005) reported that the PEG-3350 was able to increase (20%) the laccase activity, with a turnover of fivefold toward triclosan. Kim and Nicell (2006b) reported that PEG-3350 had shown to be the most effective additive for the enhancement of the conversion of bisphenol A, by preventing the laccase inactivation rather than by increasing the reaction rate. Ghosh et al. (2008) suggested that the presence of PEG and glycerol in the laccase reaction solution may reduce the destabilizing effect of the products by hindering their hydrogen bonding site with the hydroxyl end group of those additives. It is also suggested that the presence of polyols in the laccase reaction media act as a water-structure maker which depresses the hydration of the enzyme and hence its denaturation (Papinutti et al. 2008). Although the metal ions including copper play crucial role as enhancers of laccase activity, especially that laccases have four copper atoms in their catalytic center, it could inhibit it at varying degrees, yet the destabilizing mechanism was not elucidated yet (Hernandez-Monjoraz et al. (2018).

The effects of selected additives, including CuSO4, PEG-8000 and glycerol, on laccase activity at different incubation temperatures were investigated. The overall results suggest that the laccase showed a higher residual activity in the presence of the investigated additives as compared to that of the control trial after 168 h of incubation at different temperatures. Poonkuzhali and Palvannan (2011) also reported that the addition of Guar Gum, Starch, agar and agarose to the laccase Pleurotus florida at different temperatures (50, 55, 60 and 65 °C) increased the enzymatic activity when compared to the control and the reported results demonstrated the ability of those additives in converting thermolabile laccase into a thermostable one attributed to the gelling nature of polysaccharide additives.

The laccase thermal stability at 50 °C was also investigated over 7 days period of incubation. The overall findings suggest that the investigated additives conferred a close level of stabilization for the laccase activity at 50 °C, by extending their half-life time values. Papinutti et al. (2008) reported that the highest stabilization effect was obtained when laccase from F. sclerodermeus was incubated in the presence CuSO4; these authors also reported that the combination of 1.25 mM CuSO4 and 0.2% glycerol conferred an increases in the half-life time value for laccase of 114, and 9.81 h as compared to the control, when incubated at 40 °C, respectively. Baldrian and Gabriel (2002) indicated that the stability of laccase, from Pleurotus ostreatus, was increased in the presence of Cu2+ with a residual enzymatic activity of 45% as compared to 27% of that of the control, after 7 days of at 20 °C.

On the other hand, Stepanova et al. (2003) reported on the effects of dextran, lactitol and polyacrylic acid, used as additives, on the thermal stability of laccase, from C. hirsutus and C. zonatus, incubated at 40 °C for 196 h, and indicated that neither the individual additives nor their combination had provided stabilization for both laccases. The literature (Koroleva et al. 2001; Papinutti et al. 2008) suggested that the thermal inactivation of laccase may be due to the depletion of the copper ion from the enzyme center, though the stabilization of laccase by the presence of CuSO4 could be due to the effect of the Cu2+ in delaying the release of the copper center from the laccase and preventing hence its inactivation. The mechanism of laccase stabilization conferred by PEG and glycerol might be related to their effects by decreasing the water activity or by regulating the interactions between the water molecule and the proteins (Kim and Nicell 2006a).

The experimental data obtained throughout this study showed that the optimal lyophilized laccase preparation was achieved with the use of mannitol as lyoprotectant. The lyophilized laccase was stable at various storage conditions and the reconstituted enzyme was stable at different incubation temperatures. The results also indicated that the addition of selected chemicals to the reaction medium resulted by an increase in the enzyme thermal stability. The enhanced stability of the laccase enzymatic extract could provide a better use of these enzymes in various biotechnological applications.

Abbreviations

ATCC: 

American Type Culture Collection

BKSS: 

sodium benzoate, potassium sorbate and sorbitol

BSA: 

bovine serum albumin

HPL: 

hydroperoxide lyase

LOX: 

lipoxygenase

PEG: 

polyethylene glycol

RSD: 

relative standard deviation

SD: 

standard deviation

SG: 

syringaldazine

T g

glass transition temperature

Declarations

Authors’ contributions

CBM designed the study, conducted the laboratory work, analyzed the data and wrote the manuscript. SK supervised the work and critically reviewed the manuscript. Both authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

Not applicable.

Ethics approval and consent to participate

This article does not contain any studies with human participants or animals performed by any of the authors.

Funding

This research was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC).

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Faculty of Nursing and Health Sciences, Notre Dame University Zouk Mosbeh, P.O.Box: 72, Zouk Mikael, Lebanon
(2)
Department of Food Science and Agricultural Chemistry, McGill University, 21,111 Lakeshore, Ste-Anne de Bellevue, QC, H9X 3V9, Canada

References

  1. Agrawal PK, Shrivastava R, Verma J (2019) Bioremediation Approaches for Degradation and Detoxification of Polycyclic Aromatic Hydrocarbons. In: Bharagava R, Chowdhary P (eds) Emerging and eco-friendly approaches for waste management. Springer, Singapore, pp 99–119View ArticleGoogle Scholar
  2. Allison SD, Randolph TW, Manning MC, Middleton K, Davis A, Carpenter JF (1998) Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration-induced damage and its inhibition. Arch Biochem Biophys 358:171–181View ArticleGoogle Scholar
  3. Baldrian P, Gabriel J (2002) Copper and cadmium increase laccase activity in Pleurotus ostreatus. FEMS Microbiol Lett 206:69–74View ArticleGoogle Scholar
  4. Capolongo A, Barresi AA, Rovero G (2003) Freeze-drying of lignin peroxidase: influence of lyoprotectants on enzyme activity and stability. J Chem Technol Biotechnol 78:56–63View ArticleGoogle Scholar
  5. Carpenter JF, Pikal MJ, Chang BS, Randolph TW (1997) Rational design of stable lyophilized protein formulations: some practical advice. Pharm Res 14:969–975View ArticleGoogle Scholar
  6. Chansanroj K, Thanawattanawanich P (2016) Lyophilization of pharmaceutical products: from concept to reality. Asian J Pharm Sci 11:39View ArticleGoogle Scholar
  7. Chefetz B, Chen Y, Hadar Y (1998) Purification and characterization of laccase from Chaetomium thermophilum and its role in humification. Appl Environ Microbiol 64:3175–3179PubMedPubMed CentralGoogle Scholar
  8. Coll PM, Fernandez-Abalos JM, Villanueva JR, Santamaria R, Perez P (1993) Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971). Appl Eviron Microbiol 59:2607–2613Google Scholar
  9. Crowe JH, Crowe LM, Carpenter JF (1993) Preserving dry biomaterials: the water replacement hypothesis, part 1. Biopharm 6:28–37Google Scholar
  10. Dalfard AB, Khajeh K, Soudi MR, Naderi-Manesh H, Ranjbar B, Sajedi RH (2006) Isolation and biochemical characterization of laccase and tyrosinase activities in a novel melanogenic soil bacterium. Enzyme Microb Technol 39:1409–1416View ArticleGoogle Scholar
  11. Freire RS, Duran N, Kubota LT (2001) Effects of fungal laccase immobilization procedures for the development of a biosensor for phenol compounds. Talanta 54:681–686View ArticleGoogle Scholar
  12. Ghosh J, Taylor K, Bewtra J, Biswas N (2008) Laccase-catalyzed removal of 2,4-dimethylphenol from synthetic wastewater: effect of polyethylene glycol and dissolved oxygen. Chemosphere 71:1709–1717View ArticleGoogle Scholar
  13. Gianfreda L, Bollag JM (2002) Isolated enzymes for the transformation and detoxification of organic pollutants. In: Burns RG, Dick RP (eds) Enzymes and the environment: activity, ecology, and applications. Marcel Dekker Inc, New York, pp 495–538Google Scholar
  14. Gloger O, Witthohn K, Müller BW (2003) Lyoprotection of aviscumine with low molecular weight dextrans. Int J Pharm 260:59–68View ArticleGoogle Scholar
  15. Hall CE, Karboune S, Florence H, Kermasha S (2008) Stabilization of an enzymatic extract from Penicillium camemberti containing lipoxygenase and hydroperoxide lyase activities. Process Biochem 43:258–264View ArticleGoogle Scholar
  16. Hartree EF (1972) Determination of protein: a modification of the lowry method that gives a linear photometric response. Anal Biochem 48:422–427View ArticleGoogle Scholar
  17. Hernandez-Monjoraz WS, Caudillo-Perez C, Salazar-Sanchez PU, Macias-Sanchez KL (2018) Influence of iron and copper on the activity of laccases in Fusarium oxyporum f. sp. lycopersici. Braz J Microbiol 418:1–7Google Scholar
  18. Izutsu K, Yoshioka S, Terao T (1994) Effect of mannitol crystallinity on the stabilization of enzymes during freeze-drying. Chem Pharm Bull 42:5–8View ArticleGoogle Scholar
  19. Jeon JR, Chang YS (2013) Laccase-mediated oxidation of small organics: bifunctional roles for versatile applications. Trends Biotechnol 31:335–341View ArticleGoogle Scholar
  20. Kim YJ, Nicell JA (2006a) Laccase-catalysed oxidation of aqueous triclosan. J Chem Technol Biotechnol 81:1344–1352View ArticleGoogle Scholar
  21. Kim YJ, Nicell JA (2006b) Laccase-catalyzed oxidation of bisphenol A with the aid of additives. Process Biochem 41:1029–1037View ArticleGoogle Scholar
  22. Koroleva OV, Stepanova EV, Binukov VI, Timofeev VP, Pfeil W (2001) Temperature-induced changes in copper centers and protein conformation of two fungal laccases from Coriolus hirsutus and Coriolus zonatus. Biochim Biophys Acta Protein Struct Mol Enzyme 1547:397–407View ArticleGoogle Scholar
  23. Liu J, Yu Z, Liao X, Liu J, Mao F, Huang Q (2016) Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization. J Clean Prod 127:600–609View ArticleGoogle Scholar
  24. Modaressi K, Taylor KE, Bewtra JK, Biswas N (2005) Laccase-catalyzed removal of bisphenol-A from water: protective effect of PEG on enzyme activity. Water Res 39:4309–4316View ArticleGoogle Scholar
  25. Nunes CS, Kunamneni A (2018) Laccases-properties and applications. In: Enzymes in human and animal nutrition. Elsevier, Chennai, pp 133–161View ArticleGoogle Scholar
  26. Papinutti L, Dimitriu P, Forchiassin F (2008) Stabilization studies of Fomes sclerodermeus laccases. Bioresour Technol 99:419–424View ArticleGoogle Scholar
  27. Poonkuzhali K, Palvannan T (2011) Thermstabilization of laccase by polysaccharide additives: enhancement using central composite design of RSM. Carbohydr Polym 86:860–864View ArticleGoogle Scholar
  28. Poonkuzhali K, Sathishkumar P, Boopathy R, Palvannan T (2011) Aqueous state laccase thermostabilization using carbohydrate polymers: effect on toxicity assessment of azo dye. Carbohydr Polym 85:341–348View ArticleGoogle Scholar
  29. Rocha-Martín J, Martínez-Bernal C, Zamorano LS, Reyes-Sosa FM, Díez García B (2018) Inhibition of enzymatic hydrolysis of pretreated corn stover and sugar cane straw by laccases. Process Biochem 67:88–91View ArticleGoogle Scholar
  30. Saparrat MCN, Mocchiutti P, Liggieri CS, Aulicino MB, Caffini NO, Balatti PA, Martinez MJ (2008) Ligninolytic enzyme ability and potential biotechnology applications of the white-rot fungus Grammothele subargentea LPSC no. 436 strain. Process Biochem 43:368–375View ArticleGoogle Scholar
  31. Shin K, Lee Y (2000) Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus. Arch Biochem Biophys 384:109–115View ArticleGoogle Scholar
  32. Stepanova EV, Koroleva OV, Gavrilova VP, Landesman EO, Makower A, Papkovsky DB (2003) Comparative stability assessment of laccases from the basidiomycetes Coriolus hirsutus and Coriolus zonatus in the presence of effectors. Appl Biochem Microb 39:482–487View ArticleGoogle Scholar
  33. Stepanova EV, Fedorova TV, Sorokina ON, Volkov VV, Koroleva OV, Dembo AT (2009) Effect of solvent phase transitions on enzymatic activity and structure of laccase from Coriolus hirsutus. Biochemistry 74:385–392PubMedGoogle Scholar
  34. Strong PJ, Claus H (2011) Laccase: a review of its past and its future in bioremediation. Crit Rev Environ Sci Technol 41:373–434View ArticleGoogle Scholar
  35. Tanaka K, Takeda T, Miyajima K (1991) Cryoprotective effect of saccharides on denaturation of catalase by freeze-drying. Chem Pharm Bull 39:1091–1094View ArticleGoogle Scholar
  36. Taqi M (2012) Biomass Production, Purification and Characterization of Selected Microbial Laccases, Ph.D. Thesis, McGill University, Montreal, Qc, CanadaGoogle Scholar
  37. Tarasi R, Alipour M, Gorgannezhad L, Imanparast S, Yousefi-Ahmadipour A, Ramezani A, Ganjali MR, Shafiee A, Faramarzi MA, Khoobi M (2018) Laccase immobilization onto magnetic-cyclodextrin-modified chitosan: improved enzyme stability and efficient performance for phenolic compounds elimination. Macromol Res 26:1–8View ArticleGoogle Scholar
  38. Thakur K, Kalia S, Pathania D, Kumar A, Sharma N, Schauer CL (2016) Surface functionalization of lignin constituent of coconut fibers via laccase-catalyzed biografting for development of antibacterial and hydrophobic properties. J Clean Prod 113:176–182View ArticleGoogle Scholar
  39. Urena YRC, Lisboa-Filho PN, Szardenings M, Gatjen L, Noeske PL, Klaus R (2016) Formation and composition of absorbates on hydrophobic carbon surfaces from aqueous laccase-maltodextrin mixture suspension. Appl Surf Sci 385:216–224View ArticleGoogle Scholar
  40. Wang W (2000) Lyophilization and development of solid protein pharmaceuticals. Int J Pharm 203:1–60View ArticleGoogle Scholar
  41. Yaropolov AI, Skorobogatko OV, Vartanov SS, Varfolomeyev SD (1994) Laccase: properties, catalytic mechanism, and applicability. Appl Biochem Biotechnol 49:257–280View ArticleGoogle Scholar
  42. Yesilada O, Birhanli E, Geckil H, (2018) Bioremediation and decolorization of textile dyes by white rot fungi and laccase enzymes. In: Mycoremediation and environmental sustainability, 7 edn. Springer, Cham, pp 121–153Google Scholar

Copyright

© The Author(s) 2018

Advertisement