Chapter 4: Purification and Characterization of Laccase from...

Enzymological and Biotechnological Prospects in Lignolytic system of White Rot Fungi.

4.1 Ph.D Thesis: Minal K. Narkhede, North Maharashtra University, Jalgaon. (January2014)

CChhaapptteerr 44:: PPuurriiffiiccaattiioonn aanndd CChhaarraacctteerriizzaattiioonn ooff LLaaccccaassee ffrroomm BBaassiiddiioommyyccoottaa sspp.. LL--116688 4.1 Introduction

The production of laccase by BspL-168, a newly isolated white rot fungal

species, was improved several fold by medium optimization and other strategic

experimentation as mentioned in earlier chapter 3. However, the ability to

produce high titers of laccase would not be the sole factor in considering a strain

for commercial laccase production. Since, white rot fungi exhibit great diversity

in biochemical characteristics, a thorough investigation involving characterization

with respect to pH and temperature requirement, substrate specificity and stability

under different adverse conditions of pH, temperature and solvents etc. must be

undertaken. Therefore, considerable efforts are subsequently extended in this

chapter to determine biochemical properties of the enzyme.

The purification of enzyme is invariably the first imperative step in any

investigation of enzyme characteristics. In last couple of decades, a typical protein

purification protocol usually considered use of ammonium sulphate precipitation,

one or two ion exchange steps, gel filtration and finally an affinity

chromatography step (Galhaup et al. 2002; Sadhasivam et al. 2008; Gautam et

al. 2012). In fact diverse industrial applications have different requirements of

protein purity. However, from industrial perspective, the economics and scale of

operation are two important requirements expected to be met by any purification

protocol. Limited applications of purified laccase have been reported, which were

carried out mainly by chromatographic techniques. Most separation methods



reported were developed for characterization purposes rather than commercial use

(Rajeeva and Lele 2010).

Among different methods, membranes have always formed a part of the

traditional bioseparation protocol for proteins (Gautam et al. 2012). Ultra

filtration can be carried out at mild conditions of temperatures and pressures.

Moreover, since there are no phase changes and no chemical additives, it ensures

minimum degradation making it particularly suitable for highly labile biological

macromolecules like enzymes (Rajeeva and Lele 2010). Although, traditionally

considered to be a low-resolution process, the pore size of the sieves can be

adjusted to achieve desired degree of separation (Gautam et al. 2012).

Recently in industrial as well as academic sectors, there is an increasing

trend towards designing short and efficient protein purification protocol,

simultaneously considering the economics and scale up possibilities (Gautam et

al. 2012). Among the non-chromatographic methods, Three Phase Partitioning

(TPP) and it’s another version called Macro-(affinity ligand) Facilitated Three

Phase Partitioning (MLFTPP) has now a day attracted much attention (Mondal et

al. 2003; Gautam et al. 2012). The application of MLFTPP is reported for

purification of glucoamylase and pullulanase using smart polymer, alginate

(Mondal et al. 2003) and of protease (Prakash et al. 2011). However, it is

essential that the protein under study is able to bind to the polymer. In present

investigation, laccase purification was attempted by the application of TPP.

Furthermore, based on above observation, the suitability of alginate for

purification of laccase from BspL-168 was examined.



In structural terms, laccases can be either monomeric or multimeric

glycoproteins. They may exhibit additional heterogeneity because of variable

carbohydrate and copper content (Sadhasivam et al. 2008). There are many

reports on the purification and biochemical characterization of fungal laccases.

The key characteristics of laccases include pH and temperature stability, substrate

specificity, inhibition etc. Besides these, the standard redox potential and

characterization of types of copper centers (T1, T2 and T3) further help to

distinguish the enzyme.

Enzyme deactivation is found to be one of the major constrains in the

rapid development of biotechnological processes. It is defined as a process where

the secondary, tertiary or quaternary structure of a protein changes without

breaking covalent bonds (Naidu and Panda 2003). The enzymes are deactivated

by many ways to an inactive state. Since the enzymes have highly defined

structures, the slightest deviation from their native form can affect their specific

activity. Therefore, understanding the effects of different environmental factors

on enzymatic activity and molecular structure is highly useful to industrial

applications. The knowledge of the enzyme stability can facilitate an economical

production design by optimizing the profitability of enzymatic processes (Jurado

et al. 2004)

The diverse industrial applications demand the enzymes with

characteristics to suit the conditions of the given process. Since the fungal

laccases have wide range of applications in biotechnological and food processing

industries, studies on pH and thermal stability are very important. The



deactivation study and determination of thermodynamic parameters not only help

to understand the relation between structure and function of the enzyme but also

help to determine the probable mechanism of deactivation to certain extent

(Naidu and Panda 2003).

Several studies have determined the properties and kinetic parameters of

laccase, such as the deactivation rate constants (kd), half-life (t1/2), deactivation

energy and the kinetic constants Km and Vmáx (Galhaup et al 2002; Chernykh

et al. 2008; Park and Park 2008; Huang et al. 2011b). However, the data on

thermodynamic parameters is scare. Moreover, the enzyme properties such as

their kinetic and thermodynamic behavior may be modified by the processes of

purification used. The decreased stability and/or affinity of the purified enzyme

may be associated with the removal of ligand and/or proteins that had a protective

effect on the crude enzyme. On the other hand, the purification process could

improve the specificity of the enzyme (Braga et al. 2013).

Laccases catalyze the oxidation of a broad range of substrates such as

ortho and para-diphenols, methoxy-substituted phenols, aromatic amines,

phenolic acids and several other compounds (Sadhasivam et al. 2008). The

laccase catalyzed one electron oxidation is coupled to the reduction of molecular

oxygen to water. A general reaction scheme has been proposed as:

4RH + O2 → 4R + 2H2O

Laccase is the most promising enzyme of oxidoreductase group for

industrial applications since it recycles molecular oxygen as an electron acceptor

and does not require any other co-substrate (Sahay et al. 2008). However, the



substrate specificity of laccases varies from one organism to another

(Sadhasivam et al. 2008). Furthermore, the spectrum of laccase oxidizable

substrates can be expanded considerably in the presence of appropriate redox

mediators. The great potential of laccases in diverse environmental applications is

due to their broad substrate specificity. Therefore, studies involving catalytic

properties of laccases have gained considerable interest in recent years.

The effect of potential inhibitor compounds has been often investigated for

characterization of iso-enzymes and study of their role in lignin synthesis or

biodegradation (Johannes and Majcherczyk 2000). The inhibitors are

compounds which in an ideal case selectively deactivate the desired enzyme. Over

the years, numerous inhibitors have been used for the description of laccases. The

most common compounds applied are dithiothreitol (DTT), thioglycolic acid

(TGA), cysteine, EDTA, sodium fluoride, and sodium azide (NaN3) (Saito et al.

2003; Younes et al. 2007; Sadhasivam et al. 2008). In the present study, the

effect of commonly used inhibitors was tested. The methods and results of

characterization of laccase from BspL-168 are summarized in this chapter

4.2 Objectives

To purify the laccase for characterization of its kinetic properties

To analyze the biochemical and kinetic properties of the laccase

4.3 Material and methods

4.3.1Chemical

Unless otherwise stated, chemicals were purchased from M/s Hi-Media

Limited, Mumbai, and were of the highest purity available. 2,2’-Azinobis (3-



ethylbenzthiazoline-6-sulfonic acid) (ABTS) and syringaldazine was obtained

from Sigma.

4.3.2 Syringaldazine assay for laccase

This assay is based on the oxidation of 4,4’-

[azinobis(methanylylidene)]bis(2,6-dimethoxyphenol) (syringaldazine) to the

corresponding quinone, 4,4’-[azinobis(methanylylidene)]bis(2,6-

dimethoxycyclohexa-2,5-diene-1-one) (Figure 4.1). An increase in absorbance at

529 nm is followed to determine laccase activity in international units (IU) where

1 IU is defined as the amount of enzyme forming 1 µmole of product per minute.

FIGURE 4.1: The laccase-catalyzed oxidation of syringaldazine to its corresponding quinine (Source: Bar 2001)

The reaction mixture in final volume of 2 ml consisted of 0.1 M Britton-

Robinson buffer (pH 4.5) along with appropriate amount of substrate and enzyme.

Laccase activity in U/mL (µmole cation radical released.min-1ml-1) was calculated

using the extinction coefficient of syringaldazine of 6.5 x 104 M -1cm-1.

4.3.3 Protein estimation

Aliquots of different concentrations of standard protein (BSA) and sample

(0.2 ml each) were added with 2 ml freshly prepared alkaline copper reagent (see



note below). After incubation of 10 min, 0.2 ml of the folin-ciocaltaeu reagent

was added and again incubated at room temperature for 50 min. After incubation

blue colour was measured at 660 nm. A proper blank without the protein was

used. Amount of protein in culture filtrate was estimated using standard graph

(Lowry et al. 1951).

Note: The alkaline copper reagent was prepared by mixing 2 % sodium carbonate (in 0.1N NaOH) with 0.5% copper sulphate (in 1% sodium potassium tartarate) in 50: 1 ratio.

4.3.4 Laccase purification

a) Ammonium sulphate precipitation

Laccase from BspL-168 was purified from 7-day-old liquid culture. The

liquid culture was separated from mycelia by filtration on Whatman paper No 1.

The culture filtrate was then fractionated with ammonium sulphate in order to

remove unwanted proteins.

A pre-experiment was conducted to determine the degree of ammonium

sulphate saturation required to precipitate laccase from the supernatant.

Ammonium sulphate was dissolved in 20 ml of culture filtrate and centrifuged

(10000 rpm for 20 min at 4 oC). The amount of ammonium sulphate was

increased by 5 % intervals ranging from 30% to 85% saturation. The amount of

laccase activity remaining in the supernatant was determined

spectrophotometrically with the ABTS assay. The amounts of solid ammonium

sulphate required (grams per 100 ml of supernatant) to obtain different levels of

saturation were obtained from ‘Laboratory Manual in Biochemistry’ (Jayaraman

2008).



Since maximum laccase recovery was obtained at 80% saturation, in

further experiment the proteins were precipitated from liquid culture by the

addition of (NH4)2SO4 up to 80% saturation. It was followed by centrifugation at

10,000 rpm for 20 min at 4 oC. The precipitate was resuspended in 50 mmol l-1

potassium phosphate buffer pH 5.0 and extensively dialysed against the same

buffer using dialysis membrane (HiMedia) with the Molecular Weight Cut Off

(MWCO) of 12-14 kDa. The total tubing length required is calculate by the

equation as follows:

Total length = (sample volume) / (vol/length) + (additional 10-20%) + 4 cm

The additional 10 to 20% more length is required to account for head

space (air) to keep the sample buoyant. Moreover, about 2 cm at each end is

required for applying two closures.

b) Ultrafiltration and Concentration through 30 Kd cassette

As laccase is an extracellular protein, the culture broth was first filtered

through Whatmann filter paper to remove mycellial biomass and then centrifuged

for 15 min at 10000 rpm and 4 oC. For further downstream processing a cross-

flow filtration module (Pall, USA) equipped with a 30 kDa molecular weight cut-

off cassette was used to get rid of small molecular contaminants and to further

concentrate the supernatant (Plate 4.1).

c) Three phase partitioning

Principle: Three-phase partitioning (TPP) is a method in which proteins are

salted out from a solution containing a mixture of water and t-butanol. t-Butanol

is infinitely miscible with water but upon addition of sufficient ammonium



sulphate the solution splits into two phases, an underlying aqueous phase and an

overlying t-butanol phase (Dennison 2002). If proteins are present in the initial

solution, three phases are formed, proteins are precipitated in a third middle phase

between the aqueous and t-butanol phases (Figure 4.2 and Plate 4.2).

FIGURE 4.2: Protein separation in Three-phase partitioning (Source: A Guide to Protein Isolation by Dennison 2002) d) Macro affinity Ligand -Facilitated Three-Phase Partitioning (MLFTPP)

Principle

Earlier, it was shown that smart polymers like alginate also precipitate up

when subjected to TPP. In MLFTPP, a solution of a smart polymer is added to the

crude extract of protein (s). Upon addition of optimized amounts of ammonium

sulphate and t-butanol, an interfacial precipitate consisting of the smart polymer

and the protein(s) having affinity for the polymer is obtained (Mondal et al.

2003). As the precipitate floats at the interface, the need for a

centrifugation/membrane separation step is eliminated which are relatively costly

and tedious at the industrial scale (Gautam et al. 2012).

Method

The steps of MLF TPP were followed as reported by Mondal et al. (2003)

and Sharma et al. (2003) with necessary modifications (Plate 4.3). A fixed

concentration of crude laccase preparation was added to 1 ml of alginate solution.



Two different concentrations of alginate were used i.e. 1 and 3%. The total

volume was made up to 3 ml with 0.05 M acetate buffer (pH 4). The above

solution was made up to 50% (w/v) with respect to ammonium sulphate by adding

solid ammonium sulphate and vortexing the system. The percentage of

ammonium sulphate saturation was selected based on prior optimization during

TPP using RSM. This was followed by addition of 8 ml t-butanol to the above

solution. Again, the amount of butanol added was selected based on prior

optimization during TPP using RSM. After vortexing, the resulting mixture was

incubated at 37 °C for 1 h. The three phases (upper t-butanol phase, interfacial

precipitate, and lower aqueous phase) were then separately collected for assay of

enzyme and total proteins. The difference between the total enzyme activity in

the crude extract and the activity in the aqueous phase represented the amount of

enzyme bound to the alginate in the interfacial layer.

The polymer-bound enzyme was recovered by following the procedure as

described by Sharma et al. (2003). The precipitate was dissolved in 2 ml of 1 M

NaCl (in 0.05 M phosphate buffer) and incubated at 4 oC for 18 h. Enzyme was

then recovered by precipitating alginate with 0.35 ml of 1 M CaCl2 (final

concentration of CaCl2 in the solution was 0.07 M). This procedure was repeated

twice in order to recover all the bound enzyme activity. Enzyme activity and

protein in the supernatant were determined after extensive dialysis against 0.05 M

phosphate buffer, pH 5.0.

e) Optimization of parameters affecting TPP using RSM



Three parameters were optimized using response surface methodology

(RSM). Box Behnken design was used as RSM tool for optimization. The coded

and uncoded values chosen for the variables namely salt concentration, pH and

Enzyme: Butanol ratio (E:B) are presented in Table 4.1 a and b. The factors are

arranged into three levels and coded -1, 0 and +1 for low, middle and high

concentration (value) respectively. All statistical analysis was carried out using

Minitab 16 software.

TABLE 4.1a: Uncoded values low, middle and high concentration setting for three variables in Box Behnken design for TPP optimization.

Parameters Value

Salt conc. pH E:B

Low (-) 20 2.0 05 Medium (0) 50 5.5 175

High (+) 80 9.0 30 Salt – Salt concentration in %; pH- pH of the mixture; E:B – Enzyme to butanol ratio

Various measurements for each experimental set were carried out in repeat

blocks. The relationship of the independent variables (X1-X3); and their response

(Y), was calculated by the second order polynomial equation, the details of which

are discussed in chapter 3, section 3.3.6.

TABLE 4.1b: Box Behnken design matrix with the coded values chosen for the experimental variable for TPP optimization

RunOrder Salt pH E:B RunOrder Salt pH E:B

1 0 0 0 16 0 0 0 2 0 0 0 17 0 - + 3 - 0 - 18 0 + - 4 0 0 0 19 - + 0 5 + + 0 20 0 + + 6 - - 0 21 + + 0 7 0 + - 22 0 - - 8 - 0 + 23 + - 0 9 0 0 0 24 - 0 - 10 + 0 - 25 0 0 0 11 + 0 + 26 - - 0 12 - 0 + 27 0 - +



13 + 0 + 28 + 0 - 14 0 - - 29 + - 0 15 - + 0 30 0 + +

Salt – Salt concentration in %; pH- pH of the mixture; E:B – Enzyme to butanol ratio

In the present case, three independent variables viz. salt, pH, E:B are used

instead of five. The purified fraction obtained from TPP was used for further

characterization studies. For simplicity, it is denoted as Partially Purified Laccase

from Basidiomycota sp. L-168 (PPLB)

4.3.4 Laccase Characterization

a) Effect of pH on enzyme activity

The influence of pH on PPLB activity was studied spectrophotometrically.

As reported in earlier literature, laccases have variable optimum pH values for

different substrates (Baldrian 2006; Sadhasivam et al. 2008). In present study,

three substrates ABTS, Syringaldazine, and guaiacol were used and pH optima

determined over a range of pH 2 to 10. The entire pH range was maintained by

using 0.1 M Britton- Robinson buffer. It was made by mixing 0.1 M boric acid,

0.1 M acetic acid and 0.1 M phosphoric acid and the pH was adjusted with 0.5 M

NaOH. The assays for the different substrates were conducted as described under

assay methods in chapter 2 and earlier section 4.3.2. All assays were done in

triplicate. The influence of pH on the laccase activity was also determined at

different pH values ranging from 2.6 to 9.0, using 0.1 M concentrations of the

following buffer systems: citrate phosphate (pH 2.6, 3, 3.6, 4, 4.6, 5, 5.6, 6, 6.4

and 7), sodium acetate (pH 3.6, 4, 4.6, 5 and 5.6), sodium phosphate (pH 6.3, 7.3,

8.0) and Tris-HCl (pH 8.0 and 9.0).

b) pH stability



To study pH stability, Britton Robinson buffer (0.1 M) adjusted to

different pH values with 0.1M NaOH was used. The stability of the enzyme in a

pH range from 2.0 to 10.0 was tested. The PPLB enzyme solutions was incubated

at different pH values for 1, 24 and 48 h at room temperature and then the residual

enzyme activity was assayed using ABTS as substrate at the optimum pH 2.0.

% 푅푒푠푖푑푢푎푙 푎푐푡푖푣푖푡푦 =푇표푡푎푙 푒푛푧푦푚푒 푎푐푡푖푣푖푡푦 푎푓푡푒푟 푝퐻 푡푟푒푎푡푚푒푛푡푇표푡푎푙 푒푛푧푦푚푒 푎푐푡푖푣푖푡푦 푏푒푓표푟푒 푝퐻 푡푟푒푎푡푚푒푛푡 ∗ 100

c) Effect of temperature on enzyme activity

The effect of temperature on PPLB activity was spectrophotometrically

determined following the laccase-catalyzed oxidation of 1 mM ABTS at

temperatures ranging from 25 °C to 100 °C at 5 °C intervals. Britton Robinson

buffer (0.1 M) was used for all the reactions, at the optimum pH for ABTS of

BspL-168 laccase i.e. pH 2. The substrate was incubated for at least 10 minutes at

the different temperatures before the enzyme was added to start the reaction.

Optimum temperature profile was studied using a Shimadzu UV-1800

spectrophotometer (with UV probe software) with temperature control assembly

and temperature controlled cuvette holder. The Shimadzu spectrophotometer was

equilibrated to assay temperature for 10 minutes prior to assaying. Samples,

reagents and quartz cuvette used for assay, were equilibrated to assay temperature

by placing them in the water bath at the relevant temperature for 10 minutes.

The activation energy was calculated by using Arrhenius plot. It was

constructed by plotting the ln v versus the inverse temperature, 1/T (Kelvin). The

resulting negatively sloped line was used for finding the activation energy. The

slope of the line is equal to the negative activation energy divided by the gas



constant, R. The slopes were calculated by linear regression analysis using

GraphPad Prism 6 software. The Arrhenius equation is

푘 = 퐴푒

The Arrhenius equation can be rearranged by taking the logarithm of both sides

yielding the above equation in the form y=-mx+b as follows

ln푘 =−퐸푎푅푇

+ 푙푛퐴

Where y = lnk, m= -Ea/RT, x =1/T, b= lnA. R is the gas constant (8.314 J mol-1

K-1) and T is the absolute temperature (K).

d) Thermal stability

The thermal stability of PPLB was investigated at different temperatures

between 40 and 70 ºC for varying periods of time in a temperature controlled

circulating water bath (Medica Equibath #8506). Aliquots of the enzyme were

transferred to pre-warmed tubes with a micropipette. The incubation was carried

out in sealed tubes to prevent change of volume of the sample and consequently,

the enzyme concentration due to evaporation. The temperature of the water bath

was verified with a calibrated mercury thermometer. The water bath was covered

with a lid to prevent evaporative cooling. The enzyme solution was incubated at

different combinations of pH and temperature. The pH values selected to study

the deactivation of laccase were 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0. At each pH the

deactivation was carried out at temperature of 40, 50, 60 and 70 0C.

The come-up time for the tubes was determined by placing a calibrated

mercury thermometer in the solution at the center of the tube and recording the

time required for the solution in the tube to reach that of the water bath. This time



was determined to be 15s. Following heating, aliquots of enzyme were withdrawn

at regular time intervals, cooled on ice and residual activity was assayed. The

stability of the enzyme was expressed as per cent residual activity (%RA). The

data obtained from the thermal stability profile was used to analyze

thermodynamic parameters related to the laccase activity.

Thermal deactivation kinetics and estimation of the deactivation energy

The thermal deactivation rate constant (kd) for first-order reaction were

determined from the slopes of the inactivation time courses according to

following equation 1

log( ) = −(.

)푡 ……Equation 1

Where, A0 is the initial enzyme activity and A is the activity after heating

for time t. The slopes of these lines were determined by linear regression. The

calculated rate constants were plotted in Arrhenius plots. The activation energy

for deactivation (Ed) were calculated from the slopes of the Arrhenius plots of

ln(k) versus 1/T according to eq 2

ln(푘 ) = − + 푐 ……Equation 2

Where R is the gas constant (8.314 J mol-1 K-1) and T is the temperature in

Kelvin. The slopes were calculated by linear regression analysis using GraphPad

Prism 6 software.

The inactivation data, in terms of the two parameters kd and Ed, was used

to express D and z values by following calculations according to Anthon et al.

(2002). A ‘D’ value, the time required to reduce the enzyme activity to 10% of its

original value, is directly related to an inactivation rate constant kd by equation 3:



푘 = . …………………….. Equation 3

The log D is linearly related to the inverse of temperature (0C). The z value

(temperature rise necessary to reduce D-value by one logarithmic cycle) was

calculated from the slope of plot between log D versus T (ºC) using equation 4

푆푙표푝푒 = …………………….Equation 4

The half-life of the laccase (t1/2, min-1) was determined from the relationship

푡 ⁄ = ………………………Equation 5

Estimation of the thermodynamic parameters

The change in enthalpy (ΔHº, kJ mol-1), free energy (ΔGº, kJ mol-1) and

entropy (ΔSº, J mol-1 K-1) for thermal denaturation of laccase were determined

according to Pal and Khanum (2010) using the following equations.

퐻 = 퐸 − 푅푇 …………………Equation 6

퐺 = −푅푇푙푛 ..

………………Equation 7

푆 = …………………..Equation 8

Where Ed is the activation energy for denaturation, T is the corresponding

absolute temperature (K), R is the gas constant (8.314 J mol-1 K-1), h is the Planck

constant (11.04 X 10-36 J min), kB is the Boltzman constant (1.38 X 10-23 J K-1)

and kd is the deactivation rate constant (min-1).

e) Organic solvent stability

The organic solvent-tolerance was examined at room temperature for 1h

and 16 h. Various organic solvents, such as acetonitrile, acetone, ethanol,

methanol, and propanol were tested at concentration of 10, 20, 30 and 40% (v/v)



(Huang et al. 2011). The PPLB activity was assayed as described in earlier

sections with ABTS.

f) Substrate specificity

Twelve other phenolic compounds (Syringaldazine, pyrocatechol,

resorcinol, hydroquinone, pyrogallol, vanillic acid, gallic acid, tannic acid, o-

cresol, p-cresol, m-cresol, and tyrosine) were also tested as substrates for PPLB.

This was qualitatively explored by changes in the optical absorbance spectra of

the reaction mixtures at the optimum pH of each substrate. The reaction mixture

contained 100 µM potential substrates, along with fixed concentration of partially

purified enzyme in a total volume of 2 ml at respective pH. One unit of enzyme

was defined as the amount of enzyme required to convert 1 µmol of substrate to

product in 1 minute under the assay conditions employed. All assays were

performed in triplicates.

g) Kinetic Constants

The specificity of laccase towards different phenolic compounds was

investigated. Substrates were chosen according to the positions of substituents on

the phenolic ring and the type and/or length of the substituents.

Kinetic studies were conducted for two most commonly used substrates

(ABTS and guaiacol) that were oxidized by PPLB. At least eight different

substrate concentrations for each substrate were assayed for the laccase at the

optimal pH values for each. Triplicates of each assay were done. The molar

extinction coefficients for ABTS were 420 = 36,000 M-1cm-1 and 432 = 12,100 M-

1cm-1 for guaiacol. All the laccase catalytic assays were done at room temperature.



Initial velocity was measured in 3-mL glass cuvette with 1 cm path lengths.

Reactions were initiated by adding laccase. Initial rates were calculated from the

linear portion of the progress curve.

The data was subjected to nonlinear regression analysis (Graph Pad Prism

6 Software) using the Michaelis-Menten equation and the kinetic parameters (Km,

Vmax) were determined. The Lineweaver-Burk plots were also constructed for

the two substrates. The kcat for each substrate was also determined and the

kcat/Km calculated. The wavelengths for measuring laccase activity with the

above mentioned substrates were determined spectrophotometrically by allowing

a reaction with laccase, performing a spectral scan on the Shimadzu UV-1800

spectrophotometer (with uv probe software) in the visible region (300 nm to 700

nm). The wavelength where maximal absorbance increase over time was observed

was recorded and used to measure the oxidation rate during further studies.

h) Inhibition study

Seven potential inhibitors were evaluated for their activity toward PPLB

using (ABTS) as substrate. These inhibitors included: sodium azide (NaN3) that

complexes to the copper in the active site, cysteine that is a sulfhydryl organic

compound with a reducing effect on the copper-containing active site of laccase,

EDTA that exhibits metal chelating properties and three halides (I-, Cl-, F-) of

different sizes.

The inhibitory action on enzymes was tested by preincubation of the

enzyme with the inhibitor substance for 5 min to ensure complete inhibition. The

kinetic measurements were started by the addition of ABTS as substrate and the



formation of product was monitored at 420 nm. Different concentrations (see

Table 4.14) of these inhibitors were tested and the percentage of inhibition was

calculated (Younes et al. 2007).

i) Effect of metal ions

The effect of several metal ions including Cu2+, Mg2+, Mo2+, Ni2+, Co2+,

Li2+, Ca2+, Mn2+, Cd2+ and Al3+ on laccase activity was investigated (Sadhasivam

et al. 2008). The 2 ml of reaction mixture contained appropriate dilution of metal

ion so as to maintain 0.1 mg/ml concentration along with 50 µl of enzyme

solution. The reaction was initiated by adding ABTS as substrate and the residual

activity was determined. The reaction mixture containing all except the metal ion

served as a control.

j) Spectroscopic studies

The UV–vis absorption spectrum of PPLB (1 mg ml-1 in 0.1 M phosphate

buffer, pH 5.0) was recorded at room temperature on a Shimadzu UV-

spectrophotometer (model UV-1800 equipped with UV probe software) in 1 cm

path length quartz curettes (Sadhasivam et al. 2008).

k) Determination of metal content of enzyme

Zinc, copper, iron, manganese and magnesium contents of PPLB were

determined by Atomic Absorption Spectrometry at NABL accredited Laboratory,

Jain Research and Development, Jain Irrigation Systems Limited, Jalgaon,

Maharashtra.

l) Molecular Weight Determination by SDS-PAGE



Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-

PAGE), or denaturing gel electrophoresis, was used to monitor the development

of the purification process, to determine homogeneity and to determine the

relative molecular mass of the laccase enzyme. SDS-PAGE was carried out on a

12% resolving gel containing 0.1% SDS and a 4% stacking gel (details of the

method are given in Appendix I and II). The lyophilized samples were dissolved

in a minimum amount of phosphate buffer, and subjected to denaturing and non-

denaturing gels.

The approximate molecular mass of the laccase was determined by

calibration against broad range molecular weight markers (Protein pre stained

standards, Genei, Banglore), which contained the proteins myosin (200 kDa),

phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (43

kDa), carbonic anhydrase (29 kDa), soybean trypsin inhibitor (20 kDa), lysozyme

(14.3 kDa) and aprotinin (6.5 kDa). After treated with sample buffer and boiled at

100°C for 10 minutes, samples were denatured and loaded onto the gel. The gel

was installed on a Genei Mini-Vertical Electrophoresis apparatus (Genei scientific

instruments, Bangalore). A voltage of 150 V was applied though a Genei power

pack and the gels were left running for 45 minutes to achieve sufficient migration.

Proteins were visualized by staining with Coomassie Brilliant Blue-R250

(Genei). Gels were then destained with a mixture of acetic acid and ethanol (40%:

10%). The determinations of the relative molecular masses of the denatured

laccase enzyme was based on the relative distance of migration of molecular

standards.



푅푓 =Distance migrated by protein

Distance migrated by tracking dye

Molecular weight of laccase was calculated by using the standard graph

constructed by plotting Rf values of the marker proteins against the logarithm of

molecular weight of marker proteins.

m) Native PAGE and Zymogram analysis

SDS-PAGE revealed the presence of one protein. Non-denaturing PAGE

was performed to ascertain which protein correlated to laccase activity. The non-

denaturing gel with duplicate sets of samples was bisected and half was stained

with Coomassie Brilliant Blue R-250, the other half was stained with guaiacol to

determine which band correlated to PPLB activity.

Native PAGE (non-denaturing PAGE) was carried out in alkaline pH

condition, in which anionic detergent SDS was omitted from gels and the samples

were not incubated prior to the loading of the gels. Separating and stacking gels

were 9 and 4% acrylamide, respectively. Samples (15 µl) were loaded on the gel

and after electrophoretic separation the gels were stained as mentioned above.

Activity Staining- zymogram analysis PPLB activity stain was performed with 2,2’-Azino-bis(3-

ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS - Fluka) as the

substrate, using agarose to localize the ABTS oxidation by laccase. Non-

denaturing gels were allowed to stand in 100 ml of succinate-lactate buffer (100

mM, pH 4.5) for 10 minutes to allow for pH adjustment. The acrylamide gel was

removed from the buffer and placed on a clean surface. Substrate (1ml of 10 mM

ABTS) was spread evenly over the surface of the gel and allowed to stand for 2



minutes. Laccase activity bands were indicated by the development of a green

colour.



4.4 Results

When ammonium sulphate precipitation was used for purification of

laccse from BspL-168, maximum laccase recovery was obtained at 80%

saturation (Table 4.2). The enzyme activity from the precipitate was determined

by using ABTS as substrate. The total proteins were determined from calibration

curve of Bovine Serum Albumin (BSA) using Lowry method.

TABLE 4.2: Ammonium sulphate precipitation for laccase purification

Step Volume ml

Total proteins mg

Total activity Units (U)

Specific activity U/mg

Yield %

Purification fold

1 Crude laccase

50 274.3 1832.5 6.68 100 1

2 80 % saturation

2 1.4 60.908 43.51 3.32 6.512

It is evident from Table 4.2 that, ammonium sulphate precipitation about

6.5 fold purification was achieved at 80 % saturation with a yield of 3.32%.

However, significantly higher laccase purification as well as recovery was

attained by use of recent technique of Three Phase Partitioning (TPP). Therefore,

in further experiment TPP was preferred for laccase purification and the

parameters affecting TPP were first optimized with RSM technique.

Laccase Purification with TPP

The experimental results and the predicted values of Box Behnkan design

for optimization of laccase purification by TPP are presented in Table 4.3. The

experimental values are significantly close to predicted values. The standardized

residuals which are calculated as value of a residual divided by an estimate of its

standard deviation, help in detecting outliers. As evident from Table 4.3, the



values of standardized residuals fall within ±2 indicating there are no outliers in

the data set (Minitab 16 user manual).

TABLE 4.3: Analysis of laccase purification based on the Trials of the Box Behnken design

Run Order

Experimental response

Predicted response

Standardized Residuals

1 13.054 12.881 0.197 2 12.373 12.881 -0.579 3 0.937 0.318 0.814 4 12.901 12.881 0.023 5 6.213 5.982 0.303 6 0.04 0.662 -0.819 7 1.985 2.409 -0.557 8 7.082 8.156 -1.412 9 13.3 12.881 0.477

10 8.013 7.562 0.593 11 9.31 8.957 0.465 12 9.267 8.156 1.462 13 7.55 8.957 -1.850 14 4.098 4.501 -0.530 15 0.264 0.207 0.075 16 14.327 12.881 1.647 17 7.121 5.727 1.833 18 1.613 2.409 -1.047 19 0.316 0.207 0.143 20 9.148 10.415 -1.666 21 7.009 5.982 1.350 22 5.108 4.501 0.798 23 3.017 2.932 0.112 24 0.753 0.318 0.572 25 11.332 12.881 -1.765 26 0.028 0.662 -0.835 27 5.553 5.727 -0.229 28 7.074 7.562 -0.642 29 2.681 2.932 -0.330 30 11.478 10.415 1.399

Model fitting

The application of linear model suggested the need of applying a higher-

order model. When the full quadratic model was fitted, the p-value for lack of fit

was 0.207, suggesting that this model adequately fits the data. The error term, s =

0.9615, was also smaller as it reduced the variability accounted for by error



(Table 4.4). To test goodness of fit of regression equation, R2 was also

considered. As for a good statistical model, R2 should be close to one; the

tabulated results indicate a satisfactory adjustment of full quadratic model to

experimental data as compared to other models. 96.95% of variability in

dependent variables could be explained by this model, significant p value for lack

of fit and smaller error term (s) were the reasons to select the model.

TABLE 4.4: Evaluating fit model for the data for TPP optimization Sr Model R-sq % p Value for

lack of fit Error term (s) Adequacy of model

1 Linear 23.72 0.000 4.2916 Not adequate 2 Linear + Square 86.41 0.000 1.9261 Not adequate 3 Linear + interaction 32.77 0.000 4.2836 Not adequate 4 Full Quadratic 96.95 0.207 0.9615 Adequate

The ANOVA table for the Box Behnken design (Table 4.5) summarizes

the linear terms, the squared terms, and the interactions. The small p-values for

the interactions and the squared terms suggest there is curvature in the response

surface. Small p-values (<0.05) of the single factors as well as all squared effects

and interaction effects indicate that these had a significant effect on the

purification.

Multiple regression analysis of the experimental data resulted in the

following second order polynomial regression equation for the purification. The

values of regression coefficients are given in Table 4.6 and can be used for

predicting purification;

Y = -23.0077 + (0.6491*X1) + (3.9930* X2) + (5.5770* X3) + (-0.0055* X12) + (-0.4457* X2

2) + (-1.0611* X3

2) + (0.0083* X1* X2) + (-0.0430* X1* X3) + (0.3874* X2* X3)

Where, the X1, X2 and X3 represents three parameters; salt concentration,

pH and E:B respectively.



TABLE 4.5: Analysis of variance (ANOVA) for the Box Behnken design for TPP optimization

Source DF Seq SS Adj SS Adj MS F P Regression 9 587.079 587.079 65.231 70.55 0.000 Linear 3 156.686 233.174 77.725 84.06 0.000 Salt 1 64.722 178.783 178.783 193.35 0.000 pH 1 6.734 98.035 98.035 106.02 0.000 E:B 1 85.230 27.302 27.302 29.53 0.000 Square 3 380.507 380.507 126.836 137.17 0.000 Salt*Salt 1 148.958 182.771 182.771 197.66 0.000 pH*pH 1 211.249 220.164 220.164 238.10 0.000 E:B*E:B 1 20.299 20.299 20.299 21.95 0.000 Interaction 3 49.886 49.886 16.629 17.98 0.000 Salt*pH 1 6.146 6.146 6.146 6.65 0.018 Salt*E:B 1 20.756 20.756 20.756 22.45 0.000 pH*E:B 1 22.984 22.984 22.984 24.86 0.000 Residual Error 20 18.493 18.493 0.925 Lack-of-Fit 3 4.247 4.247 1.416 1.69 0.207 Pure Error 17 14.246 14.246 0.838 Total 29 605.572

DF- Degree of freedom; Seq SS - Sequential sums of squares; Adj SS- Adjusted sum of squares; Adj MS- Adjusted Mean squares; F- F statistics; P- P value

TABLE 4.6: Model Coefficients estimated by multiple linear regression

Term Coef SE Coef T P Constant -23.0077 1.99560 -11.529 0.000 Salt 0.6491 0.04668 13.905 0.000 pH 3.9930 0.38779 10.297 0.000 E:B 5.5770 1.02636 5.434 0.000 Salt*Salt -0.0055 0.00039 -14.059 0.000 pH*pH -0.4457 0.02889 -15.431 0.000 E:B*E:B -1.0611 0.22647 -4.685 0.000 Salt*pH 0.0083 0.00324 2.578 0.018 Salt*E:B -0.0430 0.00907 -4.738 0.000 pH*E:B 0.3874 0.07771 4.986 0.000

Coef- Coefficients; SE Coef – Standard error of Coefficient; P-Probability value; T- T value



FIGURE 4.3: Contour plots for laccase recovery from TPP (SA – specific activity)

The contour plot (Figure 4.3) depicts how a response variable relates to

two factors based on a model equation maintaining others at fixed level. A circle

in the square of contour plot shows that response was sensitive to that factor. As

observed from the main effect and interaction plots (Figure 4.4 and 4.5), the

laccase recovery increases with enhancement in the ratio of enzyme to butanol

ratio (E:B) while it increases with increase in both salt concentration and pH, but

up to a certain limit, and then gradually decreased in both. In both the cases, sharp

decline was noted on either sides of middle value.



FIGURE 4.4: Main effect plot for laccase recovery from TPP (SA– specific activity)

FIGURE 4.5: Interaction plot for laccase recovery from TPP (SA–specific activity)

As evident from the desirability value d= 0.98834 (Figure 4.6), the goal

for the response specific activity (SA) to maximize the laccase recovery was

achieved. Since only one response was measured, the value of individual

desirability (d) equals composite desirability (D).

805020

10

8

6

4

29.05.52.0

3.001.750.50

10

8

6

4

2

Salt

Mea

n

pH

E:B

Main Effects Plot for SAData Means

10

5

0

3.001.750.50

9.05.52.0

10

5

0

805020

10

5

0

Salt

pH

E:B

205080

Salt

2.05.59.0

pH

0.501.753.00

E:B

Interaction Plot for SAData Means



FIGURE 4.6: Optimization plot for laccase yield with TPP

(E:B–Enzyme:Butanol ratio SA- Specific activity)

The Residual plots generated for Box Behnken design through Minitab 16

software (Figure 4.7) reveals that the goodness of model fit in regression and

ANOVA as well as the least squares assumptions are met by the data in present

investigation and the residuals are normally distributed.

FIGURE 4.7: Residual plot for laccase yield in TPP. (SA- Specific activity)

The optimum values of parameters affacting TPP as obtanied from

optimization plot were used for further purification studies.

CurHigh

Low0.98834D

New

d = 0.98834

MaximumSA

y = 13.8934

0.98834DesirabilityComposite

0.50

3.0

2.0

9.0

20.0

80.0pH E:BSalt

[52.7273] [6.1717] [2.6607]

210-1-2

99

90

50

10

1

Residual

Per

cent

129630

1

0

-1

Fitted Value

Res

idua

l

1.60.80.0-0.8-1.6

6.0

4.5

3.0

1.5

0.0

Residual

Freq

uenc

y

30282624222018161412108642

1

0

-1

Observation Order

Res

idua

l

Normal Probability Plot Versus Fits

Histogram Versus Order

Residual Plots for SA



TABLE 4.7: Purificaiton of laccase from BspL-168

Step Volume ml

Total proteins mg

Total activity Units (U)

Specific activity U/mg

Yield %

Purification fold

1 Crude enzyme preparation

430 2358.98 15759.50 6.681 100 1

2 Ultra filtration 250 467.5 12360 26.439 78.4 3.96 3 Concentration 100 274 12100 44.161 76.8 6.61 4 TPP 10 0.2 4190 20950 26.6 3135.76

The laccase was purified to homogeneity from the YEMM medium

supplemented with 2 mM xylidine. The results obtained from the different steps

of laccase purification are summarized in Table 4.7. It is evident from the above

table that by TPP about 3135.76 fold purification was achieved with a yield of

26.6%.

Although, in the present study, considerable purification was attained

using salting out, this conventional technique has many disadvantages. Moreover,

the resolving power of salting out is not high. Besides many disadvantages in

salting out the dehydrated protein sinks to bottom due to more density than

solution. Therefore, it is not only tedious and time consuming to collect the

precipitate but there is possibility of loss of precipitate during the process. In

comparison, as described in method section, TPP was found to be more

convenient, rapid, simple and effective method for protein purification. The

purified fraction obtained from TPP was used for further characterization studies.

A comparison between conventional salting out using ammonium sulphate

and TPP is given in Table 4.8. Nevertheless, during the step of salting out, the

laccase activity was separated from most impurities as indicated by considerably

high specific activity (see Table 4.2).



TABLE 4.8: Ammonium sulphate precipitation vs TPP as a method of protein purification: a comparison Conventional

salting out TPP

Protein dehydration Less High

Salt content with Protein High Very less

Require desalting Yes No Dehydrated protein More dense than solution so

sink Less than so floats

Effect of temperature Yes Very less

Centrifugation Required No need

Advantage -- Denaturation of impurities by distortion of proteins

During the first step of ultra filtration (through 30 KDa cassttee), some of

the impurities were separated from laccase activity (Plate 4.1). Further removal of

impurities by concentration lead to 6 folds purification. The third step (TPP)

permitted to separate laccase from the other proteins present in the fraction,

removed almost impurities and yielded a single fraction with high laccase activity

(Plate 4.2). However, one of the limitations was appearance of a brown pigment

secreted in the secondary metabolism by the organism in xylidine induced

conditions. Surprisingly, at the end of the process, laccase was purified 3135 fold.

The overall yield of the purification was about 26%. The homogeneity of the

purified laccase was indicated by appearance of a single band on native and SDS

PAGE (Plate 4.4)

The purification of laccase from BspL-168 was also attempted by MLF-

TPP (Plate 4.3). However, in comparison to TPP, very less purification was

attained in MLF-TPP (data not shown).



The effect of pH on laccase activity

Three substrates were used to determine the effect of pH on laccase

activity, as the pH optima of laccases are highly dependent on the substrate

(Baldarian 2006). The entire pH range of pH 2 to 10 was maintained by using 0.1

M Britton- Robinson buffer. The PPLB showed the optimal pH for

syringaldazine, guaiacol and ABTS oxidation as 4.5, 2.5 and 2.0, respectively.

The pH profile for the various substrates is shown in Figure 4.8. The enzyme

exhibited a comparatively broader pH profile with syringaldazine than the other

substrates tested.

To determine the influence of buffer composition, the laccase activity was

also determined at different pH values using citrate phosphate, sodium acetate,

sodium phosphate and Tris-HCl buffer. However, no influence of buffer

composition was found on laccase activity in comparison to activity in presence

of BR buffer.

FIGURE 4.8: The pH profile for PPLB with various substrates The pH optimum for the activity of the induced laccase from Basidiomycota sp. L-168 was evaluated using ABTS, Guaiacol and Syringaldazine as the substrates. A value of 100 was ascribed to the highest laccase activity in each case and the other activities were expressed as a percentage of this value.

0

20

40

60

80

100

0 2 4 6 8

Act

ivity

% o

f max

imum

pH

Syringaldazine

Guaiacol

ABTS



pH stability

In general at all tested time durations the per cent residual activity

increased with increase in pH, maximum recorded at pH 8 and then decreased.

FIGURE 4.9: The stability of PPLB at different pH

In 1 h pH treatment, about 30% enzyme activity was lost at pH 2 (Figure

4.9); however, with the increase in pH, the per cent residual activity also

increased. At pH values ranging from 4 to 6 and 7 to 10 after 1 h, the enzyme

retained about 95% and 100% activity, respectively. PPLB exhibited pH optima

for ABTS oxidation at pH 2. However, the enzyme was found to be least stable at

this pH while showed stability at higher pH in neutral to alkaline range.

Nevertheless, even at pH 2 after 1 h, the enzyme showed 70% residual activity.

The long-term pH stability of the partially purified laccase, after 24 and 48

h incubation, was also investigated. The effect of pH on the long term enzyme

stability is illustrated in Figure 4.9. After both 24 and 48 h incubation, the

maximum stability of laccase was recorded at pH 8, retaining 93.17 and 92.56%

activity, respectively.

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10

% R

esid

ual a

ctiv

ity

pH

% Residual activity after 1 h % Residual activity after 24 h % Residual activity after 48 h



PPLB retained more than 85% and 90% of its activity in solutions with pH

from 7 to 8 after 24 h incubation at room temperature, respectively. In contrast, at

pH 2.0 the enzyme lost about 97 and 100 % of activity after 24 and 48 h

respectively.

Thus PPLB was found to be most stable at pH 8 and least stable at pH 2

(100% activity lost in 48 h). Unlike most of fungal laccases, BspL-168 laccase

was found to be active in acidic range and shows stability from neutral to alkaline

pH range.

Effect of temperature on enzyme activity

FIGURE 4.10: The effect of temperature on the activity of PPLB

The effect of temperature on the rate of laccase catalyzed reaction was

determined at temperatures between 25 to 100 oC. The PPLB enzyme activity

increased with increase in temperature with optimum at the temperature of 70 °C

(Figure 4.10). On further increase in temperature, the enzyme activity declined

gradually. The temperature range where the enzyme is active is remarkably wide,

ranging from 25 °C to 100 °C. Interestingly, even at temperature of 100 ºC

significant activity is restored (68.75% of the maximum activity).

45

55

65

75

85

95

105

20 40 60 80 100

Act

ivit

y %

of m

axim

um

Temperature o C



The activation energy (Ea) calculated from the Arrhenius plot, 2.868 kcal

mol-1 (11.999 KJ mol-1), was in the range that is characteristic of a typical

enzymatic reaction (Figure 4.11).

FIGURE 4.11: Arrhenius plot of ln v vs 1/T for determination of activation energy of laccase from BspL-168 using ABTS as substrate Thermal stability

The thermostability of PPLB at various temperature and pH combinations

is depicted in Figure 4.12. The enzyme showed no loss in activity after

incubation at 40 ºC and 50 ºC during the test period. At 60 ºC and above, the

activity decreased with increasing temperature. However, the stability of laccase

was found to be pH dependent. In presence of pH 2, at 40 ºC, 3% residual activity

was noted after 150 minutes. With increase in pH from 2 to 8, at 40 ºC the

residual activity gradually increased. The maximum stability at 40 ºC was

observed at pH 8 with 89.6% residual activity.



Note: In each graph, the the number in the legand indicates temperature and pH combination. The first two digits represents temperature while the last represents pH value.

FIGURE 4.12: First order thermal deactivation of PPLB at different pH values



Similarly, in presence of pH 2, at 50 ºC only 0.25% residual activity was

noted after 150 minutes. With increase in pH from 2 to 8, the residual activity

gradually increased. The maximum stability at 50ºC was observed at pH 8 with

99.87% residual activity. Similar effect of pH on temperature stability of laccase

was observed at 60 and 70 ºC. However, the maximum stability at 60ºC was

observed by pH 7 with 76.17% residual activity and at 70 ºC by pH 6 with

13.17% residual activity. Although the residual activity at 60 ºC was much higher

than at 70 ºC, in comparison to pH 2 the residual activity retained for 70 ºC

(98.48%) was higher than that for 60 ºC (86.9%).

The plots of the log of residual activity versus heating time reasonably

fitted to a straight line with good R2 values over most of the time courses (Table

4.9). It indicates that the thermal inactivation of laccase followed first-order

kinetics. In general with increasing temperature the t1/2 and D-value decreased

while the rate of enzyme deactivation (kd) increased (Table 4.9).

The thermostabilizing effect of pH on PPLB is evident from following

observations – i) the t1/2 and D-values at all temperatures respectively increased

with increase in pH from 2 to 8 and ii) the rate of enzyme deactivation (kd) at all

temperatures respectively decreased with increase in pH from 2 to 8 indicating the

thermostabilizing effect of pH since a lower rate constant means the enzyme is

more thermostable.



TABLE 4.9: Inactivation kinetic parameters of laccase towards thermal processes

pH Temperature R2 Kd (min-1)

D value (min)

t1/2 (min)

Ed kcal / mol

z value oC

2.0

40 0.962 0.0213782 107.73 32.42 21.254 23.403 50 0.937 0.1668785 13.80 4.15

60 0.936 0.4399333 5.23 1.58 70 0.807 0.4111214 5.60 1.69

3.0

40 0.745 0.0035315 652.13 196.27 30.542 16.120 50 0.981 0.0140063 164.43 49.49 60 0.971 0.0818478 28.14 8.47 70 0.719 0.2291845 10.05 3.02

4.0

40 0.982 0.0013813 1667.22 501.79 32.082 15.266 50 0.970 0.0040846 563.83 169.70 60 0.975 0.0152840 150.68 45.35 70 0.956 0.1357490 16.97 5.11

5.0

40 0.959 0.0010651 2162.17 650.76 30.618 15.936 50 0.787 0.0009483 2428.62 730.96 60 0.994 0.0089494 257.34 77.45 70 0.944 0.0622440 37.00 11.14

6.0

40 0.976 0.0009575 2405.16 723.90 28.131 17.201 50 0.840 0.0003739 6158.88 1853.67 60 0.915 0.0022203 1037.27 312.19 70 0.908 0.0458359 50.24 15.12

7.0

40 0.988 0.0008097 2844.38 856.09 33.351

14.497 50 0.844 0.0002308 9977.51 3002.99

60 0.961 0.0019923 1155.93 347.91 70 0.969 0.0786282 29.29 8.82

8.0

40 0.968 0.0007432 3098.84 932.67 45.151 14.573 50 0.857 0.0000079 292004.07 87886.15 60 0.987 0.0041970 548.72 165.15 70 0.980 0.1215842 18.94 5.70

Interestingly, in comparison to half life at pH two, 28.76 and 21,177.38

fold increase in half life at pH 8 for the temperatures 40 ºC and 50 ºC,

respectively were recorded. Similarly, increase in half life of 220.2 fold at pH 7

for the temperatures 60 ºC and 8.9 fold at pH 6 for the temperatures 70 ºC was

observed. This clearly revealed that as compared to acidic conditions (pH 2) at



higher pH near neutrality (pH 6, 7 and 8) the enzyme was more stable at every

temperature studied. The PPLB exhibited high stability at 50 and 60 ºC. The half-

life of laccase at 60 ºC was about 5 h whereas at 50 ºC, it was remarkably high

(1464 h). At temperature of 70 ºC, however, half life was shorter (15 min).

Also, the time needed to reduce the enzyme activity by 90%, i.e. D-value,

increased by similar fold as half life at higher pH further proving its

thermoprotecting effect on laccase. The temperature increase required to decrease

the D-value by one log cycle i.e. z-value, was calculated from the plot of Log D

vs temperature (oC) (Figure 4.13). The z values for laccase at different pH are

presented in Table 4.9.

FIGURE 4.13: Temperature dependence of the decimal reduction of partially

purified laccase preparation to calculate z-values

The activation energy for deactivation (Ed) calculated from the slopes of

the Arrhenius plots (Figure 4.14) showed significant differences with different

pH values. A larger value of Ed indicates that more energy is required to

inactivate the enzyme. It is evident from the Table 4.9 that Ed increases with rise

in pH from 2 to 8, maximum being observed at pH 8 (45.15 kcal/mol) indicating a

very stable and compact laccase that is resistant to heat denaturation. All the



above results indicate that the stabilization of laccase at neutral pH range was of

conformational origin.

FIGURE 4.14: Arrhenius plot to calculate the thermal inactivation rates constant (kd) for PPLB

In general with increase in temperature, the enzyme showed a decreasing

trend in enthalpy of denaturation (ΔHº). For example, with pH 8, the enzyme had

a range of 186.31 to 186.06 kJ/mol of ΔHº at 40-70 ºC showing a decreasing trend

with increase in temperature (Table 4.10). The fact that ΔHº value decreases with

increase in temperature reveals that less energy is required to denature enzyme at

high temperature. The ΔHº of laccase was also affected by pH. At 40ºC, the ΔHº

was 86.32 kJ mol-1 with pH 2 while at the same temperature it was 186.31 kJ mol-

1 with pH 8. Thus ΔHº in later case was 2.15 times more than in the former case.

It reveals that more energy is required for thermal denaturation of enzyme at

higher pH indicating the resistance of enzyme towards thermal unfolding at

higher temperatures which indirectly indicates its stability.

The free energy of thermal denaturation (ΔGº) for laccase with pH 8 was

38.96 kJ/mol at 40 ºC which initially increased and then decreased with increase

in temperature. The positive values of entropy of inactivation (ΔSº) at each

temperature indicate that there is no significant aggregation since negative values



of entropy indicates protein aggregation. At all temperatures studied, the value of

ΔSº at pH 8 was much higher than at pH 2. Furthermore, the ΔGº values, which

are measures of the spontaneity of the inactivation processes, are lower than the

ΔHº values. This is due to the positive entropic contribution during the

inactivation process.

TABLE 4.10: Thermodynamic studies for thermal inactivation of laccase Temperature (ºC) pH G0 kJ mol-1 H0 kJ mol-1 S0 J mol-1

40

2 30.22 86.32 179.16 3 34.91 125.18 288.29 4 37.35 131.63 301.06 5 38.03 125.50 279.33 6 38.31 115.10 245.22 7 38.74 136.94 313.57 8 38.96 186.31 470.52

50

2 25.75 86.24 187.20 3 32.40 125.10 286.85 4 35.72 131.54 296.54 5 39.64 125.42 265.45 6 42.14 115.01 225.51 7 43.44 136.85 289.08 8 52.51 186.23 413.80

60

2 23.94 86.16 186.74 3 28.60 125.02 289.40 4 33.25 131.46 294.79 5 34.73 125.34 271.96 6 38.59 114.93 229.13 7 38.89 136.77 293.79 8 36.83 186.14 448.18

70

2 24.94 86.07 178.15 3 26.61 124.93 286.54 4 28.10 131.38 300.96 5 30.33 125.25 276.63 6 31.20 114.85 243.76 7 29.66 136.69 311.89 8 28.42 186.06 459.40



At high temperature, the deactivation of enzyme occurs mainly due to

disruption of non-covalent linkages thus increasing the enthalpy. However,

disorder or randomness or entropy of inactivation increases as a result of opening

up of the enzyme structure.

Organic solvent stability

The PPLB showed good stability in the presence of 10% (v/v) organic

solvents. At a concentration of 10% (v/v) after 1h, the enzyme retained more than

97% activity in ethanol and acetone, followed by 93, 87 and 78% in methanol,

propanol and acetonitrile, respectively. Irrespective of type of solvent, as the

concentration increased from 10 to 40%, the residual activity decreased (Figure

4.15a). However, as compared to others, the magnitude of this decrease was

found to be maximum (about 43, 55 and 52% decrease in residual activity with

each successive increment of 10%) in acetonitrile followed by propanol with

about 22, 71 and 55% decrease with each successive increment of 10%.

Interestingly, considerable stability of the enzyme was noted in presence of

acetone; even at a concentration of 40%, the enzyme maintained 71% of residual

activity after 1h. Furthermore, among all solvents tested in presence of acetone,

the least decrease in residual activity of 3, 6, and 18% with each successive

increment of 10% was observed.

On the other hand, as compared to 1 h, after 16 h more than 50% reduction

in residual activity was observed at all concentrations in every type of solvent

(Figure 4.15b). Nevertheless, even after 16 h the enzyme maintained more than

40% residual activity in 10% of both acetone and ethanol.



FIGURE 4.15 The organic solvent-tolerance of PPLB after 1 h (Figure 4.15a) and 16 h (Figure 4.15 b) The laccase activity in 0.1M BR buffer (pH 2.0) without any added solvent was treated as control activity. The laccase activity after 1h and 16 h incubation of laccase with different concentrations of solvents was assayed using ABTS as substrate.

The properties of solvent such as dielectric constant, log P value are

frequently used to explain the effect of solvents on enzyme stability. These

properties of the solvents used in present study along with the stability of laccase

from our isolate in these solvents is summarized in Table 4.11

TABLE 4.11: Stability of PPLB after incubation of 1 h in presence of (10%) organic solvent - water mixtures

Solvent Polarity Dielectric constant log P % Residual activity After 1 h After 16 h

1 Acetone Dipolar 20.7 -0.24 97.90 41.98

2 Acetonitrile Dipolar 37.5 -0.34 78.25 20.76 3 Ethanol Polar 24.6 -0.30 97.04 46.85 4 Methanol Polar 32.7 -0.74 93.99 36.74 5 Propanol Polar 19.9 0.05 87.02 28.91

The data on Dielectric constant, log P and Polarity is adapted from Roy and Abraham, 2006. The % Residual activity after incubation was assayed with ABTS as substrate at optimum conditions of this assay.

0

20

40

60

80

100

Ace

tone

Ace

toni

trile

Etha

nol

Met

hano

l

Prop

anol

% R

esid

ual a

ctiv

ity

10%

20%

30%

40%

Figure 4.15a

01020304050

Ace

tone

Ace

toni

trile

Etha

nol

Met

hano

l

Prop

anol

% R

esid

ual a

ctiv

ity

10%

20%

30%

40%

Figure 4.15 b



Substrate specificity

Fourteen different aromatic compounds were chosen to study the substrate

specificity of the PPLB.

ABTS Syringaldazine Guaiacol

Pyrogallol Pyrocatechol Hydroquinone

Gallic acid Tannic acid Vanillic acid

Veratryl alcohol Tyrosine

p-Cresol o-Cresol m-Cresol

FIGURE 4.16: Structures of substrates used in the evaluation of substrate specificity (Source: Bar 2001)



The substrates were chosen according to the nature and position of

substituent on the phenolic ring (Figure 4.16). ABTS, a non-phenolic compound

and common substrate as well as mediator, was also included in the study. The

wavelengths of maximum absorbance for the oxidized substrates by the isolated

laccase are reported in Table 4.12. PPLB was able to oxidize ABTS and various

phenolic compounds including guaiacol, syringaldazine, pyrogallol, catechol,

gallic acid, o- cresol and m-cresol. However, no activity was observed using

vanillic acid, tannic acid and vanillin as substrates.

TABLE 4.12: Wavelengths of the oxidation products formed by the action of PPLB on different phenolic/aromatic compounds

Sr no. Substrate λ max nm (M–1 cm–1)

Optimum pH

1 ABTS 420 3.6 × 104 2 2 Guaiacol 432 1.21 × 104 2.5 3 Syringaldazine 529 6.5 × 104 4.5 4 Pyrogallol 370 -- 4 5 Catechol 390 -- 3 6 Hydroquinone NC -- - 7 Gallic acid 386 -- 4 8 Tannic acid NC -- - 9 Vanillic acid NC -- - 10 Veratryl alcohol NC -- - 11 Tyrosine NC -- - 12 p-Cresol NC -- - 13 o-Cresol 411 -- 4.5 14 m-Cresol 418 -- 3.5

Molar extinction coefficients were obtained from the literature NC: No changes in the absorbance spectrum

Moreover, activity of PPLB as a function of the pH was studied for

different substrates (Figure 4.17). The pH optimum varied between pH 2 and 4.5,

depending on the substrate employed. All the phenolic substrates showed a typical

bell-shaped pH activity profile with pH optima in acidic range.



FIGURE 4.17: The activity of PPLB as a function of the pH as studied for different substrates

The experimental kinetic data (average of triplicates) was fitted to single

substrate Michaelis-Menten kinetics by nonlinear least squares regression analysis

using GraphPad Prism 6 software. The data observed for kinetics including

Michaelis-Menten (MM) and Lineweavar Burk (LB) with ABTS as substrate

(Figure 4.18) and with Guaiacol as substrates (Figure 4.19) is illustrated as

follows.

10

30

50

70

90

110

1 3 5 7

% R

elat

ive

acti

vity

pH

Pyrogallol

10

30

50

70

90

110

1 3 5 7

% R

elat

ive

acti

vity

pH

Catechol

10

30

50

70

90

110

1 3 5 7

% R

elat

ive

acti

vity

pH

Gallic acid

10

30

50

70

90

110

1 3 5 7%

Rel

ativ

e ac

tivi

typH

o-Cresol

10

30

50

70

90

110

1 3 5 7

% R

elat

ive

acti

vity

pH

m-Cresol



ABTS MM ABTS LB

FIGURE 4.18: Michaelis-Menten and Lineweavar Burk plots with ABTS as substrate for laccase

Guaiacol MM Guaiacol LB

FIGURE 4.19: Michaelis-Menten and Lineweavar Burk plots with Guaiacol as substrate for PPLB

The Km values (Table 4.13) of PPLB toward the various substrates

indicates that the binding affinities toward ABTS as substrate was much higher as

compared to Guaiacol.

TABLE 4.13: Kinetic parameters for the oxidation of various substrates by PPLB

Substrate

Km

V kcat

kcat

/Km

(M) max (s

-1) (M

-1s

-1)

ABTS 6.042 x 10 -5 331.1 12.6567 209478.649

Guaiacol 8.133 x 10 -4 220.5 8.4289 10363.826

The molecular weight of laccase was 66 KDa, which was used to calculate the kcat (s−1) values. All of the values were calculated by the linear regression (Using GraphPad Prism software).



The Michaelis constant, KM, is often associated with the affinity of the

enzyme for substrate. However, precisely it is a measure of the substrate

concentration required for effective catalysis to occur. The kinetic parameters of

PPLB were studied with two different substrates ABTS and guaiacol. The Km and

Vmax values for laccase were found to be 6.042 x 10 -5 M and 331.1 µM min-1 for

ABTS and 8.133 x 10 -4 M and 220.5 µM min-1 for guaiacol, respectively. The

high KM with guaiacol reveals that higher substrate concentration is required to

achieve a given reaction velocity than with ABTS which has a low KM. Moreover,

small Km means tight binding; high Km means weak binding.

The kcat (the turnover number) which is a measure of catalytic activity is

defined as the number of substrate molecules converted to product per enzyme

molecule per unit of time, when E is saturated with substrate. If the M-M model

fits, k2 = kcat = Vmax/ [Etotal]. The values of kcat range from less than 1/sec to

many millions per sec (http://www.uvm.edu/~mcase/courses/chem205/lecture13.pdf).

The Catalytic Efficiency (kcat/Km) is an estimate of "how perfect" the

enzyme is. It measures how the enzyme performs when [S] is low. The partially

purified laccase showed notable differences in its catalytic efficiencies (kcat/Km)

when substrates were varied. Interestingly, the catalytic efficiency with ABTS

was about 20 times greater than that with guaiacol (Table 4.13). The high value

of kcat/Km for ABTS indicates very high frequency with which enzyme and

substrate molecules can collide; every encounter leading to reaction.

All the kinetic parameters suggest that the enzyme has a higher affinity

towards ABTS than guaiacol.



Inhibition studies

The effects of several laccase inhibitors were determined with ABTS as a

substrate. Inhibition studies revealed that EDTA was not an efficient inhibitor of

laccase whereas sodium azide which is an inhibitor of metallo-enzymes,

demonstrated strong inhibition of laccase from L-168. Lower concentration of

some inhibitors completely inhibited the laccase of BspL-168 while other

inhibitors required a higher concentration for complete inhibition.

TABLE 4.14: Effect of Putative Inhibitors on the PPLB

Compound Concentration µm/ml

Inhibition %

Amino acid Cysteine 50 50.42 100 73.11 200 88.31 400 97.74 600 99.68

Chelating agent EDTA 10 5.77 100 17.68

Halides NaF 0.01 15.28 0.1 37.01 0.4 68.43

1 87.74 10 97.53 50 99.17

NaCl 10 46.62 100 80.57 400 96.75 500 97.31

1000 99.10 KI 50 89.65

100 90.77 400 92.57 600 91.75

1000 92.80 Other chemical NaN3 0.001 40.62

0.01 86.60 0.1 98.21 0.2 99.17

The result is the mean value of every set of triplicate measurements, of which the variation is less than 5%. Residual activities (%) were measured using ABTS as the substrate. The assay mixture containing enzyme and inhibitors at various concentrations was incubated at 30°C for 5 min and the assay was then started by addition of substrate.

As evident from Table 4.14, sodium azide inhibited laccases completely

only at a concentration of 0.2 µm/ml, while other inhibitors, such as L-cysteine

(600 µm/ml) required at least 3000 times higher concentration than that of sodium



azide for complete inhibition. It was observed that, thioglycolic acid exhibited no

inhibition after an initial lag phase.

Halides are well-known inhibitors of laccases. The effect of four halides

(Br-, F-, Cl- and I-) was investigated for PPLB. Amongst halides, fluoride was

found to be a potent inhibitor for laccases (Figure 4.20).

FIGURE 4.20: Effect of various halides on PPLB catalyzed oxidation of ABTS

Chloride was less effective than fluoride for the inhibition of laccase. On

the contrary, both iodide and bromide exhibited no significant inhibition since

approximately 90 % activity was restored in both cases as compared to control.

The order of inhibition of laccases by halides is F- > Cl- > I- = Br-.

Effect of metal ions on laccase activity

The activity of laccase was tested in the presence of fifteen metal ions

including Zn2+, Ba2+, Ca2+, Cd2+,Co2+, Mg2+, Mn2+, Na+, K+, Fe3+, Cu2+, Pb2+,

Cr2+, As2+ and Mo2+.

0

20

40

60

80

100

KBr KI KCL NaF

% R

elat

ive

acti

vity

Type of Halides

1 um

10 um



FIGURE 4.21: Effect of various metal ions on L-168 laccase catalyzed oxidation of ABTS

It was found that, for a concentration of 0.1 mg/ml of metal ion, the

laccase activity was inhibited by all the metal ions except for Cu2+, Pb2+, Cr2+,

As2+ and Mo2+ (Figure 4.21). In particular Fe3+ in the form of FeCl3 and FeSO4

resulted in 84 and 100% inhibition of laccase. In contrast, laccase activity was

found to be increased in presence of Cu2+, As2+ and Mo2+ and remained stable in

presence of Pb2+ and Cr2+.

Spectroscopic studies

To determine the state of its catalytic center, the PPLB was characterized

spectroscopically. When compared with that of the already characterized

enzymes, a different behavior was observed.

FIGURE 4.22: UV/visible absorption spectra of PPLB

0

35

70

105

140

Zn Ba Ca Cd Co Mg

Mn

Na K

FeCl

3 Cu

FeSO

4 Pb Cr As Mo

% R

elat

ive

acti

vity

Type of ion



In the absorbance spectrum of laccase (Figure 4.22), a peak at 605- 610

nm which is typical for the type I Cu (II) and is responsible for the deep “blue”

color of the enzyme, was absent. Furthermore, it is remarkable that the shoulder at

around 330 nm which corresponds to Type III binuclear Cu (II) pair was also

lacking. These properties are representative of Yellow laccase as reported in

literature. However, EPR spectroscopy study may further confirm the state of

catalytic center of laccase from our isolate. The spectroscopic properties of so

called Blue, White and Yellow laccases in comparison to PPLB from present

investigation are summarized in Table 4.15. Moreover, when laccase was

examined for presence of different metal such as zinc, copper, iron, manganese

and magnesium by atomic absorption, the presence of four metals zinc, copper,

iron and magnesium was revealed. However further quantitative analysis was not

performed.

TABLE 4.15: The spectroscopic properties of blue, yellow and white laccases Organism Isoenzyme Peak at

605 nm Shoulder/Peak at nm

A280/A598-615 ratio

Type of laccase

Metal content Reference

Pleurotus ostreatus

POXC Y (605) 330 20 Blue - Palmieri et al. 1997 POXA1 N 400 White 1Cu,2Zn,1Fe/mol

POXA2 Less intense (605)

400 50 Blue 4 Cu/mol

Phellinus ribis - N 330 - - 1Cu,2Zn,1Mn/mol Min et al. 2001

strain I-4 - Y(611) 333 Blue - Saito et al. 2003

Pleurotus ostreatus

- N N 106.2 Yellow - Pozdnyakova et.al., 2006

Steccherinum ochraceum

Laccase I Y(611) - 10.4 - - Chernykh et al. 2008 Laccase

II Y(611) - 9.2 - -

Laccase III

Y(611) - 10.5 - -

Trichoderma harzianum

Y( 608) 325 - Blue - Sadhasivam et al. 2008

Pichia pastoris

- N N 36 Yellow - Huang et.al. 2011

Myrothecium verrucaria

- N N - White 3Cu,1 Fe Zhao et al. 2012

Basidiomycota sp L168 (PPLB)

-- N N 13.74 Yellow Cu, Zn, Fe, Mn Present study



Molecular Weight Determination

PPLB was homogenous according to size with SDSPAGE (Plate 4.4) as

well as according to charge with Native-PAGE.

FIGURE 4.23: Calibration curve for the determination of molecular mass of PPLB, relative to the migration of protein standards on denaturing gel electrophoresis.

The relative molecular mass of the laccase protein was determined to be

approximately 66 kDa relative to the molecular mass markers (Figure 4.23).

Zymogram analyses were conducted for laccase activity on the non-denaturing

electrophoresis gels. Activity staining of the laccase with Guaiacol as substrate

revealed a single protein band corresponding to the position of the laccase activity

(Plate 4.5).

R² = 0.973

0

0.2

0.4

0.6

0.8

1

1.2 1.4 1.6 1.8 2 2.2

Dis

tanc

e m

igra

ted

Log of molecular weight



4.5 Discussion

Being an extracellular enzyme, the purification for laccase is a simple. In

the present investigation, the simple composition of culture medium (see chapter

3) and the fact that laccase was the sole ligninolytic enzyme produced by BspL-

168 facilitated the purification.

In most of the studies on purification, the commonly used methods include

ammonium sulphate precipitation, ultra filtration and a combination of different

chromatographic steps (see Table 1.6). However, it is evident from this Table that

as compared to many other studies, in present investigation, highest specific

activity as well as purification fold is achieved that too with a simple purification

protocol.

Laccase from Trametes pubescens was purified by ion-exchange and Gel

filtration chromatography with a final purification of 1.7 fold (Galhaup et al.

2002) whereas, laccase purified from Trametes trogii employing ultra filtration

followed by Hittrap, Superdex and MonoQ Lac1, Lac2 chromatography had a

specific activity of 53.56, a yield of 13.6% with the purification of 6.6 fold

(Mechichi et al. 2006).

In another case, laccase purified from Cerrena unicolor by ultra filtration,

size exclusion and anion exchange chromatography showed a specific activity of

10918, yield of 17% and a purification factor of 33 fold (Ticlo et al. 2009b). A

recombinant laccase, obtained by cloning and expression of laccase gene from

white-rot fungus Ganoderma fornicatum in Pichia pastoris, was purified by ultra

filtration, Q-Sepharaose XL and Sephacryl S-200 chromatography with a specific



activity of 59.23 and a yield of 16.3% (Huang et al. 2011). The purification of

laccase produced by Ganoderma sp. by use of TPP is reported (Rajeeva and Lele

2010). However, in the present investigation, with a simple two step approach,

3135.76 fold purification with a specific activity of 20950 U mg-1 was obtained

which is comparatively much higher.

During TPP, in presence of butanol the protein may acquire a greater

proportion of α-helices. As a result the protein conformation may become

distorted leading to denaturation of many proteins (Dennison 2002). However,

the laccase from BspL-168 is able to survive TPP. Therefore, TPP resulted in

effective purification not only by its fractionating ability but also removed

denatured impurities.

Typical fungal laccase is a protein of approximately 60–70 kDa (Baldrian

2006). However, laccase with molecular weight as high as 97 kDa is reported in

Trametes versicolor (Han et al. 2005) and 90 KDa in Pleurotus sajor-caju (Sahay

et al. 2008). In the present study, appearance of a single protein band on SDS-

PAGE in denaturing conditions suggested that the isoform was monomeric

(Farnet et al. 2002).

The molecular mass determined for laccase from PPLB compares well to

that reported for typical laccase enzymes. For example, 63kDa in Perenniporia

tephropora (Younes et al. 2007), 63 kDa in Steccherinum ochraceum (Chernykh

et al. 2008), 65 kDa in Marasmius quercophilus (Farnet et al. 2002), 65 kDa in

Trametes pubescens (Galhaup et al. 2002), 62 kDa in Trametes trogii (Mechichi

et al. 2006), 64 kDa in Pleurotus ostreatus (Pozdnyakova et al. 2006).



In white-rot fungi, laccases are typically produced as multiple iso enzymes

encoded by gene families. It has been suggested that genes encoding various

isoenzymes are differentially regulated with some being constitutively expressed

and others being inducible (Galhaup 2002). The different strains of the same

species produce laccases with different properties and characteristics (Dantan et

al. 2008). Furthermore, different isoenzymes produced by the same organism

show different properties with respect to pH and temperature optima and stability,

substrate specificity, inhibition properties, kinetic properties, molecular weight,

isoelectric pH etc. In the present study, however, BspL-168 produces only one

form of enzyme of laccase under the growth conditions used.

The use of purified enzymes for industrial applications is not economically

feasible. On the other hand, crude preparations with high enzyme titer and

exhibiting stability and desired specificities are always preferred. Since,

isoenzymes produced by same organism frequently show different substrate

specificities, it may hinder the application of such crude preparation. The

specificity for a particular compound of crude preparation containing only one

isoenzyme is likely to surpass the cumulative specificity of a preparation

containing many isoenzymes. BspL-168, therefore, may serve as a good candidate

for industrial applications in view of the facts that it not only secrets laccase as the

sole lignin degrading enzyme but exhibits only one isoenzyme. Furthermore, the

simple production medium, use of lignocellulosic waste for cost effective

production and high titer of laccase attained are the added advantages.



Fungal laccases typically exhibit pH optima in the acidic pH range. The

pH optima for the oxidation of ABTS are generally lower than 4.0 while for

phenolic compounds like DMP, guaiacol and Syringaldazine, pH values are

higher between 4.0 and 7.0 (Baldrian 2006). The pH optima of the laccase

purified from Trichoderma harzianum for guaiacol is reported to be 4.5

(Sadhasivam et al. 2008). In contrast, in the present investigation very low pH

optima for guaiacol oxidation (pH 2.5) was recorded.

Furthermore, laccase from BspL-168 showed optimum pH in acidic range

(pH 2) and optimum temperature of 70 oC for ABTS oxidation. This observation

is corroborated with reports on laccase from Steccherinum ochraceum showing

the highest activity with ABTS at extremely low pH (≤ 2) (Chernykh et al.

2008). They reported lowest optimum pH of 1.2 for one of the laccase isoforms

and high temperature optima in range of 70 to 80 oC for the three laccase

isoforms.

As reviewed by Baldrian (2006), the bell-shaped pH profile of phenolic

compounds is formed by two opposing effects. The oxidation of phenols depend

on the redox potential difference between the phenolic compound and the T1

copper. The E0 of a phenol decreases when pH increases due to the oxidative

proton release. The enzyme activity at higher pH is decreased by the binding of a

hydroxide anion to the T2/T3 coppers of laccase that interrupts the internal

electron transfer from T1 to T2/T3 centers.

Temperature optima obtained for BspL-168 laccase agreed well with

values reported for Marasmius quercophilus of 80 °C (Farnet et al. 2002);



Fomitella fraxinea 70 °C (Park and Park, 2008), Steccherinum ochraceum 70,

75 and 80 °C for isoforms I,II and III respectively (Chernykh et al. 2008). And in

other organism i.e. Cerrena unicolor it is 70 °C (Ticlo et al. 2009b)

The activation energy, Ea, is the minimum energy molecules must possess

in order to form a product. The activation energy for laccase from BspL-168

(11.999 KJ mol-1) is higher than that reported for Cerrena unicolor (8.15 KJ mol-

1) (Ticlo et al. 2009b).

The stability of fungal laccases is generally higher at acidic pH (Baldrian

2006). Laccase from Fomitella fraxinea is reported to be stable within an acidic

pH range from 3.0 to 5.0 (Park and Park, 2008). However, the stability of the

laccase in this study was highest at pH of 8.0. Stability of laccase at non-optimal

pH has been reported in previous studies (Min et al. 2001). Similarly more

stability of laccase at alkaline values than at acidic pH is reported in Perenniporia

tephropora at pH 8.0 (Younes et. al. 2007); in Cerrena unicolor at pH 9.0 (Ticlo

et al. 2009b); in Pycnoporous coccineus at pH 7.5–10 (Jaouani et al. 2005).

Stability of the laccase from BspL-168 is quite high over a broad pH range. This

could be a very useful characteristic for various industrial applications.

The recombinant laccase (lac gene from Ganoderma fornicatum cloned

and expressed in Pichia pastoris) is reported to retain more than 80% of its

activity in solutions with pH ranging from 2.5 to 10.0 after a 24 h incubation

(Huang et. al. 2011b). In comparison, BspL-168 laccase retained more than 85%

and 90% of its activity in solutions with pH from 7 to 8 after 24 h incubation at

room temperature respectively.



According to Huang et al. (2011b), most of the currently known laccases

are stable at pH between 6.0 and 7.0, but experience a tremendous loss in their

enzyme activity, if subjected to a more-acidic or more-alkaline environment. In

earlier studies, Peniophora sp. laccases are also reported to retain full activity at

pH 6.0–7.0 at 20 0C during 22 h (Niku-Paavola et al. 2004). However, laccase in

present study retained about 95 to 100% of activity in pH ranging from 3 to 10

after 1 h. Moreover, even at pH 2 after 1 h, the enzyme showed 70 % residual

activity. In another study, Pycnoporous coccineus laccase is reported to show 90–

100% activity after 1 h of incubation in the pH range 2.5 - 10 while after 24 h

incubation >80% of activity is reported at pH 7.5 - 10 (Jaouani et al. 2005). In

contrast, PPLB retained more than 85% and 90% of its activity in solutions with

pH from 7 to 8 after 24 h incubation at room temperature respectively.

The stability of enzymes to different conditions is a desirable

characteristic for industrial applications. However, though some fungal laccases

are thermo stable, most of the white-rot fungal laccases are not stable above 50 °C

(Dantan et al. 2008). The temperature stability varies considerably with laccase

and its producing organism (Baldrian 2006). The half life of 2.9 h at 50 °C, 1.5 h

at 60 °C and 3.7 min at 70 °C is reported in laccase from Trametes pubescens

(Galhaup et al. 2002). In contrast, laccase from our isolate exhibited more

stability with half life of 1464 h at 50 °C, 5.7 h at 60 °C and 15 min at 70 °C.

Stability studies of laccase from our isolate at 50, 60 and 70 °C show better

results than that of Galhaup et al. (2002) findings. This observation suggests a

very high potential for biotechnological applications.



Moreover, the data on stability of laccase from our isolate at 60oC agreed

well with observations by Chernykh et al. (2008). They reported high stability of

laccase from Steccherinum ochraceum with half life of 5 to 6 h. The recombinant

laccase (lac gene from Ganoderma fornicatum cloned and expressed in Pichia

pastoris) is reported to maintain at least 80% residual activity after 9 h incubation

at 50 oC (Huang et al. 2011b). The stability data recorded for L-168 laccase is in

agreement with reports by Huang et.al. (2011b), since it had a half life of 1464 h

at 50 oC with pH 8. Furthermore, the observation that the decrease in stability at

70 oC in comparison to 60 oC noted for BspL-168 laccase is also supported by

Huang et al. (2011b).

In another study on laccase from Trametes versicolor, Roy and Abraham

(2006) reported the half-life of 123 min and 24 min at 60 oC, while 453 min and

112 min at 50 oC for immobilized and free enzyme respectively. In the present

study however, the laccase exhibited a half-life of 347 min at 60 oC which is

about 2.8 fold higher than half life of immobilized enzyme and about 14.46 fold

higher than free enzyme from Trametes versicolor. In addition, the half life of

87886 min at 50 oC exhibited by laccase from our isolate is about 194 fold higher

than half life of immobilized enzyme and about 784 fold higher than free enzyme

from Trametes versicolor.

Jaouani et al. (2005) with Pycnoporous coccineus reported a half life of 8

and 2 h at 50 and 60 oC, respectively. As compared to this, PPLB showed a half-

life of about 183 and 2.85 fold higher, at 50 and 60 oC, respectively. The half-life

of laccase B isoenzyme from Trametes sp. is reported to be 14 min at 60 oC and



was found stable at 50 oC for more than 50 min. On the contrary, the half life of

5.7 h at 60 oC and 1464 h at 50 oC by laccase from our isolate is significant

higher.

A strain of Peniophora species is reported to produce a thermo stable

laccase with the half-life of approximately 5 h at 60 oC and 15 min at 70 oC

(Niku-Paavola et al. 2004). Furthermore, the Peniophora laccases were reported

to be more thermo stable than some of the thermophilic fungi such as

Myceliophthora thermophila and Coprinus cinereus. This observation supports us

to the report as thermo stable nature of laccase from our isolate which exhibited

the half life of 5.7 h at 60 oC and 15 min at 70 oC.

The high magnitudes of z-values mean more sensitivity to the duration of

heat treatment and lower z-values mean more sensitivity to increase in

temperature. In present study, the lesser z-value noted at pH 3 to 8 as compared to

pH 2 indicates that pH makes the enzyme more sensitive to increase in

temperature rather than its duration. Studies pertaining to this aspect of laccase

characterization are lacking to compare and discuss our findings. However,

similar type of studies is reported with other enzyme xylanase by Pal and

Khanum (2010).

Besides pH and temperature, different solvent may also affect the activity

of enzyme. The solvent may directly interact with the enzyme by acting as an

enzyme inhibitor or may causes conformational changes in the enzyme, thereby

changing its catalytic properties. In addition, the solvent may indirectly affect an

enzymatic reaction by influencing the amount of water bound to the enzyme



thereby causing inactivation of enzyme due to dehydration (Adlercreutz 2008).

The direct interaction between solvent and enzyme can affect both enzyme

stability as well as activity. The polar solvent like methanol is able to dissolve the

enzyme, resulting in inactivation of the enzyme due to disruption of its tertiary

and sometimes also secondary structure (Adlercreutz 2008).

The recombinant laccase is reported to exhibit good stability in the

presence of 10% (v/v) of various organic solvents such as acetonitrile, acetone,

ethanol, methanol, DMF and DMSO whereas its activity was <20% in 50%

acetone and acetonitrile (Huang et al., 2011b). In comparison, BspL-168 laccase,

at a concentration of 10% (v/v) after 1h, retained more than 97% activity in

ethanol and acetone, followed by 93, 87 and 78% in methanol, propanol and

acetonitrile respectively. On the other hand, as compared to 1 h, after 16 h more

than 50% reduction in residual activity was observed at all concentrations in every

type of solvent. This observation in the present study is corroborated with the fact

that, though enzyme can express catalytic activity even in the organic media, it is

several orders of magnitude lower than those in aqueous solution. However, a

careful selection of the type of enzyme preparation and the reaction conditions

can help to solve this problem (Adlercreutz 2008).

Since dehydration inactivates the enzymes, the residual catalytic activity

after exposure to different solvents can be correlated well with the log P value of

the solvent. In general, solvents having log P values above 4 (water immiscible/

hydrophobic solvents) cause negligible inactivation, while those with log P values

below 2 (polar solvents) are highly inactivating (Adlercreutz 2008). In a study on



effect of organic solvents on stability of immobilized laccase from Trametes

versicolor, the enzyme is reported to exhibit higher activity in non-polar organic

solvents such as hexane, toluene, isooctane and cyclohexane due to their higher

log P values (2.73–4.0) whereas, medium activity is exhibited in organic solvents

such as ethyl acetate, chloroform, isopropanol and acetone having log P values

between 0.73 and −0.24 (Roy and Abraham 2006). Furthermore, they reported

very low activity in solvents such as ethanol, methanol and acetonitrile due to

their lower log P values. The observation in this study is in good agreement with

these reports; however, stability studies reported by Roy and Abraham (2006)

were carried out on immobilized laccase whereas in present study, free enzyme

was used.

Although, log P (P = partition coefficient between octanol and water) is

the most frequently used solvent descriptors of organic solvents, a single solvent

descriptor cannot give good prediction for all combinations of solvents and

enzymes. For more accurate prediction of inactivation of enzymes by solvents,

several other solvent descriptors must also be considered such as Sw/o (solubility

of water in solvent, wt%) So/w (solubility of solvent in water, wt%) and ε

(dielectric constant) values, ET (empirical polarity parameter by Reichardt-

Dimroth) and HS (Hildebrand solubility parameter) (Adlercreutz 2008). The

discussion on use of these descriptors in stability studies is beyond the scope of

this investigation. Thus, further detailed studies with this respect are needed for

more accurate prediction of solvent stability of laccase from our isolate.



It is difficult to define laccase by its ability to oxidize different substrate

due to its broad substrate range. Moreover, the substrate range for laccases

frequently overlaps with the substrate range of other enzymes particularly

tyrosinase (Baldrian 2006). Syringaldazine [N,N’-bis(3,5-dimethoxy-4-

hydroxybenzylidene hydrazine)] is often considered to be a unique laccase

substrate. The ability to oxidize a range of phenolic compounds (guaiacol,

pyrogallol, catechol, gallic acid, o- cresol and m-cresol) including syringaldazine

and inability to oxidize tyrosine proves that the enzyme activity from BspL-168

originated from laccase.

Laccases can be grouped according to their preference for ortho-,

meta- or para- substituted phenols. In general, ortho-substituted compounds are

better substrates than para-substituted compounds and the lowest reaction rates

are obtained with meta-substituted compounds (Baldrian 2006). In the present

investigation, all ortho-substituted compounds (guaiacol, catechol, gallic acid and

pyrogallol) were better oxidized by laccase along with some meta-substituted

compounds (m-cresol) while para-substituted compounds (p-cresol and

hydroquinone) were not oxidized. In contrast, laccase from Trametes trogii

although is reported to oxidize gallic acid, catechol and pyrogallol, all the three p-

cresol, m-cresol as well as o-cresols were not oxidized (Mechichi et al. 2006).

Generally introduction of OH, OCH3, or CH3 groups into the aromatic

system renders the compound more easily oxidized by laccase (Min et al. 2001).

However, substrates containing these groups such as tannic acid and



hydroquinone with OH, veratryl alcohol with OCH3, vanillic acid with OH and

OCH3 were not oxidized by laccase from BspL-168.

Fungal laccases are known to possess very broad substrate affinities

generally with greater affinity for ABTS than for other substrates (Dantán et al.

2008). The laccase from BspL-168 also seems to follow this generalization.

The kinetic investigation of laccase from BspL-168 showed that its

catalytic efficiency expressed as kcat ⁄Km for both the studied substrates are much

higher. These results are parallel with studies on laccase from Steccherinum

ochraceum (Chernykh et al. 2008). The kcat ⁄Km ratio is often thought of as a

measure of enzyme efficiency. Either a large value of kcat (rapid turnover) or a

small value of Km (high affinity for substrate) makes kcat/Km large.

The Km values of 270 µM, 180 µM, 250 µM, 177 µM, 238.55 µM for the

laccase from Fomitella fraxinea (Park and Park, 2008), Trichoderma harzianum

(Sadhasivam et al. 2008), Pleurotus sp. (More et al. 2011), Trametes sp. (Xiao

et al. 2004) and Pycnoporus sanguineus (Dantán et al. 2008) respectively are

reported using ABTS as the substrate. In comparison, the Km value (60.42 µM) of

laccase from our isolate was much lower indicating very high affinity to ABTS.

Although enzyme efficiencies exactly parallel to laccase in present

investigation are reported with studies on laccase (isoforms l and lll) from

Steccherinum ochraceum (Chernykh et al. 2008), there is a notable difference in

affinity of the enzyme for substrate. The affinity of PPLB to ABTS is much

higher (Km = 6.0 x 10-5 M-1) than laccase l from Steccherinum ochraceum (Km =



1.0 x 10-4 M-1). Moreover, the affinity of PPLB to guaiacol is also higher (Km =

8.1 x 10-4 M-1) than laccase l from Steccherinum ochraceum (Km = 1.3 x 10-3 M-1).

The Km value for guaiacol as substrate of 1249 µM for laccase from

Trametes sp. (Xiao et al, 2004) and 1236.4 µM for the recombinant laccase from

Pichia pastoris (Huang et al. 2011b) is reported. In comparison, the Km value

(813.3 µM) of laccase from the present isolate is much lower indicating higher

affinity to guaiacol.

In another study on immobilized laccase from Trametes versicolor, the Km

of 0.859 mM and a catalytic efficiency (kcat/Km) of 3.73×103 for ABTS as

substrate is reported (Roy and Abraham 2006). In comparison, the Km value

(0.06042 mM) of PPLB was significantly lower indicating very high affinity to

ABTS. Furthermore, kcat/Km (2.09×105) of PPLB was also significantly higher

indicating very high catalytic efficiency. Thus, the molecular weight, Km, Vmax

and kcat/km for the enzyme characterized in this study collate well with laccases

previously reported in literature.

The complete inhibition of laccase from unidentified strain I-4 (Saito et al.

2003), Fomitella fraxinea (Park and Park 2008) by cysteine is reported.

Similarly, Younes et al. (2007) reported complete inhibition of laccase from

Perenniporia tephropora by l-cysteine at a concentration of 5 mM and by EDTA

at 0.1 mM. On the other hand, laccase from BspL-168 required 600 mM

concentration of l-cysteine for complete inhibition. Moreover, EDTA even at 100

mM concentration did not inhibit the enzyme significantly.



Similarly, no inhibition of laccase by EDTA is reported in an unidentified

strain I-4 (Saito et al. 2003); in Phellinus ribis (Min et al. 2001); in Marasmius

quercophilus (Farnet et al. 2002); in Trametes pubescens (Galhaup et al. 2002);

Sodium azide is found to be the most potent inhibitor leading to complete

inhibition of laccase in Perenniporia tephropora (Younes et al. 2007), in

Trametes versicolor (Johannes and Majcherczyk 2000-); Phellinus ribis (Min

et al 2001); Marasmius quercophilus (Farnet et al. 2002); Trametes pubescens

(Galhaup et al. 2002);

Saito et al. (2003) demonstrated little inhibition (54%) of the laccase even

at the much higher concentration of sodium azide (10 mM). On the contrary,

laccase in present study was completely inhibited only at a concentration of 0.2

mM. Among the halides, bromide exhibited no significant inhibition of laccases

from BspL-168. This observation is parallel with the reports on Perenniporia

tephropora laccase (Younes et al. 2007).

Inhibition by thioglycolic acid can be correlated with the presence of

copper in the catalytic centre of the enzyme (Baldrian 2006). The inhibition of

laccase from BspL-168 by thioglycolic acid therefore, confirms presence of

copper. However, it was observed that, thioglycolic acid exhibited no inhibition

after an initial lag phase. The lag phase was thought to be due to the oxygen

chelating activity of the thioglycolic acid, and since oxygen is the final electron

acceptor for the laccase-catalyzed reaction, the reaction pathway could not be

completed until the thioglycolic acid was oxygen saturated. These observations

are parallel to the reports by Jordaan (2005).



Younes et al. (2007) observed 82% inhibition of laccase from

Perenniporia tephropora by the ion Mo2+. However, laccase activity from BspL-

168 was increased in presence of this ion. Although laccase activity from BspL-

168 was inhibited by many ions, the concentration of these ions used in present

study was very high. The inhibition of laccase at high concentration of ions is

reported in Perenniporia tephropora (Younes et al. 2007). On the other hand in

present study, increased laccase activity was obtained in presence Cu2+. This

observation is parallel with that of laccase from Perenniporia tephropora

(Younes et al. 2007). In contrast, activity of laccase from Fomitella fraxinea is

reported to be unaffected by copper (Park and Park, 2008). Laccase from L-168

was inhibited in presence of K+ and Ca 2+. On the quantrary, the stimulatory effect

of K+ and Ca 2+ is reported in Fomitella fraxinea (Park and Park 2008).

Laccases typically contain four copper atoms, which are organized into

three types of copper sites. These copper sites can be distinguished by UV/visible

and Electron paramagnetic resonance (EPR) spectroscopy. Type I copper gives a

blue color to the protein from an absorbance at about 600 nm and is EPR

detectable. Type II copper confers no color, but is EPR detectable. Type III

copper is a pair of copper atoms that gives a weak absorbance in the near UV and

has no EPR signal (Leontievsky et al. 1997) Similar enzymes lacking the Cu

atom responsible for the blue color are called ‘yellow’ or ‘white’ laccases

(Baldrian 2006).

The lack of the typical absorbance peak at 610 nm as well as no shoulder

around 330-400 nm is reported in a recombinant laccase expressed in Pichia



pastoris (Huang et al. 2011b). This laccase is therefore, regarded as yellow

laccase. The spectroscopic characteristics of laccase from BspL-168 are exactly

parallel to those reported for recombinant laccase from Pichia pastoris. However,

the A280/A610 ratio 13.74 of BspL-168 laccase was much lower than the ratio of 36

reported by Huang et.al. (2011). Similar spectroscopic characteristics as noted in

the present study as well as laccase from Pichia pastoris were found to be

reported in another study on yellow laccase from Pleurotus ostreatus D1 but with

A280/A610 ratio of 106.2 (Pozdnyakova et al. 2006).

The A280/A610 ratio within the range of 92.6 to148.0 for yellow laccases

and 15 to 20 for typical blue laccases was reported in a study on different white

rot fungi (Leontievsky et al. 1997). Thus, according to the features discussed

above, laccase from BspL-168 may be classified as so-called “yellow laccase”

with reference to that recently reported in Pleurotus ostreatus (Pozdnyakova et

al. 2006) and Pichia pastoris (Huang et al. 2011b).



4.5 Conclusion

Effective purification of laccase from BspL-168 was achieved by

relatively least accounted technique of Three Phase Partitioning without any

chromatographic steps. The technique was found to be more convenient, rapid,

simple and effective for laccase purification. Under the culture conditions

employed the organism produced single enzyme form, which was purified to

electrophoretic homogeneity by TPP. The protein was monomeric with a

molecular mass of 66 kDa (SDS–PAGE) and was capable of oxidizing wide

variety of substrates. The spectroscopic study indicated that the enzyme may be

Yellow laccase. The optimum pH of the enzyme varied and was substrate

dependent. It was 4.5, 2.5 and 2.0 for Syringaldazine, Guaiacol and ABTS

respectively. Under standard assay conditions, Km values of the enzyme were

6.04 x 10-5 and 8.13 x 10-4 M towards ABTS and Guaiacol, respectively. The

laccase was inhibited by Cysteine, NaF, NaCl, KI and NaN3 but not by EDTA and

thioglycolic acid. This inhibition was however concentration dependent. Laccase

was stable in the presence of some metal ions such as Cu2+, Pb2+, Cr2+, As+2 and

Mo2+. It showed optimum temperature with ABTS at 70 oC and was stable at

temperatures as high as 50, 60, 70 oC with a half life of 1464 h, 5.7 h and 15 min

respectively.

Thus laccase is thermostable, metal and organic solvent tolerant. This

enzyme is, therefore, practically robust under most physical and chemical

conditions. This study thus prompts to extend and magnify its applications in

biotechnological and allied industries.

Chapter 4: Purification and Characterization of Laccase from...

Documents

Transcript of Chapter 4: Purification and Characterization of Laccase from...