3_manole 8pag

8/13/2019 3_manole 8pag

1/8

SCIENTIFIC ANNALS OF ALEXANDRU IOAN CUZA DIN IAI UNIVERSITY

Tomul I, s. Biomaterials in Biophysics, Medical Physics and Ecology 2008

LACCASE ACTIVITY DETERMINATION

Alina Manole1, D. Herea2, H. Chiriac2, V. Melnig1

KEYWORDS: enzyme, activity, specific activity, turnover number.

Enzymes catalyze most biochemical reactions, governing the chemical changes which wecall metabolism. We can gain some understanding of enzyme behaviour through the study

of rates of enzyme catalyzed reactions. In this paper, syringaldazine oxidation to the

corresponding quinone, catalyzed by laccase is studied to determine laccase activity.

1. INTRODUCTIONEnzymes speed up the rate of a reaction by a definite amount, proportional to

quantity of enzyme present. Simple measurements of enzyme reactions include

activity, specific activity (activity per unit mass) and turnover number, activity per

mole of enzyme. Turnover number also represents the actual number of times an

enzyme molecule reacts per second. To measure reaction rate, some property

difference between reactant and product must be identified. Rate can be measured as

disappearance of reactant or accumulation of product [1].

Quantitative description of enzyme catalysis

Rate of reaction = concentration of substrate disappearing per unit time (mol L1

sec1

)

= concentration of product produced per unit time (mol L1

sec1

)

Enzyme activity = moles converted per unit time

= rate reaction volume

The SI unit is the katal, 1 katal = 1 mol s-1

, but this is an excessively large unit.

A more practical value is 1 international unit (IU or U), defined by necessary enzyme

quantity for consuming 1 mol of substrate or producing 1 mol of product per

minute.

Specific activity = moles converted per unit time per unit mass of enzyme

= enzyme activity / actual mass of enzyme present

SI units: katal kg-1; Practical units: mol mg-1min-1or mol g-1min-1. Specific

activity is a measure of enzyme efficiency, usually constant for a pure enzyme. An

1Al.I. Cuza University, Faculty of Physics, Carol I Blvd., No.11, 700506, Iasi2National Institute of Research and Development for Technical Physics 47 Mangeron Blvd., 700050, Iasi

8/13/2019 3_manole 8pag

2/8

Alina Manole, D. Herea, H. Chiriac, V. Melnig18

impure sample has lower specific activity because some of the mass is not actuallyenzyme and if the specific activity of 100% pure enzyme is known the purity of an

impure sample can be calculated with the formula:

activityspecificenzymepure

activityspecificenzymetestxpurity %100% = .

Turnover number = moles of substrate converted per unit time / moles of enzyme

(usually per second)

= specific activity molar mass of enzyme

(with necessary unit conversions!)

Multiplying mass by molar mass converts specific activity (per unit mass) into

activity per mole. If n moles of substrate are catalyzed by one mole of enzyme per

second, then n molecules of substrate are catalyzed by each molecule of enzyme per

second. Hence turnover number represents the number of times per second that the

enzyme completes a reaction cycle.

Laccases (E.C. 1.10.3.2, benzenediol: oxygen oxidoreductase) are either mono

or multimeric copper-containing oxidizes that catalyze the one-electron oxidation of a

vast amount of phenolic substrates. Molecular oxygen serves as the terminal electron

acceptor and is thus reduced to two molecules of water [2]:

O2+4e-+4H

+2H2O.

Three types of copper can be distinguished Type 1 copper is responsible for the

blue color of the protein at an absorbance of approximately 600 nm, Type 2 copper

does not confer color and Type 3 copper consists of a pair of copper atoms in a

binuclear conformation that give a weak absorbance in the near UV region. The Type

1 Cu is usually coordinated to two nitrogens from two histidines and sulphur from

cysteine (figure 1). It is the bond of Type 1 Cu to sulphur that is responsible for thecharacteristic blue color of typical laccase enzymes. The geometry is described as a

distorted trigonal bipyramidal coordination with a vacant axial position where the

substrate docks. The coordination is unusual as it is intermediate between the

preferred coordination states for Cu (I) and Cu (II) species. A leucine residue is

present but is too far away to be directly coordinated. The Cu is therefore only

coordinated to three atoms. The structure showed that type 2 and type 3 coppers are

close together in a trinuclear centre. The copper atoms of the T2/T3 sites are

coordinated to eight histidines, which are conserved in four His-X-His motifs. The

two T3 atoms are coordinated to six of the histidines (figure 1) while the T2 atom is

coordinated to the remaining two. A hydroxide ligand bridges the pair of T3 atoms

(figure 1 in red), resulting a strong anti- ferromagnetic coupling. The cloned

sequences of various laccases also show that the 10 histidine and 1 cysteine residues

are copper ligands conserved in all laccase sequences known to date except one from

Aspergillus nidulans that has a methionine ligand of type 1 copper. These conservedcysteine and histidine residues serve as a pathway for the transport of electrons from

the T1 Cu site where electrons are extracted from phenolic substrates to the trinuclear

site that serves as the binding site of dioxygen where the electrons are required for

dioxygen reduction [3].

8/13/2019 3_manole 8pag

3/8

LACCASE IMMOBILISED ON HYDROTALCITES 19

Fig. 1 A ball-and-stick model depicting the coordination of copper atoms and

ligands in the active site of Cu-2 depleted Coprinus cinereus laccase. Copper atoms

are in green, sulphur in yellow and oxygen in red.

Laccase is regarded as the simplest enzyme that can be used to define thestructure-function relations of copper containing proteins. Only laccase presents the

possibility to oxidize activated metoxiphenols like syringaldazine [4].

In this paper, we study the oxidation of 4,4-[azinobis(methanylylidene)]bis

(2,6-dimethoxyphenol) (syringaldazine) to corresponding quinone, 4,4-[azinobis

(methanylylidene)]bis(2,6-dimethoxycyclohexa-2,5-diene-1-one) (Fig. 2). The

increase in absorbance is followed at 530 nm and 25oC, to determine the laccase

activity in international units.

Fig. 2 Catalyzed oxidation of syringaldazine by laccase to the corresponding

quinone (Sanchez-Amat and Solano, 1997).

2. MATERIALS AND METHODSLaccase (from trametes versicolor), syringaldazine, hydroquinone, tannic acid

and vanillic acid were purchased from Sigma Aldrich.. All other chemicals are of

analytical grade. Absolute methanol and ethanol were purchased from Chemical

Company. 0.1M Britton buffer solutions were obtained by mixing the same amount of0.1M acetic acid, boric acid and phosphoric acid. The desired Britton buffer solutions pH

was adjusted by different amounts of NaOH solution. All solutions were made using

deionised water.

8/13/2019 3_manole 8pag

4/8


Laccase activity determination using syringaldazineThe specific activity of laccase was assayed spectrophotometrically by monitoring

the absorbance increase from oxidation of syringaldazine at 530 nm (=65 mM1cm

1)

with a Perkin-Elmer Lambda UV-VIS spectrophotometer at room temperature (light path

1 cm). The assay mixture for the test cuvette consisted in 2.20 ml Britton buffer, 0.3ml of

syringaldazine 0.216 mM in absolute methanol and 0.5 ml of laccase 1mg/ml solution.

The assay mixture for the blank cuvette consisted in 2.20 ml Britton buffer, 0.3ml of

syringaldazine 0.216 mM in absolute methanol and 0.5 ml of deionised water. The

reaction was started by the addition of syringaldazine solution and immediate mixing by

inversion [5].

The absorbance of a sample is directly proportional to concentration (Beer's

Law) and to sample thickness (Lambert's Law). When these two relationships are

combined, we get the Beer-Lambert equation:

clA = ,

where = extinction coefficient, a characteristic constant for a given absorbingsubstance; c = concentration of substrate in mol/L; l = thickness of the sample in cm

(usually 1.00 cm for standard sample cuvettes).

The effect of pH on laccase activity and substrate specificity

Four substrates, syringaldazine, hydroquinone, vanillic acid and tannic acid (1mM

in ethanol 98%) were used to determine the effect of pH on laccase activity, as laccase

enzymes tend to react differently to pH with different substrates [6]. The pH optima were

determined over a range of pH 3 to 8. A 0.1 M Britton buffer was used for the entire pH

range. The assays for the different substrates were conducted in the same manner

described in the previous paragraph. The four different aromatic compounds were also

chosen to study the substrate specificity of laccase.

In (Table 1) are the wavelengths of maximum absorbance for the oxidized

substrates.

3. RESULTS AND DISCUSSIONSTable 1. Wavelengths of maximal absorption of laccase-oxidisable aromatic compounds.

Substrate (max. change, nm)

Syringaldazine 530

Hydroquinone 390

Vanillic acid 390

Tannic acid 458

Once the assays have been run, the absorbance values versus time are then

plotted and a regression analysis performed on the data. The slope of the graph is the

crude velocity in absorbance/second. Then that crude velocity is converted to the

change in concentration per minute, and then converted again into the change innumber of moles/mL/minute. Often the turn over number is then computed from the

knowledge of the velocity and the concentration of the enzyme that was used in the

reaction.

8/13/2019 3_manole 8pag

5/8


0 1 2 3 4 5 6 7 8 9 100,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,82,0 pH=3

pH=4

pH=5

pH=6

pH=7

pH=8

Absorbance(a.u.)

Time (min) Fig. 3 Absorbance values for syringaldazine oxidation catalyzed by laccase, at

different pH values.

1:1 1:2

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

1,2

1,3

1,4

1,5

Absorbance(u.a.)

Dilution

pH=5pH=6

Fig. 4 Absorbance values for syringaldazine oxidation catalyzed by laccase (initialconcentration 1:1 and dilution 1:2), at pH=5 and pH=6.

0 1 2 3 4 5 6 7 8 9 10

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0

Absorbance(a.u.)

Time (min)

tg = 0,4 A/min

Fig. 5 Initial rate of syringaldazine oxidation reaction catalyzed by laccase, at pH = 5.

8/13/2019 3_manole 8pag

6/8


Initial rate, v0= 0.4 A/min. To determine the concentration variation in timethe Beer law is used. The cuvette dimension is 1 cm. A = cl, then A/min = cl/min

by rearranging is obtained A/(minl) = c/min. Is known that =65 mM1cm

1 then

concentration variation/minut = 0.4/ 65 mM/min = 0.6 10-2

mM/min = 0.6 10-2

moli/mL/min.

The optimum pH values, for which the enzyme activity is maxim, are

characteristic for each enzyme. In this pH optimum domain the proton acceptor and

proton donor groups of the active centre are in ionized state necessary for the enzyme

to be active. Outside this domain the binding of substrate is not possible and if the pH

value exceeds o certain limit value the enzyme can be irreversible denaturized. This

pH optimum depends on the environment composition, temperature and enzyme

stability in acid and alkaline environment. The pH stability domain doesnt necessary

coincide with reaction rate optimum domain [6].

3 4 5 6 7 8

0,080

0,085

0,090

0,095

0,100

0,105

0,110

0,115

Absorbance(a.u.)

pH Fig. 6 Absorbance values for tannic acid oxidation catalyzed by laccase, function of

pH (after 8 minute from the reaction start).

3 4 5 6 7 80,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

0,45

Absorbance(a.u.)

pH Fig. 7 Absorbance values for vanillic acid oxidation catalyzed by laccase, function ofpH (after 8 minute from the reaction start).

8/13/2019 3_manole 8pag

7/8


3 4 5 6 7 80,05

0,10

0,15

0,20

0,25

0,30

Absorbance(a.u.)

pH Fig. 8 Absorbance values for hydroquinone oxidation catalyzed by laccase, function

of pH (after 8 minute from the reaction start).

3 4 5 6 7 8

0,0

0,5

1,0

1,5

2,0

Absorbance(a.u.)

pH

Fig. 9 Absorbance values for syringaldazine oxidation catalyzed by laccase, functionof pH (after 8 minute from the reaction start).

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0

0,100

0,102

0,104

0,106

0,108

0,110

0,112

0,114

0,116

0,118

0,120

Absorbance(u.a.)

Time (min)

hidroquinonevanillic acid

tannic acid

Fig. 10 Absorbance values for the three different aromatic compounds oxidation

catalyzed by laccase, at pH=5.

8/13/2019 3_manole 8pag

8/8


From the measurements done it can be observed that the optimum pH forsyringaldazine oxidation and tannic acid respectively, by laccase, is 5 and, for

hydroquinone and vanillic acid oxidation is 4.

4. CONCLUSIONSFrom the enzymatic assay of laccase, in case of using syringaldazine as

substrate, according to the procedure indicated by the producer, resulted that laccase

has an activity of 1,8 10-2 mols/min. Also, by using the continuous

spectrophotometric rate determination method the optimum pH determined for this

oxidation reaction, catalyzed by laccase, at 25oC, is 5. In the case of using tannic acid

as substrate the optimum pH is also 5, and for vanillic acid and hydroquinone is 4.

From the measurements regarding laccase substrate specificity for the 4

different phenolic compounds (syringaldazine, hydroquinone, vanillic acid and tannic

acid) can be observed that the enzyme is catalyzing the oxidation of all thiscompounds. Also, it can be observed that the activity of laccase presented in the case

of syringaldazine oxidation is superior to that presented for the other 3 phenols.

REFERENCES

1. D.L. Nelson, M.M. Cox, Lehninger: Principles of Biochemistry, 2004.2. A. Ghindilis, Direct electron transfer catalysed by enzymes: application for biosensor

development, Biochemical Society Transactions, Vol. 28, part 2, p. 84-89, 2000.

3. Marille Bar, Kinetics and physico-chemical properties of white-rot fungal laccases, 20014. C.F. Thurston, The structure and function of fungal laccases, Microbiology, 140, p. 19-26, 1994.5. J.P. Ride, Physiological Plant Pathology, Vol. 16, p. 187-196, 1980.6. F. Xu, Effects of redox potential and hydroxide inhibition on the pH activity, profile of fungal

laccases, The Journal of Biological Chemistry, 272 (10), p. 924-928, 1997.

3_manole 8pag

Documents