Indian Journal of Biochemistry & Biophysics Vol. 36, February 1999, pp. 39-43

n-Propanol as a substrate for assaying the ligninperoxidase activity of Phanerochaete chrysoporium

V Shanmugam, Meera Kumari and K D S Yadav*

Department of Chemistry, DDU Gorakhpur University, Gorakhpur 273 009, Ind ia

Received 2 September 1997; revised 5 OClober 1998

The steady state kinetics of ligninperoxidase catalysed reaction using II-propanol as the organic substrate and monitoring the formation of propanaldehyde at /... = 300 nm spectropbotometericall y as functions of different reaction parameters has been studied . It has been concluded that n-propanol can be used as a substrate for analysing the activ ity of li gninperoxidase. The turnover number of Iigninperoxidase of Phallerocfla ele cflrysosporium using n-propanol as substrate has been fou nd to be higher approximately by a factor of 103 as compared to th at using veratryl alcohol as the substrate. The method works in assaying the activity of Iigninperoxidase produced by Aspergillus jUfll iga Ills indicating that it can be used for assayi ng the ligninperoxidase activities produced by other microorganisms also and is not limited to assaying the ligninase activity produced by Phanerochaele chrysosporium alone. Under identical experimental conditions, horseradish peroxidase does not show peroxidase activity using n-propanol as substrate indicating that the method does not interfere with the acti vi ti es of other peroxidases.

Ligninperoxidases are enzymes of great research interest '-

6 due to their role in delignification of lignocellulosis material s7

,8 and degradation of recalcitrant organic pollutants9

" o. A number of methods for assaying the activity of ligninperoxidase have been developed I I which use monomeric and dimeric lignin model compounds, polymeric dyes and 14C and I3C labeled lignins , However, these methods ei ther involve rare and expensive s ubstrates like 14C and I3C labe led lignins or lack wide applicability. The most convenient alTXlng the avai lable assay methods is based on using 3,4-dimethoxy benzyl alcohol (veratryl alcohol) as substrate and monitoring the formation of corresponding aldehydes at 310 rnM spectrophotometrical I y ,

Even this substrate is not available in pure form commercially and the available form needs vacuum distillation be fore use l2

, We searched for an alternative to thi s. Lignin consists of aryl propyl unit and major reaction in its degradation seems to occur in propyl unit2 An enzyme assay method for ligninperoxidase based purely on propyl unit has been attempted so far. We used n-propyl alcohol in place of veratry l alcohol and monitored the formation of propanaldehyde at 300 rnM instead of veratraldehyde at 3 10 nm. Our finding is that n-propanol can be used for assaying the activity of ligninperoxidase from Phanerochaele chrysosporium. The method is also

·To whom all correspondence should be addressed

CH 30yCH;PH + H;P2

C H30

Lignin . ,.

Pcro.\1dc C H30yCHO

C HJO

applicable for assaying the ligninperoxidase activity produced by Aspergillus fumigatus indicating that it can be used for assaying the ligninperoxidase activities of other mocroorganisms also, Horseradish peroxidase does not show peroxidase activity using n-propanol as substrate under the experimenta l conditions identical to those used for ligninperoxidase assay indicating that the method does not interfere with the activities of other peroxidases,

Materials and Methods

Veratryl alcohol (Aldrich, USA), n-propanol (E. Merck, Germany), dimethyl succinate, guaicol , horseradish perOXIdase and nitrilotriacetate (Sigma, USA:) were used in thi s work. A ll other chemicals were either from CDH (Delhi) · or Loba Chemie (Bombay) and were used without further purification ,

Crude preparation of ligninperoxidase fro m Phanerochaete chrysosporium strain BKM-F-1767 [ATCC 24725] has been used in this work which was prepared in our laboratory by modification of a reported method 13 . Our method consists of immobilising the mycelia of Phanerochaete chrysosporium on nylon net which is used for making

40 INDIAN J. BIOCHEM. BIOPHYS., FEBRUARY 1999

mosquito net. The immobilised mycelia were dlipped in liquid culture media l2 under the stationary culture .condition. The liquid culture media was kept saturated with oxygen.

The ligninperoxidase activity has been assayed in 50 mM tartaric acidldisodium tartrate buffer, pH 3.0 which was 0.41 mM with respect to H20 2 and temperature was kept at 30°C. UVNIS Spectrophotometer Hitachi Model U-2000 fitted with electronic temperature control has been used for spectrophotometric measurements. The sensitivity of the equipment is 0.00 I 0.0. unit. The data points are average of duplicates. The molar extinction coefficients of 20 MI cm-I at A = 310 and 9300 M lcm·1 at A = 310 have been used for monitoring the formation of propanaldehyde and veratraldehyde respectively.

Results and Discussion Fig, I(a) shows the time course of prop anal de hyde

formation using enzyme saturating concentration of n­propanol in presence ofH20 2• In separate experiments no formation of propanaldehyde was detected either in absence of H20 2 or in absence of ligninperoxidase which confirms the enzymatic nature' of the reaction in presence of H20 2 only. Fig. l(b) represents the corresponding production of veratraldehyde using enzyme saturating concentration of veratryl. In both these experiments, all other conditions except the organic substrates and their concentrations hav(: been ~ept the same. It is obvious from the figures thalt 0.0. change/minute in case of n-propanol as the organic

(I) (b)

0.12

0.01

::5 gl ,..,

8 0.04

JO 60 90 JO 60 90

Time (sec)

Fig. 1-0.0. vs time plots of Jigninperoxidase catalysed reactions [(a), Propanol 3.25 mM; (b), Veratryl alcohol \.8 nm; 50 mM tartaric acid/disodium tartrate buffer pH 3.0; H20 2 0.4 1 mM; Temperature 30°C; 0.5 ml crude extract of ligninperoxidase (activity 0.055 Ulml) in 3 ml reaction solution]

substrate is larger than that in case of veratryl alcohol showing that it is more convenient to follow the steady state production of n-propanaldehyde as compared to the steady state production of veratraldehyde in the ligninperoxidase catalysed reaction. The enzym.atic production of propanaldehyde has been monitored at A = 300 rnM using molar extinction coefficient value of 20 MI em-I whereas the formation ·of veratraldehyde has been monitored at A = 310 mM using molar extinction coefficient value of 9300 MI em-I (ref. 15). In both the cases, the enzyme is saturated with the organic substrates and the same concentration of H20 2 has been used. This allowed us to calculate the ratio of turnover number which is approximately 103 times higher in case of n-propanol as the organic substrate as compared to veratryl alcohol. Thus though the molar extinction coefficient of propanaldehyde, 20 MI cm-t

, is much smaller than the molar extinction coefficient of veratraldehyde, which is 9300 MI cm-I, the turnover number of ligninperoxidase in case of n­propanol as the substrate is much higher than in case of veratryl alcohol and makes it feasible to follow the steady state kinetics of ligninperoxidase catalysed reaction spectrophotometrically.

The steady state velocities of ligninperoxidase catalysed reaction at varying concentrations of n-propariol as the organic substrate are plotted ill

:j" '--' 3 0 > .

I

(b) 30

'2 120 's Ul cU '0 E s:: '-'

>

0.4 0.8 1.2 1.6

[S] (mM)

Fig. 2~ichaelis-Menten plots [Ca), Concentration of propanol yaried; (b), concentration of veratryl alcohol varied. Other conditions are same as mentioned in the legend to Fig. I]

SHANMUGAM et 01.: n-PROPANOL AS SUBSTRATE FOR ASSAYING L1GNINPEROXIDASE 41

Fig.2(a) which clearly establishes the Michaelis­Menten behaviour. The cOlTesponding plot using veratryl alcohol as the variable organic substrate is shown in Fig. 2(b) for the sake of comparison. The Km values calculated from the plots ar~ approximately 500 ~ for n-propanol and 50 ~ for veratryl alcohol. The reported l2 Km value for veratryl alcohol is 60 ~ which is nearly the same as determined in our laboratory. This gives confidence in the determination of Km value of approxim~tely 500 ~ for n-propanol. The double reciprocal plots and Eadie-Hofstee plots of the data shown in Fig. 2(a) are shown in Figs. 3 and 4 respectively. These plots confirm the linear steady state kinetics of the reaction catalysed by ligninperoxidase usingn-propanol as the organic substrate in the concentration range in which the reaction has been. studied.

Variation of steady state velocity of ligninperoxidase catalysed reaction with pH using n­propanol as the organic substrate is shown in Fig. 5(a) which clearly indicates a pH optimum value of around 3 which agrees well with the value obtained from Fig. 5(b) using ver~tryl alcohol as the organic substrate in our laboratory and also with the value reported in the literature l 6

.17 using veratryl alcohol as the substrate.

The level of agreements clearly establishes the fact that n-propanol and veratryl alcohol both react with enzyme apparently in similar way and n-propanol can substitute veratryl alcohol as the organic substrate for assaying the activity of ligninperoxidase in preseJ\ce ofH20 2.

0.3 ,-----------------.

~ 0.2

~ '0 E 2-

;; 0.1

0.8 1.6 2.4

Fig. 3-Double reciprocal plots using n-propanol as the substrate

Fig. 6(a) shows the vanatton of steady state velocity of ligninperoxidase catalysed reaction with temperature using n-propanol as the substrate. The corresponding curve using veratryl alcohol is shown in Fig. 6(b). The values of temperature optima calculated from these figures are around 27°C and 26°C using n-propanol and veratryl alcohol as the

12.0 r------------------,

0

C 8.0

~ 0 '0 0 E 2->

4.0

o~---~~----~----~ 4.0 8.0 12.0 V

[S] [Ii moles/min] [mM}-1

Fig. 4-Eadie-Hofstee plots using n-propanol as the substrate

12.0

'2 e 8.0 -;;, u '0 E 2. >

4.0

2.0

(b) -----~-l

I

>

4.0 6.0 2.0 4.0 f) .0

pH

Fig. S"--Variations of ligninperoxidase activity with pH [(a), Propanol as the substrate, 1.33 mM; (b), veratryl alcohol as the substrate, 0.9 mM. Other conditions are same as given in the legend to Fig. I except pH]

42 INDIAN 1. BIOCHEM. BIOPHYS., FEBRUARY 1999

substrates respectively. This also shows the similarity of n-propanol and veratryl alcohol as substrates' for assaying the activity of ligninperoxidase.

The steady state velocities of ligninperoxidase catalysed reaction using n-propanol as substrate at different enzyme concentrations are plotted in Fig. 7 which show the linear variation of steady state velocity wi th the ligninperoxidase concentration.

Thus the results of steady state velocity measurements as functions of different parameters of Jigninperoxidase catalysed reaction in presence of H20 Z using n-propanol as the organic substrate and their comparison with the corresponding results using veratryl alcohol indicate clearly that n-propanol can be used for assaying the activity of Iigninperoxidase of Phanerochaete ohrysosporium .. In order to ensure that the method is not limited to assaying the ligninperoxidase activity produced by Phanerochaete chrysosporium alone but is also applicable for assaying thc Jigninperoxidase activity produced by other microorganisms, ligninperoxidase activity of culture fi ltrate of A5pergillus Jumigatus was determined using n-propanol as the substrate. The same filtrate was assayed using veratryl alcohol as the substrate also to confirm the Iignolytic activity of the filtrate. Though the level of enzyme produced in

10.0 r----------r-----..., (a) (b)

8.0

C :s ·s 6.0

e "£ <II .., v "0 "0 e e ..:. 2; > >

4.0

2.0

10 20 30 10 20 30

Temp ·c

Fig. 6--Variation of ligninperoxidasc activity with temperature [(a), Propanol as the substrate 1.33 rnM; (p), Veratryl alcohol as the substrate 0.9 rnM. All other conditions are same as given in the legend to Fig. I]

culture filtrate of A. Jumigatus is very low, the variation of absorbances as a function of time as

9.0r-------------__

6.0

~ "" Q)

"0 8 2->

3.0

o "-------::--':------'----_ _ ---.l 0.2 0.4 0.6

Volume of extract (ml )

Fig. 7-Variation of steady state velocity with Iigninperoxidase concentration [n-Propanol (concentration fixed at 1.33 mM) and different volumes of crude lignin peroxidase preparation (from 0.1 ml to 0.4 ml) in 3 ml of reaction solution. All other parameters are same as in the legend to Fig. 1]

0.012 r-----------------;(.:::-)--,

o 0

~

o 60 110 110

Time (sec)

Fig. 8--Variation of absorbance with time using A. jumigalus Iigninperoxidase [(a), n-Propanol 3.25 rnM at A = 300 rnM; (b), veratryl alcohol 1.8 mm at ,. = 310 rnM. Other experimental conditions same as mentioned in the legend to Fig. I except ligninase which was 0.001 unit and I ml of the reaction mixture was used]

SHANMUGAM et a/. : n-PROPANOL AS SUBSTRATE FOR ASSAYING L1GNINPEROXIDASE 43

shown in Fig . 8 clearly indicates that n-propanol like veratryl alcohol is a substrate for Iigninase of A. Jumigatus.

The idea that other peroxidases do not react with n-propanol under the experimental conditions used for the assay of ligninperoxidase was verified using horseradi sh peroxidase as a typical enzyme and n­propanol as the substrate. Since no absorbance change with time was observed in this case, the results are not shown. This observation clearly indicates that horseradish peroxidase cannot oxidise n-propanol to propanaldehyde under the experimental conditions identical to the assay .conditions of lignin peroxidase. The point that the used sample of horseradish peroxidase was really active and was not a completely deactivated sample was tested using guaicol as the substrate as reported by Montellano et al. 18

Since n-propanol worked as a better substrate for assaying the Iigninperoxidase activity in comparison to veratryl alcohol , it was tempting to investigate the relative merits of other alcohols lying just above and below n-propanol in the homologous series as the substrates for assaying the Iigninperoxidase activity. Optical density changes (not presented here) per unit time in case of n-butanol monitored at A = 280 mM corresponding to butanaldehyde formation were half of the optical density changes per unit time obtained in case of n-propanol monitored at A = 300 mM corresponding to propanaldehyde formation ; in both cases alcohols being present in enzyme saturating conditions. In case of ethanol as the organic substrate, no optical density change per unit time was observed at A = 270 mM corresponding to acetaldehyde formation indicating that ethanol is not a substrate for Iigninperoxidase .

This find ing may have a wider implication not only in assaying the Iigninperoxidase activity produced by other microorganisms but also in understanding the molecular mechanism of catalysis by ligninperoxidase

which in tum will lead to the mechanism of degradation of recalcitrant organic pollutants.

Acknowledgement Authors are thankful to Dr. P. J. Harvey, Imperial

College, London, UK for providing the Phanerochaete chrysosporium strain BKM-F- 1767 (ATCC 24725). This work has been supported by the Department of Science and Technology, Govt. of India, New Delhi through the grant no. SP/S01D45/90 .

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