Changing of the activity of the exoenzyme Manganese peroxidase after laser radiation

Olga Tsivunchyk1, Rainer Haas2, Eberhard v. Loew3, Eugeny P. Kremlev1, Katrin Scheibner4

1: Yanka Kupala Grodno State University, Ozheshko str. 22, Grodno, 230023, Republic of Belarus

2: gaiasafe Ltd., Stadtwaldstrasse 45a, D-35037 Marburg, Germany

3: Institute of Immunology, Pilgrimstein 2, D-35037 Marburg, Germany

4: JenaBios GmbH, Löbstedter Strasse 78, D-07749 Jena, Germany


The influence of low laser radiation at the activity of Manganese peroxidase (MnP) was investigated with photometrical studies. Different kinds of laser (YAG, He-Ne, Ar) and Xenon lamp (at wavelength of 355 nm) were used. It was found, that radiation with ultraviolet light sources (Xenon lamp and YAG laser radiation at 355 nm) and Ar laser reduced the activity of MnP. Radiation with visible light (HeNe- and YAG-laser (533 nm)) had no influence at the MnP activity. These results are according to the UV-Vis spectrum of Manganese peroxidase, MnP shows only absorbance in and near the UV part of the spectrum.

Key Words: activity, enzymatic system, laser, low laser radiation, manganese peroxidase

1 Introduction

Manganese peroxidase (MnP) is one of exoenzymes of lignin destroying mushrooms. It is well known that MnP is able to destroy the high persistent lignin. Because of it MnP is able to destroy high persistent organical pollutants too. Former investigations of Scheibner and Haas showed, that it was possible to destroy persistent arsenoorganic compounds with this MnP enzymatic system [1,2,3].

During the last twenty years there are a lot of results obtained about influence of laser radiation on biological tissues and biological responses of different biochemical systems [4,5].

Former results from Tsivunchyk and Khomich showed, that different kinds of low laser radiation had influence on biological systems and in particular on enzymes of the antioxidant system of animals [6,7].

Aim of our investigations was to detect influence of different kinds of low laser radiation on the activity of the Manganese peroxidase system of Nematoloma frowardii. As a model reaction the photometrical MnP activity test was used by the method of Wariishi et al. (1992) [8].

2 Experiments

Investigations were done to show the influence of different sources of light on the activity of the isolated enzymatic MnP system. Four sources of light were included in the experiments: Argon laser irradiation, YAG laser irradiation, He-Ne laser irradiation and irradiation by a Xenon lamp:

a) YAG laser: ultraviolet light (355 nm), energy 10 mW, pulse rate 10 Hz

b) Argon laser: blue light (458 nm), energy 500 mW

c) Xenon lamp: white light (355 nm), energy 100 µW.

d) YAG laser: green light (533 nm), energy 100 mW, pulse rate 10 Hz

e) He-Ne laser: red light (633 nm), energy 10 µW.

The exposition time was varied from 10 min, 20 min and 40 min. Light expositions were done with 1 ml aqueous solution of Manganese peroxidase (stock solution, activity = 60 U/ml) in 1 cm kuvettes. The beams were centered (Xenon lamp) or spreaded (lasers) to 1 * 1 cm beam with lenses.

The MnP activity was determined with the method of Warishi et al. (1992) [8]. The principle of the method is the photometrical detection of chelate complexes, which are build by Manganese(III) ions and malonate. Manganese peroxidase produces Mn(III) ions from Mn(II) ions.

The measuring of the MnP activity was done in the following way:

1 ml Na-malonate buffer (c = 50 mmol/l, pH 4.5) in water, 50 µl MnCl2 solution (10 mmol/l) in water and 100 µl MnP solution (stock solution with an activity of 60 U/ml is diluted with dest. water 1:50) were mixed in 1 cm UV-kuvette. The extinction was measured photometricaly at a wavelength of 270 nm.

At the first the extinction of this solution was measured and set to zero. Then 20 µl H2O2 solution (c = 300 mg/l) was added, the solution was mixed and a timer was started.

After reaction times of 1 min, 1.5 min, 2 min, 2.5 min and 3 min the extinction was measured. Calibration was done with unradiated MnP solution in different dilutions. The MnP activity of the all radiated MnP solutions were measured in the same way. The results of the radiated samples were counted in percentage of unradiated Manganese peroxidase activity.

3 Results and discussion

The results of the radiation experiments are shown in table 1. The activity of unradiated MnP was set to 100%.

Table 1: Effect of light radiation of the activity of manganese peroxidase;

Results are given in % of unradiated MnP

light source

activity in % after 10 min radiation

activity in % after 20 min radiation

activity in % after 40 min radiation













YAG, green








The most remarkable results, strong reduction of MnP activity, were obtained after radiation with YAG laser with wavelength 355 nm, energy of 100 mW and pulse rate of 10 Hz.

The activity was reduced to 34.8 % of the original activity after 10 min radiation, to 18.0 % after 20 min radiation and to 11.2 % after 40 min radiation.

Remarkable results, reduction of MnP activity, were obtained after Ar laser radiation at a wavelength at 458 nm and an energy of 500 mW.

The activity was reduced to 73.5 % of the original activity after 10 min radiation and to 20.6 % after 40 min radiation.

Visible results were obtained after radiation with Xenon lamp at a wavelength of 355 nm and an energy of 100 µW.

The activity was reduced to 83.0 % of the original activity after 10 min radiation, to 70.5 % after 20 min radiation and to 64.6 % after 40 min radiation.

In all three experiments similar effects were observed: as long as radiation time was, as less activity of MnP was detected.

The other radiation experiments, it means YAG green light (533 nm) and He-Ne laser (633 nm) didn´t show lower MnP-activity after radiation as zero control (unradiated MnP).

The results show a dependence of wavelength of light radiation and UV-Vis-spectrum of MnP: MnP has maximum of absorbance at 214 nm, in the visible region with wavelength higher as 500 nm there is nearly no absorption of the molecule. Taken energy from ultraviolet light is able to decrease the activity of MnP, but not visible light with wavelength higher than 500 nm.

The MnP system is used for the destruction of organic pollutants in the environmental surrounding. The practical applicance of these experiments allows to utilize MnP more effectively and under certain conditions, one of such conditions is daylight.

4 References

[1] M. Hofrichter, K. Scheibner, R. Haas, J. Nüske, W. Fritsche: Das Mangan-Peroxidase-System ligninolytischer Pilze: Eine innovative Strategie zur Oxidation persistenter Verbindungen. in.: Heiden, Erb, Warrelmann, Dierstein (Ed.): Biotechnologie im Umweltschutz. pp. 108 - 116. Erich Schmidt Verlag, Berlin 1999.

[2] M. Hofrichter, K. Scheibner, I. Schneegass, W. Fritsche: Enzymatic combustion of aromatic and aliphatic compounds by Manganese peroxidase from Nematoloma frowardii. Applied and environmental microbiology 64 (1998), 399 - 404.

[3] M. Hofrichter, K. Vares, K. Scheibner, S. Galkin, J. Sipilä, A. Hatakka: Mineralization and solubilization of synthetic lignin by Manganese peroxidases from Nematoloma frowardii and Phlebia radiata. J. Biotechnology 67 (1999), 217 - 228.

[4] N. Dewiatkow, S. Zubkova, I. Laprun, N. Makeeva: Physical and chemical mechanisms of biological influence of laser radiation. Successes of up-date’s Biology 1 (1987), 31 - 41.

[5] I. Liandres: Photobiological effects of low laser radiation. Mechanisms of biostimulation of low laser radiation 2 (1998), 19 - 28.

[6] O. Tsivunchyk, I. Osakowitsch, T. Khomich, S. Anufrick: Low laser radiation as a modulator of antioxidant system of animals. in: 2nd conference of quantum electronic Minsk, 2000. pp. 190 - 193. Minsk 2000.

[7] O. Tsivunchyk, T. Khomich, I. Osakowitsch: Effects of low intensity laser radiation on animal antioxidant system. in: Grodno seminar: Applicance of low laser radiation in biology and medicine. pp. 21 - 23. Grodno 2001.

[8] H. Wariishi, K. Valli, M.H. Gold: Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 267 (1992), 23688 - 23695.

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