This document summarizes research on purifying and characterizing manganese peroxidase (MnP) enzyme produced by the white-rot fungus Irpex lacteus. The MnP enzyme was purified using ion exchange and size exclusion chromatography. Characterization found the molecular mass to be 38.3 kDa by mass spectrometry and 53 kDa by SDS-PAGE. The isoelectric point was 3.7. Spectroscopy showed it is a heme-containing glycoprotein. Mass spectrometry identified tryptic peptides. The purified MnP effectively decolorized textile dye wastewater and oxidized Mn2+ to Mn3+, with optimal activity at pH 6 and 35°C.

1. Name: Sugandhi Hansini
Admission ID :LUC000000259093052021
MSc Biotechnology
Lincoln University College
Purification & Characterization of
Manganese Peroxidase of the White-
Rot Fungus Irpex lacteus

2. Contents
• Introduction
• Objectives
• Methodology
• Results and Discussion
• Future Perspectives
• References

3. White-Rot Fungus - Irpex lacteus
Belong to
Basidiomycota
Usually whitish in color
and fibrous in texture.
Decolorization of textile
industry waste water
Secretion of extracellular
ligninolytic enzymes
(Laccase/ Lignin peroxidase/
Manganese peroxidase)
Colonize plants or
plant residues (e.g.,
wood)
Promising
bioremediation
agents
Most efficient lignin
degraders in nature
Introduction

4. Manganese Peroxidase (MnP)
Belongs to the family
of oxidoreductases
Glycosylated heme
containing enzyme
Produced by
Ligninolytic fungi
Degradation of Lignin,
Polycyclic aromatic
hydrocarbons, Polychlorinated
biphenyls
Catalyzes the oxidation of
Mn2+ to Mn3+ in the presence of
hydrogen peroxide (H2O2)
Decolorization of
Textile industry waste
water

5. Purpose of the Study
• To purify the Manganese Peroxidase (MnP) enzyme produced by White-Rot
Fungus, Irpex lacteus
• To determine the characterization of MnP enzyme

6. Methodology
• Culture of the Organism
• Enzyme Purification
• Enzyme Characterization
Molecular mass
Isoelectric point
Spectroscopic properties
MS characterization
• Enzyme Assay
Catalytic properties of the enzyme
Effect of pH and Temperature

7. Culture of the organism
Irpex lacteus
Strain- KR 35W
Maintained on
100ml MGPY Agar slant (solid)
(1% malt extract,1% glucose, 0.5% peptone,
0.5% yeast extract)
For 7 days at 4°C
Four Mycelial agar
discs (0.9cm) for use as
inocula
Fungus + MGPY liquid
inocula (homogenized)
10ml of
inocula
90ml MGPY
broth
10% v/v diluted
inocula culture
fluid

8. Enzyme Purification
Culture fluid
Filtration
Whatman No 1
filter paper
Culture
filtrate
Cold Acetone (-
10°C) Precipitated
proteins
Remove
acetone Precipitate dissolve in
20mM sodium phosphate
buffer at pH 8
Protein
sample
mixture

9. Enzyme Purification
Protein
sample
mixture
Anion Exchange
Chromatography
(HiPrep 16/10 Q XL
column)
Enzyme fractions
containing MnP
collected and
concentrated
Size Exclusion
Chromatography/ Gel
Filtration (HiPrep 26/60
Sephacryl S-200)
Homogeneity of
the enzyme
confirmed by SDS
-PAGE
Figure 1: Ion Exchange Chromatography Figure 2: Size Exclusion Chromatography

10. Enzyme Characterization
Molecular mass
Estimated with SDS-PAGE 12.5% polyacrylamide
gel –
An analytical technique to separate proteins
based on their molecular weight
Estimated with MALDI-TOF mass
spectrometry –
Mass spectrometry is an analytical technique
in which samples are ionized into charged
molecules and ratio of their mass-to-charge
(m/z) can be measured
Estimated by Gel filtration
chromatography on a Superose
12 HR 10/30 column mounted
with FPLC system
Figure 3: FPLC System Figure 4: MALDI-TOF mass spectrometry Figure 5: SDS-PAGE Technique

11. Enzyme Characterization
Isoelectric point
Determined using isoelectric focusing polyacrylamide gel
electrophoresis (IEF-PAGE)
A method of separating proteins according to their Isoelectric
points in a pH gradient.
Stain – Coomassie brilliant blue G-250
Figure 6: IEF-PAGE Technique

12. Enzyme Characterization
Spectroscopic properties and Mass Spectrometry (MS) characterization
• An absorption spectrum of the purified enzyme MnP, was observed with the addition of hydrogen
peroxide
• MS characterization was determined by using MALDI-MS and ESI Q-TOF MS/MS after the digestion
of the purified enzyme with trypsin.
• Resulted protein mixture was analyzed using MASCOT peptide Mass Fingerprint software.

13. Enzyme Assay
Laccase
Activity
Lignin
Peroxidase
Activity
Manganese
peroxidase
Activity
Determined via the
oxidation of ABST
Assayed using veratryl alcohol
as substrate
Assayed by measuring the oxidation of
Mn(II) to Mn(III) at 270nm.
Assay mixture contained : 1mM
MnSO4 in 50mM sodium malonate(pH
4.5), 0.2mM Hydrogen peroxide
Purified
Protein
One enzyme unit:
Amount of enzyme
required to oxidize
1µmol of substrate per
min, under the assay
conditions

14. Enzyme Assay Continue…
MnP Catalytic Properties- Textile Industry Waste Water Decolorization
Culture filtrate
20ml of Textile
industry waste
water at 28ºC
Decolorized water
Figure 7: Catalytic cycle of MnP
With the
presence of
Hydrogen
peroxide

15. Results
• Molecular mass
• Isoelectric point
• Spectroscopic properties
• MS characterization
• Catalytic properties of the enzyme
• Effect of pH and Temperature

16. Molecular mass
Gel filtration chromatography on a
Superose 12 HR 10/30 column
mounted with FPLC system
Estimated with MALDI-TOF
mass spectrometry –
Estimated with SDS-PAGE 12.5%
polyacrylamide gel
Apparent
molecular mass of
the purified
enzyme was
observed as 53
kDa
Figure 8: MALDI-TOF mass spectrum of the native MnP.
Molecular mass was observed as 38.3kDa
Figure 9: SDS-PAGE of the purified MnP.
Molecular mass was observed as 53.2kDa
standard protein-bovine serum albumin

17. Isoelectric point
Figure 10: Isoelectric focusing of the purified MnP.
Isoelectric point of the enzyme was observed as 3.7

18. Spectroscopic properties
Figure 11: Absorption spectra of the MnP
 Absorption spectrum of the purified enzyme
showed the peaks at about 407, 500 and
640nm.
 Addition of Hydrogen peroxide to the enzyme,
Resulted in the shift from 407 and 500 to 409
and 525nm with decreased absorbance

19. Mass Spectrometry (MS) characterization
Figure 12: MALDI-TOF MS spectrum of tryptic digest of MnP
Figure 13: Identification of tryptic peptides from I. lacteus MnP by
MASCOT PEPTIDE Mass Fingerprint software
 Three peptides m/z 1039.6,1631.9 and 1854.0 were further
analyzed with ESI Q-TOF MS/MS
 Amino acid sequences for each peptide were obtained

20. Catalytic properties of the enzyme
Figure 14: MnP and Laccase activity on textile industry waste water
20ml of Textile
industry waste
water at 28ºC
With the
presence of
Hydrogen
peroxide
Purified protein
solution
decolorization.
Decolorization rate was determined by measuring the
absorbance at 600nm
Decolorized water
 Graph indicates that the MnP activity of the culture
filtrates was shown correlate with the decolorization
of textile industry waste water

21. Catalytic properties of the enzyme
Figure 16: Production of Mn(III) malonate from the oxidation of Mn (II) by the MnP
Reaction mixture contains 0.5mM MnSO4, 0.5mM sodium
malonate(pH 4.5), 0.2mM Hydrogen peroxide
(spectra recorded every 2 min)
Figure 15: Decolorization of various dyes by MnP
 The azo dye, methyl orange was the most rapidly
decolorized

22. Effect of pH and Temperature
Figure 17: Effect of pH and Temperature on the MnP activity
 Enzyme Activity was assayed at various pHs
and Temperature under the standard
conditions
 Optimum pH for the enzyme activity was
estimated to be around 6.0
 Optimum temperature for the enzyme activity
was about 35°C.

23. Discussion
 Enzyme purification
Purification of MnP from the culture filtrate of Irpex lacteus
 Molecular Mass
• Mass spectrometry showed 38.3kDa of molecular mass while gel filtration and SDS-PAGE showed
the molecular mass of 53kDa.
• It was suggested that, since the MnP is a glycoprotein, carbohydrate content of the enzyme affected
by its migration in gel filtration.
• These data indicate that the enzyme contains about 28% total carbohydrate and it is monomeric,
similar to other fungal MnP
• Enzyme had been purified 11.0 fold with a yield of 24.3%

24. Discussion
 Isoelectric point ( Net charge =0)
• Depending on the point at which the sample is loaded,
protein starts to migrate.
• If a protein is at a pH range which is below its isoelectric
point, protein is (+) charged and move towards the cathode.
• If a protein is at a pH range which is higher than its
isoelectric point , protein is (-) charged and move towards
the anode
• Isoelectric point of the enzyme was 3.7. Usual
feature among fungal MnPs, which exhibit acidic isoelectric
point

25. Discussion
 Spectroscopic properties
• After the addition of hydrogen peroxide to MnP, decreased
absorbance and red shift of wavelength was observed.
• This suggested that, with the presence of hydrogen
peroxide, MnP enzyme is oxidized and it is a heme protein
with iron protoporphyrin ix as the prosthetic group
Absorption spectra of the MnP

26. Discussion
 Mass Spectrometry (MS) characterization
• Peptide1039.6 showed 78% similarity to AY217015
• Peptide1631.9 showed 67% similarity to AY677128
• Peptide 1854.0, no significant similarity found with any other
white rot fungal peroxidase
• This results suggested that amino acid sequences of
the tryptic peptides of purified MnP were shown to
have little similarity to those of other fungal MnPS
MALDI-TOF MS spectrum of tryptic digest of MnP Identification of tryptic peptides from I. lacteus MnP

27. Discussion
 Catalytic properties of the enzyme
• LiP activity was negligible
• Laccase enzyme activity was not significantly related to
decolorization of waste water
• The MnP activity of the culture filtrate was shown to
correlate with the decolorization of textile industry waste
water
MnP and Laccase activity on textile industry waste water decolorization

28. • The azo dye, methyl orange was the most rapidly decolorized
 Catalytic properties of the enzyme
Discussion
• Reaction mixture contains 0.5mM MnSO4, 0.5mM sodium
malonate(pH 4.5), 0.2mM Hydrogen peroxide
• This suggests that, In the presence of Hydrogen peroxide, enzyme
readily oxidized Mn(II) and form Mn(III) malonate (267nm)
• Absorbance increases with the time
Decolorization of various dyes by MnP Production of Mn(III) malonate from the oxidation of Mn (II) by the MnP (spectra
recorded in evry 2min)

29. Future perspectives
Molecular biological properties of the MnP enzyme requires further studies

30. References
 Baborová, P., Möder, M., Baldrian, P., Cajthamlová, K., & Cajthaml, T. (2006). Purification of a new manganese
peroxidase of the white-rot fungus Irpex lacteus, and degradation of polycyclic aromatic hydrocarbons by the
enzyme. Research in Microbiology, 157(3), 248-253.
 Chowdhary, P., Shukla, G., Raj, G., Ferreira, L. F. R., & Bharagava, R. N. (2019). Microbial manganese peroxidase: a
ligninolytic enzyme and its ample opportunities in research. SN Applied Sciences, 1(1), 1-12.
 Shin, K. S., Kim, Y. H., & Lim, J. S. (2005). Purification and characterization of manganese peroxidase of the white-rot
fungus Irpex lacteus. Journal of microbiology, 43(6), 503-509.
 Tripathi, A., & Dixit, S. (2016). Bioremediation of phenolic compounds by higher fungi—a review. Int J Adv Res, 4(7), 14-
35.
 Wesenberg, D., Kyriakides, I., & Agathos, S. N. (2003). White-rot fungi and their enzymes for the treatment of industrial
dye effluents. Biotechnology advances, 22(1-2), 161-187.
 Xu, H., Guo, M. Y., Gao, Y. H., Bai, X. H., & Zhou, X. W. (2017). Expression and characteristics of manganese peroxidase
from Ganoderma lucidum in Pichia pastoris and its application in the degradation of four dyes and phenol. BMC
biotechnology, 17(1), 1-12.

31. THANK YOU!!!