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Keywords:

  • biodegradation;
  • enzymes;
  • fermentation;
  • fungi

Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References
  5. Supporting Information

Aims:  To overproduce manganese peroxidase (MnP) using Phanerochaete chrysosporium. Effects of carbon sources, agricultural by-products and small molecules were tested to enhance the MnP productivity.

Methods and Results:  Various carbon sources and agricultural by-products were compared as the basal medium. A MnP activity of 4·7 ± 0·31 U ml−1 was obtained using mannose as a carbon source. The enzyme productivity further reached 7·36 ± 0·05 and 8·77 ± 0·23 U ml−1 when the mannose medium was supplemented with cyclic adenosine monophosphate (cAMP) and S-adenosylmethionine, respectively.

Conclusions:  This study revealed the highest MnP productivity obtained by optimizing the carbon sources and supplementing small molecules. It can provide insight for: (i) making high quantities of enzymes by optimizing media resources and (ii) engineering the global regulatory genes in P. chrysosporium for the onsite production of MnP.

Significance and Impact of the Study:  As MnP is an important enzyme to hydrolyse lignin polymers which protects the plant cell wall from exposing of cellulose fibres, the production of the enzyme is a current demand for biofuel biotechnology. The knowledge generated from this study can help to advance the technology for stable production of MnP.

Manganese peroxidase (MnP) (E.C.1.11.1.13) is known to catalyse the oxidation of Mn2+ to Mn3+, which in turn oxidizes a variety of phenolic substrates degrading and/or modifying the lignin polymers in the lignocelluloses (Hatakka 1994). The degradation and/or modification of lignin in the biomass may increase the accessibility to cellulases to hydrolyse and release mono-sugars. Thus, the enhanced MnP production is important for helping to create environmentally sound biomass degradation alternatives and reduce the cost of energy and chemicals in the biofuel industries. MnP also has potential application in other industrial processes such as biopulping, biobleaching and bioremediation (Young and Akhtar 1998). It has been realized that the production of MnP is unstable. To date, organism that produces sufficient enzyme for the industrial applications has not been reported.

Manganese peroxidase expression has been shown to be dependent on the depletion of nutrient nitrogen (Pribnow et al. 1989) and Mn2+ concentration (Gettemy et al. 1998). Attempts to overexpress mnp genes in homologous hosts were not very successful in terms of enhancing MnP productivity (Mayfield et al. 1994; Sollewijn Gelpke et al. 1999). Recently, addition of haemoglobin to the fermentation medium for mnp2-overexpressing Pichia pastoris was also studied to increase MnP activity, speculating that the haemoglobin could help to supply porphyrin biosynthesis in this peroxidase (Jiang et al. 2008). In regard to discovering the regulatory pathways for MnP production, studies have reported the expression behaviour of mnp genes in synthetic media containing glucose as a major carbon source (Singh and Chen 2008). However, it may need further investigation to design and implement a simple medium for MnP secretion.

An early study in Phanerochaete chrysosporium demonstrated that cyclic adenosine monophosphate (cAMP) plays a key role in the regulation of production of lignin peroxidase (LiP) and MnP. Furthermore, LiP and MnP gene expression appeared to be differentially regulated on the basis of intracellular level of cAMP (Boominathan and Reddy 1992), which clearly indicated that these classes of enzymes can possibly be regulated by a signal transduction cascade and triggered by cAMP-like small group of molecules. However, the role of cAMP to maximize MnP productivity has not been addressed yet. Like cAMP, there could be possibilities of other small molecules that may trigger the MnP productivity. It was established that molecules like S-adenosylmethionine (SAM), S-adenosyl homocysteine, ppGpp, γ-butyrolactone and cytokinin act as inducers for the secondary metabolic pathways in Streptomyces (Kim et al. 2003). SAM-dependent 4-O-methyltransferase from P. chrysosporium has been isolated and demonstrated that whole mycelia of the fungus readily took up and utilized SAM in the methylation of acetovanillone (Coulter et al. 1993).

This study focused on to overproduce MnP from P. chrysosporium. To optimize the media conditions, influence of simple carbon sources, agricultural by-products and small molecules were tested. Figure 1(a) illustrates MnP activities profiling in media (Data S1) containing various nutrient sources (glucose, malt extract, xylose, mannose, sorbitol, soy flour, cotton seed flour, corn steep solids, haemoglobin, hemin and succinic acid). Among the carbon sources used in this study, glucose, malt extract, xylose and sorbitol gave the respective MnP activities of 2·3 ± 0·17, 2·5 ± 0·2, 2·67 ± 0·14 and 0·9 ± 0·4 U ml−1. While replacing the carbon source with mannose, the activity was considerably increased up to 4·7 ± 0·31 U ml−1 (Fig. 1a and Table S1), which is among the highest levels reported in the literature. We identified that 2% mannose supplement to the medium demonstrated the best MnP productivity. Compared to our results, Karimi et al. 2006 reported 0·17 U ml−1 MnP activity using glucose (1%) as a carbon source. Likewise, the steam-exploded straw (0·5%) helped to demonstrate 1·375 U ml−1 MnP activity (Fujian et al. 2001). Kapich et al. (2004) have reported 1·8 U ml−1 of MnP activity from P. chrysosporium ME-446 as the highest MnP activity by using wheat straw. From the heterologous expression of MnP in P. pastoris, the highest MnP activity was 2·4 U ml−1 (Jiang et al. 2008).

image

Figure 1.  Manganese peroxidase (MnP) activity in various media conditions. (a) Effect of various nutrient sources on MnP activity: in media containing mannose (•), xylose (○), malt extract (bsl00072), glucose (bsl00084), haemoglobin (bsl00066), hemin (▽) and succinic acid (inline image); (b) MnP activity profiling in cAMP fed Phanerochaete chrysosporium pellets. Various concentration combinations of cAMP/3-isobutyl-1-methylxanthine (IBMX) used in the mannose medium. In figure, control (•), 1 mmol l−1/0.1 mmol l−1 (◊), 1 mmol l−1/0.01 mmol l−1 (bsl00072), 0.1 mmol l−1/0.1 mmol l−1 (bsl00084), 0.1 mmol l−1/0.01 mmol l−1 (inline image), 0.05 mmol l−1/.01 mmol l−1 (□), 0.05 mmol l−1/0.001 mmol l−1 (♦). The feeding of cAMP/IMBX to the culture was performed at 3 days. 0.05 mmol l−1/0.1 mmol l−1 and 0.1 mmol l−1/0.1 mmol l−1 of cAMP/IBMX showed the highest MnP activity and (c) MnP activities shown by different S-adenosylmethionine (SAM)-amended cultures. 0.01 mmol l−1 concentration of SAM found to activate the MnP productivity.

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In addition to the use of direct carbon sources, the agricultural by-products such as soy flour, cotton seed flour and corn steep solids were also tested to supplement the medium. However, none of those sources displayed the comparable MnP activity (Table S1). In addition, the use of porphyrin biosynthetic components to the mannose medium, hemin and haemoglobin, attained the level of MnP productivity closely similar to the MnP activity given by mannose medium; 4·5 ± 0·16 and 4·8 ± 0·17 U ml−1, respectively. However, the MnP activity appeared earlier (fourth day), and the activity was decreased significantly after the sixth day of fermentation. Targeting heme biosynthetic component of MnP, supply of hemin and haemoglobin did not improve the MnP productivity significantly; the activities were 4·5 ± 0·16 and 4·8 ± 0·17 U ml−1,, respectively. Supplementing heme biosynthetic components could be necessary to biosynthesize core porphyrin in the MnP which could possibly enhance the accumulation of the enzyme in the medium earlier, whereas mannose alone might need longer time to support the MnP biosynthesis. Likewise, the quick decline in the enzyme activities could be related to the degradation of the enzymes by proteases simultaneously secreted in the medium. Compared to the MnP yield in our study, Jiang et al. (2008) with the addition of 0·5 g l−1 haemoglobin to mnp2-overexpressing P. pastoris strain reached 2·4 U ml−1 of MnP activity. We also supplemented succinic acid/glycine to the culture, speculating that it may contribute to the formation of 5-Aminolevulinic acid (an intermediate in the biosynthesis of tetrapyrrole found in porphyrins, heme) (Sasaki et al. 2002). The succinic acid/glycine-amended culture also showed similar MnP activities as the haemoglobin- and hemin-supplemented culture did (Fig. 1a), and the highest MnP activity in this condition was appeared during the fourth day (4·1 ± 0·16 U ml−1). According to the results from the supplementation of porphyrin biosynthetic precursors, MnP productivity was observed not very significant compared with the mannose medium alone, suggesting that the MnP productivity may not be triggered by the porphyrin biosynthesis in P. chrysosporium.

In the small molecule feeding experiments, we used various concentrations of cAMP/3-isobutyl-1-methylxanthine (IBMX) (1 mmol l−1/0·1 mmol l−1, 1 mmol l−1/0·01 mmol l−1, 0·1 mmol l−1/0·1 mmol l−1, 0·1 mmol l−1/0·01 mmol l−1, 0·05 mmol l−1/0·1 mmol l−1 and 0·05 mmol l−1/0·01 mmol l−1) (also see Table S2 for more information) and SAM in the medium. Figure 1(b) shows the MnP activity profiling of cAMP-amended P. chrysosporium culture. Upon the analysis of various concentrations of cAMP versus IBMX concentration, 0·05 mmol l−1/0·1 mmol l−1 and 0·1 mmol l−1/0·1 mmol−1 of cAMP/IBMX demonstrated surprisingly higher MnP activity of 7·36 ± 0·05 and 7·12 ± 0·25 U ml−1, respectively. The experiment revealed that the IBMX concentration is important to attain the elevated MnP activity by P. chrysosporium cells. Our findings with cAMP feeding to the P. chrysosporium culture suggested that the MnP can be a part of cAMP signalling cascade. In fact, signal transduction pathways have been evolved with many secondary metabolic biosynthetic pathways that are triggered by small molecules distributed in its natural environmental surrounding. In eukaryotic cells, the secondary messenger cAMP is produced in response to extracellular stimuli such as hormones and regulates a variety of physiological processes (Kolb et al. 1993).

S-adenosylmethionine triggered surprisingly the highest MnP activity given by P. chrysosporium in mannose medium as a background. Figure 1(c) shows the MnP activities supplemented with SAM. It demonstrated the highest enzyme activity in mannose medium; 8·77 ± 0·23 U ml−1 in 6 days. In fact, in all organisms investigated, SAM serves as the major methyl group donor for numerous highly specific methyltransferase reactions. These reactions involve a large variety of acceptor molecules, such as phenylpropanoid derivatives, cyclic fatty acids, proteins, polysaccharides and nucleic acids. In addition, SAM has regulatory functions, e.g., in the allosteric stimulation of threonine synthase. In plants, SAM has been studied primarily in relation to the biosynthesis of various phenylpropanoid derivatives and as an intermediate in the biosynthesis of the phytohormone ethylene (Kawalleck et al. 1992).

Dry cell weight (dcw) in the highest MnP yield phase in all the cultures was also examined. Malt extract- and glucose-supplemented media showed higher biomass accumulation which have comparatively lower MnP yield. It suggests that faster primary growth may adversely affect the MnP yield in P. chrysosporium (Fig. S1).

This research has demonstrated an effective way to optimize MnP productivity by P. chrysosporium. The outcome of this research will direct us to investigate on producing other potential lignocellulolytic enzymes such as cellulases, hemicellulases and LiPs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References
  5. Supporting Information

The authors would like to thank Dr Chenlin Li for her support obtaining the preliminary data for this work and also thank Dr Ben Lucker for discussion while designing this experiment.

References

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References
  5. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References
  5. Supporting Information

Data S1 Supplementary Materials.

Figure S1 Dry cell wt was measured from the cells in the highest enzyme production phase.

Table S1 MnP activities in various nutrient sources.

Table S2 Ratios of cAMP and IBMX used in this experiment.

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LAM_3070_sm_FigS1-TableS1-S2.doc81KSupporting info item

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