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Comparative production of ligninolytic enzymesby Phanerochaete chrysosporium and Polyporussanguineus
Paramjit Kaur Bajwa and Daljit Singh Arora
Abstract: The aim of the present study was to compare the effect of a wide range of culture conditions on production ofligninolytic enzymes by Polyporus sanguineus and Phanerochaete chrysosporium. Lignin peroxidase production by P. san-guineus was comparable with that of P. chrysosporium, although the culture conditions giving the highest yield variedgreatly between the two fungi. Highest yield of manganese peroxidase by P. sanguineus obtained in 0.5% malt extract me-dium and peptone or malt extract supplemented mineral salts broth could not be surpassed by P. chrysosporium in any ofthe optimization experiments. In addition to lignin peroxidase and manganese peroxidase, P. sanguineus also produced lac-case, which was best expressed in malt extract medium supplemented with sugarcane bagasse.
Key words: ligninolytic enzymes, laccase, LiP, MnP, optimization, Phanerochaete chrysosporium, Polyporus sanguineus
Resume : Le but de la presente etude etait de comparer les effets d’un large spectre de conditions de culture sur la pro-duction d’enzymes ligninolytiques par Polyporus sanguineus et Phanerochaete chrysosporium. La production de lignineperoxydase par P. sanguineus etait comparable a celle de P. chrysosporium, quoique les conditions de culture permettantun rendement optimal variaient grandement entre ces deux especes de champignons. Le meilleur rendement de productionde manganese peroxydase obtenu par P. sanguineus dans un milieu contenant 0.5 % d’extrait de malt et de peptone oud’extrait de malt supplemente par des sels minerauxne pouvait etre surpasse par P. chrysosporium dans aucune des expe-riences d’optimisation. En plus de la lignine peroxydase et de la manganese peroxydase, P. sanguineus produisait aussi dela laccase, qui etait le mieux exprimee dans le milieu contenant de l’extrait de malt supplemente avec de la bagasse decanne a sucre.
Mots-cles : enzymes ligninolytiques, laccase, LiP, MnP, optimisation, Phanerochaete chrysoporium, Polyporus sanguineus
[Traduit par la Redaction]
Introduction
The lignin-degrading ability of white rot basidiomyceteshas sparked great scientific interest in developing eco-friendly technologies in the pulp and paper industry (Akhtaret al. 1998) and animal feed production (Arora and Sharma2009). Besides lignin, white rot fungi are also capable of de-grading textile dyes (Chander and Arora 2007), polycyclicaromatic hydrocarbons (Arun et al. 2008), pesticides(Bending et al. 2002), etc. Enzymes involved in ligninolysisinclude laccase, lignin peroxidase (LiP), manganese peroxi-dase (MnP), and glyoxal oxidase (Cullen and Kersten2004). Until now, Phanerochaete chrysosporium has beenthought to be one of the best ligninolytic enzyme producers.However, alterations of the physicochemical parameters orinclusion of some supplements and (or) inducers in the me-
dia may increase the production of such enzymes by otherfungi to levels much higher than that reported for P. chryso-sporium.
The fungal culture of P. chrysosporium (BKM 1767) wassupplied by Prof. T.W. Jeffries, Institute of Microbial andBiochemical Technology, US Department of Agriculture,Madison, Wisconsin, and Polyporus sanguineus (MTCC137) was obtained from the Microbial Type Culture Collec-tion, Institute of Microbial Technology (IMTECH), Chandi-garh, India.
Production profile of ligninolytic enzymesProduction of ligninolytic enzymes was studied in nitro-
gen-limited mineral salts broth (MSB) as described by Aroraand Gill (2005). Laccase was assayed according to Sandhu
Received 19 May 2009. Revision received 7 July 2009. Accepted 11 September 2009. Published on the NRC Research Press Web site atcjm.nrc.ca on 17 December 2009.
P.K. Bajwa. School of Environmental Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada.D.S. Arora.1 Department of Microbiology, Guru Nanak Dev University, Amritsar 143005, Punjab, India.
1Corresponding author (e-mail: [email protected]).
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and Arora (1985). The enzyme activity was expressed ascolorimetric units per millilitre of the enzyme extract. LiPactivity was assayed using two methods: veratryl alcohol ox-idation and Azure B assays (Arora and Gill 2001). In the en-zyme profile study over a period of 20 days, LiP wasassayed by oxidation of veratryl alcohol to veratraldehyde,and the enzyme activity was expressed as micromoles of ve-ratraldehyde formed per millilitre of the enzyme extract per
minute. In the rest of the experiments, the Azure B assaywas adopted as a standard procedure, and the enzyme activ-ity was expressed as an absorbance decrease of 0.1 unit�mLenzyme extract–1�min–1. MnP activity was assayed accordingto Orth et al. (1991). One unit of activity was defined asequivalent to an absorbance increase of 0.1 optical densityunits per minute per millilitre of the enzyme extract. Whengrown in MSB for a total period of 20 days, P. sanguineusproduced all three enzymes, i.e.,laccase, LiP, and MnP,while P. chrysosporium produced only the peroxidases(Figs. 1, 2, and 3). LiP appeared earlier than MnP in boththe fungi, achieving maximum yields on day 6 and day 10in P. chrysosporium and P. sanguineus, respectively(Fig. 1). A second LiP peak was noted on day 16 to day 18in both fungi. A marked feature was the high peak of spe-cific LiP activity in the two fungi. MnP appeared in the cul-ture broth on day 6 in P. chrysosporium and day 8 in P.sanguineus. Rate of MnP production was relatively slow inP. sanguineus, and the highest level was achieved only onday 18. In P. chrysosporium, however, MnP productionpeaked on day 6 (Fig. 3). Similar to the production of LiP,laccase was produced to detectable levels by P. sanguineuson day 2 itself and peaked on day 14 (Fig. 2). Maximummycelial dry mass was noted on days 6–8 (Fig. 3).
Fig. 1. Effect of incubation period on lignin peroxidase (LiP) pro-duction by (a) Phanerochaete chrysosporium and (b) Polyporussanguineus. &, LiP activity, units�mL–1�min–1; ^, specific LiP ac-tivity, units�mg protein–1.
Fig. 2. Effect of incubation period on laccase production by Poly-porus sanguineus. &, laccase activity, cu�mL–1; ^, specific laccaseactivity units�mg protein–1.
Fig. 3. Effect of incubation period on manganese peroxidase (MnP)production by (a) Phanerochaete chrysosporium and (b) Polyporussanguineus. &, MnP activity, units�mL–1�min–1; ^, specific MnPactivity, units�mg protein–1.
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Effect of various supplements andphysicochemical parameters on enzymeproduction
To study the effect of various nutritional supplements, theexperiments were carried out at 25 8C during an 8 day incu-bation period. Comparative ligninolytic enzyme productionwas studied in three basal media: nitrogen-limited MSB, ni-trogen-rich 0.5% malt extract broth (MEB), and mineral saltsupplemented malt extract broth (MS-MEB). Alterations invarious culture conditions markedly affected the productionof different enzymes. Enzyme production in the three basal
media revealed MSB and MEB to be the best for LiP pro-duction by P. sanguineus and P. chrysosporium, respec-tively. MnP was best produced by P. chrysosporium in MS-MEB medium. But in sharp contrast, the enzyme was not atall produced by P. sanguineus in the same medium. For thelatter fungus, the other nitrogen-rich medium, i.e., MEB,was the best for MnP production. It appears that it is notonly the nitrogen concentration but some other factors aswell that seem to affect MnP production by P. sanguineusbecause in the MS-MEB medium, the enzyme productionwas negligible. Some component of MSB must be interfer-ing with its production. Laccase was best produced by P.sanguineus in one or the other nitrogen-rich media, i.e.,MEB or MS-MEB (Table 1). It can be attributed to the fact
Table 1. Effect of different supplements on ligninolytic enzyme production by Phanerochaete chrysos-porium (PC) and Polyporus sanguineus (PS).
Laccase (cu�mL–1) LiP (U�mL–1�min–1) MnP (U�mL–1�min–1)
Medium PS PC PS PC PSMSB 0.13±0.01 0.23±0.01 0.13±0.01 0.02±0.002 0.01±0.001MEB 0.67±0.1 0.33±0.01 0.03±0.001 0.02±0.001 0.43±0.016MS-MEB 0.50±0.07 0.05±0.003 0.04±0.002 0.03±0.002 0.00MSB + VA 0.05±0.01 0.13±0.03 0.04±0.01 0.03±0.01 0.00MSB + G 0.64±0.1 0.10±0.02 0.10±0.01 0.03±0.01 0.12±0.04MEB + VA 1.74±0.08 0.37±0.04 0.00 0.06±0.02 0.00MEB + G 4.77±0.1 0.22±0.05 0.00 0.04±0.01 0.07±0.02MS-MEB + VA 0.23±0.07 0.12±0.06 0.13±0.02 0.04±0.01 0.14±0.05MS-MEB + G 1.62±0.06 0.00 0.16±0.03 0.03±0.02 0.00MSB + Indulin AT 1.36±0.08 0.00 0.17±0.04 0.03±0.01 0.00MSB + Polyphon H 3.12±0.4 0.00 0.00 0.11±0.04 0.01±0.01MSB + Reax 80 2.80±0.2 0.00 0.00 0.13±0.01 0.00MSB + Orezon 1.04±0.05 0.00 0.00 0.10±0.01 0.00MSB + WS 2.74±0.3 0.00 0.45±0.01 0.00 0.00MSB + RS 3.05±0.4 0.01±0.01 0.00 0.00 0.00MSB + SB 1.72±0.09 0.02±0.01 0.00 0.00 0.00MEB + WS 8.33±0.5 0.02±0.01 0.36±0.01 0.00 0.00MEB + RS 4.50±0.6 0.02±0.01 0.00 0.00 0.00MEB + SB 8.99±0.8 0.01±0.01 0.00 0.00 0.00
Note: Values are means ± SD. LiP, lignin peroxidase; MnP, manganese peroxidase; cu, colorimetric units; MSB,mineral salts broth; MEB, malt extract broth; MS-MEB, mineral salt supplemented malt extract broth; VA, veratrylalcohol; G, guaiacol; WS, wheat straw; RS, rice straw; SB, sugarcane bagasse.
Fig. 4. Effect of carbon sources on lignin peroxidase (LiP) produc-tion by Phanerochaete chrysosporium and Polyporus sanguineus.
Fig. 5. Effect of carbon sources on laccase production by Polyporussanguineus.
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that these media provide a complete pool of amino acids re-quired for enzyme synthesis (Sandhu and Arora 1985).
To study the effect of different carbon sources on enzymeproduction, MSB was supplemented with a 1% simple sug-ars (glucose, maltose, and lactose) or cellulose and xylan. Ofthe various carbon sources tested, glucose supported maxi-mum LiP and MnP production in both fungi. Polyporus san-guineus produced higher LiP levels as compared with thoseproduced by P. chrysosporium in the presence of maltoseand lactose as the carbon sources. Even cellulose supportedLiP production by P. sanguineus, although it totally sup-pressed enzyme production by P. chrysosporium. Moreover,all of the carbon sources (except xylan) supported a higherspecific activity of LiP by P. sanguineus as compared withthat of P. chrysosporium, supporting its superiority forlarge-scale purification of the enzyme. Consequently, xylanturned out to be a better carbon source for laccase produc-tion by P. sanguineus (Figs. 4 and 5). The suitability of dif-ferent carbon sources for the production of differentenzymes clearly revealed the high impact of the carbon tonitrogen (C/N) ratio on enzyme titers (Rothschild et al.1995). Higher laccase production by P. sanguineus on xylanpoints towards the secretion of other enzymes as well, sincethe metabolism of xylan as a carbon source requires initialhydrolysis to simple sugars in the extracellular medium, as
the large size of polysaccharides prevents their passagethrough fungal cellular membranes (Zacchi et al. 2000).
To study the effect of nitrogen sources on enzyme pro-duction, malt extract, yeast extract, and peptone were sup-plemented separately in MSB at a concentration of 0.5%.The effect of inorganic nitrogen sources was studied by re-placing 0.02% ammonium tartrate in MSB with an equalamount of either sodium nitrate or sodium nitrite. Amongthe different nitrogen sources tested, peptone supportedmaximum laccase production (1.39 units�mL–1) by P. san-guineus (Fig. 6). LiP was best produced by P. sanguineusin an ammonium tartrate based medium, while P. chrysospo-rium gave the highest LiP titers in MEB (Fig. 7). Sodiumnitrite suppressed LiP production in both fungi. Peptoneturned out to be the best nitrogen source for MnP productionby P. chrysosporium and for P. sanguineus MEB wasequally effective (Fig. 8). The stimulatory effect of malt ex-tract, yeast extract, and peptone on laccase production hasbeen reported for Trametes trogii and Lentinula edodes(Garzillo et al. 1998; Savoie et al. 1998), and the effect ofthese nitrogen sources on MnP production has been reportedfor Bjerkandera sp. strain BOS55 (Mester et al. 1996).
Different lignin preparations, e.g., Indulin AT, Polyfon H,Reax 80, and Orzan S, were added to MSB at a concentra-tion of 0.1%. Supplementation of lignin preparations toMSB medium stimulated laccase production (8- to 24-fold)by P. sanguineus, and polyfon H turned out to be the bestsource of carbon. Stimulation of laccase production by lig-nin preparations is in line with studies on other white rotfungi (Arora and Gill 2000, 2005; Savoie et al. 1998). Incontrast with that of laccase, LiP and MnP production wasselectively enhanced or suppressed in the two fungi in thepresence of lignin preparations. While Indulin AT couldslightly raise the LiP titers in P. sanguineus, all other ligninsseverely repressed its production. Moreover, in P. chryso-sporium, all lignins completely suppressed LiP production.MnP production was enhanced (1.3- to 6.5-fold) by ligninpreparations in P. chrysosporium, with Reax 80 being thebest. But in P. sanguineus, lignins suppressed MnP produc-tion partially or completely (Table 1). This variable effectcan be attributed to the extent of sulphonation of differentlignins. The observed suppression of LiP and MnP by ligninsupplementation can be explained by the fact that lignins areknown to stimulate the production of extracellular H2O2, theexcess of which can inactivate several enzymes (Faison andKirk 1985). Suppression of LiP production by P. chrysospo-rium in the presence of lignins contradicts earlier reportswhere the addition of natural or synthetic lignins was re-ported to increase LiP production by the same fungus(Keyser et al. 1978). In this study, this might have been dueto a higher concentration of lignins used in comparison withprevious studies.
Enzyme production was also studied by supplementingMSB and MEB with different agricultural residues at a finalconcentration of 1% (w/v). Supplementation of MSB andMEB with agricultural residues greatly enhanced (6- to 23-fold) laccase production by P. sanguineus, and the stimula-tory effect varied with the medium as well as the residue.The highest stimulation (23-fold) occurred in MSB mediumsupplemented with rice straw, although maximum enzymeyields (8.99 units�mL–1) were detected in MEB medium sup-
Fig. 6. Effect of nitrogen sources on laccase production by Poly-porus sanguineus.
Fig. 7. Effect of nitrogen sources on lignin peroxidase (LiP) pro-duction by Phanerochaete chrysosporium and Polyporus sangui-neus.
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plemented with sugarcane bagasse. On the other hand, pro-duction of LiP and MnP was suppressed by these residues.However, in P. sanguineus, wheat straw increased the LiPtiters 3- and 13-fold, respectively when used to supplementMSB and MEB, respectively. LiP yield (0.450 unit�mL–1)by P. sanguineus in MSB medium supplemented with wheatstraw was not exceeded by P. chrysosporium in any of thewide range of experimental conditions (Table 1). Highlymedia-specific induction and (or) suppression of enzymeproduction by agricultural residues is in line with our earlierstudies (Arora and Gill 2000, 2001). Supplementation withagricultural residues alters the C/N ratio of the media, andsince each enzyme has a specific C/N ratio requirement,this accounts for the variable effect of these residues oneach enzyme.
Finally, this study examined the effect of veratryl alcoholon LiP activity. Veratryl alcohol and guaiacol, after filtersterilization, were added to all three basal media separatelyat a final concentration of 0.4 mmol�L–1. Supplementationof veratryl alcohol and guaiacol to the three basal media af-fected enzyme production in a highly variable media-de-pendent manner. Veratryl alcohol raised laccase productionby P. sanguineus twofold in MEB, while in the other twomedia, it decreased the laccase titers. Guaiacol, on the otherhand, enhanced laccase production by P. sanguineus (three-to sevenfold) in all media. Veratryl alcohol stimulated LiPproduction (threefold) in MS-MEB, while in the other twomedia, it suppressed LiP in both fungi. Guaiacol, on theother hand, suppressed LiP production by P. chrysosporiumin all of the media and LiP production by P. sanguineus inMSB and MEB . The latter fungus could produce MnP inMS-MEB only in the presence of veratryl alcohol. But inMSB and MEB, inclusion of veratryl alcohol drastically de-creased MnP production to negligible levels. Guaiacolstimulated MnP production by P. sanguineus 12-fold inMSB, while in MEB, it repressed the enzyme and could notinduce enzyme production in MS-MEB (Table 1). Most ear-lier studies investigated the effect of veratryl alcohol only innitrogen-limited MSB. But as is evident from the presentstudy, the effect of veratryl alcohol on enzyme productionis highly variable in different media. Moreover, the effectof veratryl alcohol on LiP has received much more attention
as compared with its effect on laccase and MnP (Tonon andOdier 1988). However, the present work clearly revealedthat the enhancement by veratryl alcohol was best expressedfor laccase, and this could be achieved using a lower con-centration of veratryl alcohol (0.4 mmol�L–1) than was usedin earlier studies.
AcknowledgementP. K. Bajwa is thankful to Guru Nanak Dev University,
Amritsar, India, for providing the Fellowship.
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