Basidiomycetes laccase and manganese peroxidase activity in submerged fermentation of food industry wastes

Enzyme and Microbial Technology 41 (2007) 57–61

Basidiomycetes laccase and manganese peroxidase activity in submerged fermentation of food industry wastes Giorgi Songulashvili a,b , Vladimir Elisashvili b,∗ , Solomon P. Wasser a , Eviatar Nevo a , Yitzhak Hadar c a Institute of Evolution, University of Haifa, Mt. Carmel, Haifa 31905, Israel Durmishidze Institute of Biochemistry and Biotechnology, Academy of Sciences of Georgia, 10 km Agmashenebeli Kheivani, 0159 Tbilisi, Georgia c Department of Plant Pathology and Microbiology, Faculty of Agricultural and Environmental Quality Sciences, The Hebrew University, P.O. Box 12, Rehovot 76100, Israel b

Received 21 November 2005; received in revised form 30 November 2006; accepted 30 November 2006

Abstract The evaluation of eighteen strains of basidiomycetes laccase and manganese peroxidase (MnP) activity in submerged fermentation of mandarin peelings and ethanol production waste showed that the expression of enzyme activity is species- and strain-dependent. While all species of the genus Trametes expressed comparatively high laccase activity, the activity of this enzyme among species of the genus Ganoderma varied from 192 to 61,488 U l−1 . Phellinus robustus 250 appeared to be a promising producer of MnP, accumulating more than 4000 U l−1 of enzyme activity. It has been shown for the first time that Omphalotus olearius 174 is capable of producing high levels of laccase and MnP, while Hypsizygus marmoreus produces only laccase. Laccase and MnP production proved to be very much dependent on the lignocellulosic growth substrate. Of eight complex substrates examined in submerged fermentation by Ganoderma lucidum 447, wheat bran and soy bran gave the highest laccase activity with a maximum value of 93–97 U ml−1 . Proof that both the titre and time of maximal enzyme activity are influenced by nutrient nitrogen is presented. © 2006 Elsevier Inc. All rights reserved. Keywords: Basidiomycetes; Laccase; Lignocellulose; Manganese peroxidase; Nitrogen sources; Screening; Submerged fermentation

1. Introduction Basidiomycetes comprise very different ecological groups of white rot, brown rot, and leaf litter fungi that may insure their nutrition in different ways. Some of them are edible and/or medicinal fungi; some have important biotechnological and environmental applications. White-rot basidiomycetes are the only group of organisms capable of degrading all basic wood polymers due to their capability to synthesize relevant hydrolytic (cellulases and hemicellulases) and unique oxidative (ligninolytic) extracellular enzymes, which are responsible for the degradation of substrate major components, i.e., cellulose, hemicellulose, and lignin into low-molecular-weight compounds that can be assimilated for fungi nutrition [1,2]. The ligninolytic enzyme complexes of white-rot fungi significantly differ in

∗

Corresponding author. Tel.: +99532 528129; fax: +99532 528129. E-mail address: [email protected] (V. Elisashvili).

0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.11.024

their composition. These fungi secrete one or more of three extracellular enzymes that are essential for lignin degradation: lignin peroxidase (EC 1.11.1.14), Mn-dependent peroxidase (EC 1.11.1.13), and a copper-containing phenoloxidase, laccase (EC 1.10.3.2). The potential application of ligninolytic enzymes in biotechnology has stimulated the investigation of their production with the purpose of selecting promising enzyme producers and increasing of their yield [3–7]. In addition, the understanding of physiological mechanisms regulating enzyme synthesis in lignocellulose bioconversion could be useful for improving the technological process of edible and medicinal mushroom production. The physiology of ligninolytic enzymes has been extensively studied using submerged and solid-state fermentation of lignocellulosic substrates [3,5,8–10]. Many previous studies have proved that both the nature and concentration of nitrogen sources are powerful nutrition factors regulating ligninolytic enzyme production by wood-rotting basidiomycetes [4,11,12]. It is worth noting that the effect of these compounds depends not only on fungi physiology, but also on the composi-

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tion of cultivation medium. For example, it is well known that the model ligninolytic organism, Phanerochaete chrysosporium, cultivated in synthetic medium, produces lignin peroxidase and MnP only under nitrogen-limited conditions [13]. However, it has been demonstrated that in the presence of lignocellulosic substrate, a high concentration of organic nitrogen stimulates these enzymes production [9]. Since biotechnological applications require large amounts of low-cost enzymes, one of the appropriate approaches for this purpose is the utilization of the potential of lignocellulosic wastes/by-products, some of which may contain significant concentrations of soluble carbohydrates and inducers of enzyme synthesis ensuring efficient production of ligninolytic enzymes [3,9,14,15]. In addition, the data received proved that the type and composition of lignocellulosic substrate appear to determine the type and amount of enzyme produced by the wood-rotting basidiomycetes [12,16]. This paper describes the ligninolytic enzyme production by known and newly isolated wood-rotting and leaf-litter fungi under submerged fermentation of different wastes from the food industry and also reports on the effects of nitrogen sources on enzyme production. 2. Materials and methods 2.1. Organisms and inoculum preparation Cerrena maxima (Fr.) Ryvarden 681, Fomes fomentarius (L.: Fr.) Fr. strains 649 and 938, Funalia trogii (Berk. upud. Trog.) Bond. et Singer. 146, Ganoderma adspersum (S. Schulz.) Donk. 845, G. applanatum (Pers.: Wallr.) Pat 604, Ganoderma lucidum (W.Curt.: Fr.) P. Karst. 447, Hypsizygus marmoreus (Peck) H. E. Bigelow strains 129 and 571, Omphalotus olearius (DC.: Fr.) Singer´a. 174, Phellinus robustus (P. Karst.) Bourd. et Galz. 250, Phlebia radiata Fr. 511, Pycnoporus cinnabarinus (Jacq.:Fr) Fr. 310, Trametes hirsuta (Wulf: Fr.) Pil´at 71, T. versicolor (L.: Fr.) Pil´at strains 235, 428, 775, T. zonata (Nees.: Fr.) Pil´at 540 are maintained on malt extract agar slants at 4 ◦ C in the Culture Collection of the Institute of Evolution (HAI), University of Haifa, Israel. The majority of these strains were recently isolated from different ecological niches of Israel and Georgia. The inoculum was prepared by growing mushrooms on a rotary shaker at 140 rpm and 25 ± 2 ◦ C in 250-ml flasks containing 100 ml of following defined medium (DM) (g l−1 ): glucose 10; NH4 NO3 1; KH2 PO4 0.8; Na2 HPO4 0.2; MgSO4 ·7H2 O 0.5; yeast extract 2. The medium was adjusted to pH of 6.0 with 2 M NaOH prior to sterilization. After 5–7 days of fungi cultivation mycelial pellets were harvested and homogenized with a Waring laboratory blender, three times 20 s with 1 min interval.

2.2. Culture conditions Different wastes/by-products from the food industry were used to stimulate ligninolytic enzyme production by the selected fungi: corn bran, soy bran, wheat bran, banana, and mandarin (MP) peelings, residue of ethanol production from wheat grains (REP), kiwi fruits, and chicken feathers. All residues were dried at 60 ◦ C and milled to powder. Submerged fermentation of these growth substrates has been carried out on a rotary shaker at 140 rpm and 25 ± 2 ◦ C in 250-ml flasks containing 100 ml of the abovementioned medium with residues’ concentration at 40 g l−1 instead of glucose. To study the effect of nitrogen sources on G. lucidum 447 growth and enzyme production, the same medium containing 50 g l−1 of wheat bran as growth substrate was used, but all nitrogen containing inorganic and organic compounds were added to the medium in final concentrations equal to 10 mM of nitrogen. Ammonium tartrate, KNO3 , (NH4 )2 SO4 , NH4 NO3 , peptone from soy and bacteriological peptone were used as nitrogen sources. A control without a nitrogen source was run in parallel. The initial pH of the medium was adjusted to 6.0 prior to sterilization by adding 2 M NaOH.

Mycelial homogenates (7–10 ml) were used to inoculate the flasks containing media. After 3, 5, 7, 9, 11, and 14 days of mushroom cultivation the samples (1 ml) were taken from flasks and the solids were separated by centrifugation (14,000 rpm; 5 min) at 4 ◦ C.

2.3. Biomass estimation After 14 days of fungi cultivation the mycelia with fermented substrates were separated by centrifugation (6000 rpm; 15 min) at 4 ◦ C. The biomasses were dried at 60 ◦ C to constant weight, and the total nitrogen was determined according to Kjeldahl method with Nessler reactive after pre-boiling of samples in 0.5% solutions of trichloroacetic acid for 15 min to remove the non-protein content. True protein was calculated as the total nitrogen multiplied by 4.38.

2.4. Enzyme assays The supernatants received after the biomasses’ separation were analysed for pH and enzyme activity. Laccase activity was determined by monitoring the A420 change related to the rate of oxidation of 1 mM 2,2 -azino-bis-[3ethyltiazoline-6-sulfonate] (ABTS) in 25 mM Na-acetate buffer (pH 3.8). Assays were performed in 1-ml cuvette at 20 ± 1 ◦ C with adequately diluted culture liquid. One unit of laccase activity was defined as the amount of enzyme, which leads to the oxidation of 1 ␮mol of ABTS per minute. Manganese peroxidase (MnP) activity was measured by oxidation of Phenol Red [17]. The 1-ml reaction mixtures contained 0.89 ml lactate–succinate buffer (50 mM, pH 4.5) with manganese sulphate (0.1 mM), phenol red (0.1 mM), egg albumin (0.1%) and 0.1 ml of appropriately diluted enzyme preparation. The reaction was initiated with 0.01 ml H2 O2 (0.1 mM), mixtures were incubated for 1–5 min at 20 ± 1 ◦ C, then terminated with 50 ␮l 4M NaOH, and absorbance was read at 610 nm. One unit of enzyme activity was expressed as the amount of enzyme required to oxidize 1 ␮mol of Phenol Red in 1 min. Activities in the absence of H2 O2 were subtracted from the values obtained in the presence of hydrogen peroxide to establish true peroxidase activity. The experiments were performed at least two times using three replicates. The data presented in the tables correspond to mean values with a standard deviation less than 12%.

3. Results and discussion 3.1. Basidiomycetes enzyme activity in fermentation of MP and REP Eighteen basidiomycetes strains were tested for ligninolytic enzyme production in submerged fermentation of MP and REP. Both plant residues ensured excellent growth of all fungi in the form of pellets. Their laccase and MnP activities varied in wide ranges depending on species, strain, and growth substrate (Table 1). 3.1.1. Laccase production Ganoderma spp. strains have been extensively studied as medicinal mushrooms and little is known on the ligninolytic systems of these fungi [6,18]. In this study, when MP were used as the growth substrate, G. adspersum 845 and G. lucidum 447 appeared to be the most active producers of laccase accumulating in culture liquid 27,000 U l−1 of this enzyme activity on day 14. In contrast to previously mentioned mushrooms, another representative of the genus Ganoderma, G. applanatum 604, appeared to be a very poor producer of laccase. No activity of this enzyme was detected during 10 days of MP fermentation by the fungus and only 200 U l−1 of laccase activity was revealed on day 14. It is interesting that in the study made by D Souza et

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Table 1 Basidiomycetes laccase and manganese peroxidase activity in submerged fermentation of MP and REP Fungi

Maximal activity of laccase (U l−1 ) MP

C. maxima 681 F. fomentarius 649 F. fomentarius 938 F. trogii 146 G. adspersum 845 G. applanatum 604 G. lucidum 447 H. marmoreus 129 H. marmoreus 571 O. olearius 174 P. cinnabarinus 310 P. radiata 511 P. robustus 250 T. hirsutus 71 T. versicolor 235 T. versicolor 428 T. versicolor 775 T. zonata 540

13500 8970 2280 7820 27380 190 27040 1140 760 8000 5950 5980 4130 18280 17470 20360 17140 9470

Maximal activity of MnP (U l−1 )

REP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1190(5) 720(8) 241(10) 590(10) 2974(14) 20(14) 2887(14) 108(14) 72(10) 903(8) 518(12) 614(7) 370(10) 1475(14) 1893(7) 1705(10) 1483(7) 979(7)

13440 960 5970 6390 4620 580 61490 680 490 1330 1860 340 15930 2040 9540 490 19020 440

MP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1280(3) 102(5) 624(5) 560(12) 447(10) 51(7) 5751(10) 55(9) 48(10) 137(7) 160(10) 31(14) 1486(14) 193(14) 891(5) 44(5) 1827(7) 47(7)

70 ± 8(5)

30 ± 4(10) 0 70 ± 6(10) 50 ± 6(14) 0 40 ± 6(7) 0 0 550 ± 51(10) 0 290 ± 23(10) 2160 ± 175(10) 0 710 ± 64(10) 60 ± 4(7) 160 ± 13(7) 360 ± 42(10)

REP 460 ± 43(5) 0 0 20 ± 2(5) 10 ± 1(5) 0 80 ± 9(7) 0 0 70 ± 8(10) 0 370 ± 35(10) 4350 ± 397(10) 0 1200 ± 117(5) 0 910 ± 80(7) 30 ± 3(10)

The numbers in parentheses indicate the day of peak activity.

al. [18], four Ganoderma spp. strains possessed highly distinguished ligninolytic systems, accumulating 0.6–49.5 U l−1 of laccase activity; among them two strains exhibited MnP and lignin peroxidase activities. In this study, all strains and species belonging to the genus Trametes were distinguished by their comparatively high production rate of extracellular laccase producing 9000–20000 U l−1 of enzyme activity after 7–14 days of MP submerged fermentation. This observation confirms other data showing that the Trametes species produce significant levels of laccase [4,5,10]. The substitution of MP by REP as a growth substrate caused a significant decrease of laccase activity produced by 11 basidiomycetes strains. C. maxima 681, F. trogii 146, and T. versicolor 775 accumulated almost the same levels of the enzyme in both media, while the enzyme yield in REP fermentation by F. fomentarius 938, G. lucidum 447, and P. robustus 250 increased by 2.6–3.9 times. Among basidiomycetes tested, G. lucidum 447 was remarkable for laccase activity, accumulating 61,000 U l−1 of enzyme activity within 10 days of REP fermentation.

of producing laccase in submerged fermentation of MP and REP, while O. olearius 174 produced comparatively high levels of both laccase and MnP in fermentation of MP. The tested fungi were distinguished by the kinetics of enzyme accumulation. During fermentation of MP by the best laccase producer, G. adspersum 845, enzyme activity appeared on day 8 and gradually increased till day 14. In culture of other good enzyme producer, T. versicolor 428, two peaks of laccase activity were revealed. On the contrary, C. maxima 681 began enzyme production much earlier than other fungi and very high laccase activity (11,000–13,000 U l−1 ) was detected in the culture on day 3. In fermentation of MP the level of enzyme was almost constant till day 7 and then it sharply decreased, while in fermentation of REP laccase activity gradually disappeared after three days of fungus cultivation. Finally, in the culture of Trametes versicolor 775 laccase activity gradually increased till days 7–9 and afterwards gradually decreased. In general, MnP production started later, being detected in culture P. robustus 250 on day 5 and reached a maximal value on day 10.

3.1.2. MnP production While the majority of tested fungi accumulated comparatively high levels of laccase in fermentation of plant raw materials, the production of MnP was quite low in most of the cultures tested. Among the eighteen mushroom strains, only seven produced noticeable levels of this enzyme. Especially high MnP activity was detected in submerged fermentation of MP (2160 U l−1 ) and REP (4350 U l−1 ) by P. robustus 250. No MnP activity was detected in cultures of F. fomentarius 938, G. applanatum 604, H. marmoreus 129 and 571, P. cinnabarinus 310 and T. hirsutus 71. To our knowledge, there is no information on ligninolytic enzyme production by the medicinal mushroom H. marmoreus, and only one publication [6] exists on O. olearius. In this study, both strains of H. marmoreus 129 and 571 appeared to be capable

3.2. G. lucidum 447 enzyme activity in submerged fermentation of food industry wastes Many studies have shown that the ligninolytic enzyme production depends on the lignocellulosic substrate used in preculture and in submerged fermentation [5,9,10,19]. G. lucidum 447 was selected for the subsequent study. All media containing various residues from the food industry supported abundant growth of fungus; biomass concentration at the end of the fermentation process varied between 83 and 113 mg protein/flask. However, fungus enzyme activity was affected distinctly by growth substrate in the medium (Table 2). According to data received, defined medium, with glucose as the carbon source, supported very low laccase production whereas the substitution of glucose with complex substrates highly stimulated

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Table 2 G. lucidum 447 protein accumulation and enzyme activity in submerged fermentation of food industry wastes Substrate added

Protein/flask (mg)

Laccase (U l−1 )

MnP (U l−1 )

Glucose Corn bran Soy bran Chicken feathers Wheat bran Kiwi fruits Banana peels MP REP

91 ± 6 78 ± 8 83 ± 8 86 ± 7 73 ± 8 84 ± 7 66 ± 7 77 ± 6 65 ± 6

295 ± 22(5) 5576 ± 613(14) 93840 ± 9566(11) 7310 ± 641(9) 97340 ± 9460(11) 9520 ± 1036(14) 3145 ± 370(11) 35980 ± 3616(11) 57740 ± 6008(11)

0 0 26 ± 3(9) 0 135 ± 14(9) 0 0 102 ± 11(9) 50 ± 6(9)

The numbers in parentheses indicate the day of peak activity.

enzyme accumulation by G. lucidum 447. The maximum laccase activity was obtained after fermentation of wheat bran and soy bran (93,000–97,000 U l−1 ). The presence of wheat bran in the culture medium stimulated ligninolytic enzyme production by Ganoderma sp. strain GASI3.4 [18]. By contrast, corn bran, chicken feathers, kiwi fruits, and banana peelings were rather poor substrates for the laccase production. In addition, our results confirm the data of other authors [5,9] that showed the substrate-dependent ligninolytic enzyme production by white rot fungi. The time courses of laccase activity in fermentation of different plant raw materials are given in Fig. 1. They show that in all media laccase production started 3 days after inoculation and continued to increase till day 11. Afterwards, enzyme activity gradually diminished till the end of mushroom cultivation. As for MnP production, G. lucidum 447 seems to be a poor producer of this enzyme. Among all growth substrates tested, wheat bran followed by MP supported the highest MnP production. No MnP could be detected within 14 days of fungus cultivation in presence of glucose, corn bran, kiwi fruits, BP, and chicken feathers. It is interesting that in the study of D’Souza et al. [18] laccase was the only ligninolytic enzyme produced by G. lucidum in defined medium, and enzyme levels were substantially higher in defined media than in wood-grown cultures. In addition, MnP activity was observed only in cultures containing poplar as the growth substrate but not in pine cultures. One may suppose that some specific components exist in materials like poplar, wheat bran, and MP, or some specific compounds appear during their fermentation triggering MnP production by G. lucidum.

Fig. 1. Profile of laccase activity of G. lucidum 447 in submerged fermentation of food industry wastes.

3.3. Effect of nitrogen sources on enzyme activity of G. lucidum 447 Many previous studies have proved that both the nature and concentration of nitrogen sources are powerful factors regulating ligninolytic enzyme production by wood-rotting basidiomycetes [3,4,10,20]. To improve enzyme production by G. lucidum 447, several inorganic and organic nitrogen sources were tested in submerged fermentation of wheat bran. All supplemented nitrogen sources enhanced fungal biomass yield by 41–69% comparing to a control medium containing nitrogen only in substrate and yeast extract (Table 3). The evaluation of fungus enzyme activity showed that the control medium ensured high levels of laccase and MnP production. The maximal value of laccase activity was revealed in supplementation of culture medium with KNO3 . The same compound slightly stimulated MnP accumulation. In this case, the laccase and MnP specific activities of G. lucidum 447 increased by 75 and 27%, respectively, by the addition of nitrogen to the control medium. We suppose that this positive effect of KNO3 might be due to the prevention of culture medium acidification. Among organic compounds, only bacteriological peptone appeared to be the appropriate nitrogen source for laccase accumulation. However, in this case, the comparison

Table 3 Effect of nitrogen source on G. lucidum 447 enzyme activity in submerged fermentation of wheat bran Nitrogen source

Protein (mg/flask)

Final pH

Laccase (U l−1 )

Control KNO3 (NH4 )2 SO4 NH4 NO3 Peptone from soy Peptone bacteriological Ammonium tartrate

41 ± 4 57 ± 7 64 ± 7 66 ± 6 62 ± 6 64 ± 7 61 ± 5

5.0 ± 0.1 5.4 ± 0.2 4.7 ± 0.1 5.2 ± 0.2 5.0 ± 0.2 5.0 ± 0.1 4.9 ± 0.1

63240 110840 50320 68000 56780 89020 54400

The numbers in parentheses indicate the day of peak activity.

± ± ± ± ± ± ±

4832(9) 12163(5) 5203(9) 7357(5) 4970(8) 8145(7) 5040(9)

MnP (U l−1 ) 94 119 46 86 66 97 86

± ± ± ± ± ± ±

8(7) 14(5) 5(9) 10(5) 8(7) 10(7) 8(9)

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References

Fig. 2. Profile of laccase activity of G. lucidum 447 grown in media containing wheat bran and different nitrogen sources.

of specific laccase activity (1542 U mg−1 versus 1391 U mg−1 ) evidences that the stimulating effect of additional nitrogen on enzyme accumulation is simply due to the higher biomass yield. Moreover, our calculations showed that the addition of other nitrogen sources repressed production of both enzymes by G. lucidum 447. In contrast, D’Souza et al. [18] working with defined media, with glucose as the carbon source, showed that laccase production by G. lucidum is not inhibited in N-rich medium. The data presented in Fig. 2 show that the nitrogen source in nutritional medium effects on laccase accumulation kinetics. The supplementation of control medium with KNO3 provided very early appearance and rapid accumulation of enzyme with maximum activity on day 5 and subsequent its gradual decrease. When (NH4 )2 SO4 or ammonium tartrate were used as nitrogen source the maximal laccase activity was revealed on day 9, while in presence of NH4 NO3 two peaks of enzyme activity were revealed. In conclusion, this study emphasizes the need to explore more organisms and lignocellulosic substrates with different composition to evaluate the real potential of fungi producing ligninolytic enzymes. The selection of appropriate plant residue adequate for fungus growth and target enzyme synthesis may play an important role in the development of an efficient technology. The results obtained here allow us to conclude that G. lucidum 477 and P. robustus 250 are good candidates for scale up ligninolytic enzyme production. However, further studies are required to elucidate the reason by which some complex substrates stimulate enzyme production.

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