Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2011
Volume 14
Issue 3
Topic:
Biotechnology
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Moharib S. , Abdel-Rahman T. , Abdel-Maugod Moussa T. , Yehia R. 2011. EFFECT OF MEDIA COMPOSITION ON LACCASE PRODUCTION BY PLEUROTUS OSTREATUS IN BATCH CULTURE, EJPAU 14(3), #03.
Available Online: http://www.ejpau.media.pl/volume14/issue3/art-03.html

EFFECT OF MEDIA COMPOSITION ON LACCASE PRODUCTION BY PLEUROTUS OSTREATUS IN BATCH CULTURE

Sorial Adly Moharib1, Tahany Mohammed Abdel-Rahman2, Tarek Abdel-Maugod Moussa2, Ramy Sayed Yehia2
1 Biochemistry Department, National Research Center, Dokki, Cairo, Egypt
2 Botany Department, Faculty of Science, Cairo University Egypt

 

ABSTRACT

Laccases, enzymes involved in lignin degradation are produced by various organisms, specially fungi. The addition of inducers to the culture medium of microorganisms can enhance laccase production and facilitate its purification and utilization. Eighteen media were prepared using full L18 (21 x 37) factorial design to maximize the production of laccase by Pleurotus ostreatus. When wheat bran was used as solid substrate, maximum laccase activity was recorded in medium no. 7, while wheat bran extract was added highest enzyme activity was in medium no. 15. Generally, solid wheat bran substrate induced high laccase activity while, bran extract induced higher protein content in culture filtrates and dry biomass of P.ostreatus in all media. The biochemical parameters (protein and sugars) and growth parameters (mycelial growth rate and dry biomass) varied significantly with media composition and nature of wheat bran added as substrate (solid or extract). Maximum of extracellular laccase activity was observed in medium no.7 together with high amount of protein, reducing (DRS) and non-reducing sugars (NRS). Promising degradability action of P.ostreatus especially in presence of solid bran substrate was observed in this medium.

Key words: laccase, Pleurotus ostreatus, biochemical parameters, growth, biomass.

1. INTRODUCTION

White-rot fungi are known to secrete lignin modifying enzymes such as laccase and manganese peroxidase in cultivation medium [6, 46]. Laccases (EC 1.10.3.2) are phenol oxidases that contain four copper ions per molecule [67]. Laccase (benzendiol:oxygen oxidoreductase) is a multi-copper containing proteins that are widely distributed in many plant and fungal species [16]. Laccase catalyze the oxidation of phenolic and non-phenolic compound [2,72]. Madhavi et al. [30] reported an increase in laccase production by P. ostreatus after supplementing various phenolic substrates to the growth culture.

Fungal laccase catalyzes the oxidation of monophenols, diphenols and aromatic amines, to free radicals that subsequently undergo further enzymatic oxidation to quinine and their oxidative coupling [13, 21, 59, 67]. Fungal laccases are implicated in several physiological roles including morphogenesis and inter-specific fungal interactions [34, 55, 58]. The induction of laccase by copper (150 µM CuSO4) has already been documented for P. ostreatus [30,45]. Currently, positive regulation of laccase by copper has been reported for Tramates pubescens [15. Other investigators reported that the production of laccase by Trametes versicolor and other white-rot fungi increased or was induced when an aromatic compound or Cu2+ was added to the growth medium [40,67]. Several investigators studied the productivity and recovery of laccase produced by some white-rot fungus [12, 35, 40, 56,57, 67, 71].

The production of laccase are affected by several factors including the composition of growth medium, pH, carbon: nitrogen ratio, temperature  and aeration rate [18,19, 20, 23, 27, 37, 38]. Medeiros et al. [36] optimized laccase production by P. ostreatus DM-1513 using factorial design and they found that the low pH and high yeast extract concentration had positive effects on laccase production without the use of an inducer.

In this study we try to optimize the culture parameters for  maximal induction of laccase production by P. ostreatus NRRL 0366. The biochemical changes in the mycelia and culture filtrate were monitored to deduce the behaviour of fungus in different cultural conditions.

2. MATERIALS AND METHODS

2.1. Organism

The white-rot fungus Pleurotus ostreatus, NRRL0366 (oyster mushroom) was kindly provided by National Regional Research Laboratory, National Center for Agricultural Utilization Research Service, USDA, USA. The fungus was maintained on PDA medium [51].

2.2. Wheat bran extract preparation

Wheat bran was thoroughly washed by distilled water, dried in hot air oven (50°C for 24 h) and subsequently sieved using a metal mesh of size 0.278 inches. Wheat bran extract was prepared by soaking the wheat bran in distilled water for 2 h and then filtered.

2.3. Optimization of culture for laccase production

Eight factors: pH, glucose, wheat bran, urea, inoculum size, yeast extract, inducer and KH2PO4,   were considered in the present experiment at three levels (except pH)  (Table 1).

Table 1. Selected culture condition factors and assigned levels.

Serial no.

Factor

Level 1

Level 2

Level 3

1

pH

5.0

5.5

-

2

Glucose (% w/v)

1.0

1.5

2.0

3

Wheat bran (% w/v)

1.0

2.0

3.0

4

Urea (% w/v)

0.50

0.75

1.0

5

Inoculum  (discs )

5

7

10

6

Yeast extract (% w/v)

1.0

1.50

2.0

7

Inducer (mM)

0.50

1

1.5

8

KH2PO4 (% w/v)

0.15

0.20

0.25

Orthogonal array of the designed experiment was established according to Taguchi DOE method used earlier by Prasad et al. [52], which involves establishment of large number of experiments to reduce experimental errors and to enhance their efficiency and reproducibility in the laboratory (Table 2). The three levels of factors variation and the size of experimentation was represented by symbolic arrays of L18 (21×37),  which indicates 18 experimental media in which seven factors have been assigned with three levels (37), except pH (21).

 Table 2. L18 orthogonal array of the designed experiment.
Media  no. Factors levels
 
1
2
3
4
5
6
7
8

1

1

1

1

1

1

1

1

1

2

1

1

2

2

2

2

2

2

3

1

1

3

3

3

3

3

3

4

1

2

1

1

2

2

3

3

5

1

2

2

2

3

3

1

1

6

1

2

3

3

1

1

2

2

7

1

3

1

2

1

3

2

3

8

1

3

2

3

2

1

3

1

9

1

3

3

1

3

2

1

2

10

2

1

3

3

3

2

2

1

11

2

1

1

1

1

3

3

2

12

2

1

2

2

2

1

1

3

13

2

2

1

2

3

1

3

2

14

2

2

2

3

1

2

1

3

15

2

2

3

1

2

3

2

1

16

2

3

1

3

2

3

1

2

17

2

3

2

1

3

1

2

3

18

2

3

3

2

1

2

3

1

Batch cultures were carried out in 250 ml Erlenmeyer flasks containing 100 ml of production medium composed of different levels of the following compounds (g/ L): glucose (10.0, 15.0 and 20.0 ); yeast extract (5.0, 10.0 and 20.0 ); urea (5.0, 7.5 and 10.0 ), KH2PO4 (1.5, 2.0 and 2.5); wheat bran (10.0, 20.0 and 30.0); 10 mL of stock salt solution (in 100 ml of distilled water: MgSO4×7H2O, 0.5 g ; CaCl2, 0.1 g; KCl, 0.5 g; CuSO4×5H2O, 0.25g) and pH adjusted to 5.0 or 5.5. The syringic acid (laccase inducer) was added at 0.5, 1.0 and 1.5 mM after 96h of cultivation (to reduce its effect during initial phase of fungal growth). Five millimeter diameter fungal discs from 8-days-old P. ostreatus culture were added at different number: 5 , 7 or 10 discs for each flask. The flasks were incubated at 28±2°C for 8 days at 100 rpm on orbital shaking incubator (New Brunswick Scientific Co., Inc. Edison, N.J. USA). Mycelial mats were collected by filtration and centrifugation at 10.000 rpm for 15 min at 4°C. Obtained filtrates were used for enzyme assay and biochemical tests.

2.4. Analysis

2.4.1. Laccase assay

Extracellular laccase activity in the culture filtrate was measured spectrophotometrically as described by Niku-Paavola et al. [43]  using 2,2'-azino-bis-(3-ethylebenzothiazoline-6-sulfonic acid) as substrate (ABTS, Sigma, USA). The reaction was monitored by measuring the change in absorbance at 436 nm for 5 min using Jenway (6300) spectrophotometer. The activity of laccase was calculated according Leonowicz et al. [24, 25] using the following formula:

The specific activity calculated by the following relationship:

Where: ε = 29300 M-1 cm-1 (molar absorption coefficient of oxidized ABTS), ΔE = increase in absorbance at 436 nm, Δt = reaction time in second, p = mg protein.

One unit of enzyme activity was expressed as the amount of enzyme releasing 1µmol of oxidized product per minute.

2.4.2. Carbohydrate, Reducing Sugars and Protein content

Carbohydrate content of mycelial mat was estimated as described by Nemecet al. [42]. Direct Reducing Sugars (DRS) and Total Reducing Sugars (TRS) were measured spectrophotometrically using Nelson's solution as modified by Naguib [41]. Results are expressed as µg reducing sugars in 1g of dry biomass. 

Extraction of protein from mycelial mat was carried out as described by Osherov and May [44]. Protein was determined by the method of Lowry et al. [29].

2.4.4. Growth rate and dry biomass measurement

For growth rate determination P. ostreatus was cultivated on the different agar media according to Prasad et al. [52] using solid wheat bran and its extract as substrate. In the middle of Petri dishes 10 mm discs were placed and the diameter of fungal colony was measured at two day intervals for 12 days incubation at 28°C. Three replicate were performed and the growth rate was calculated.

For dry biomass measure  P. ostreatus was cultivated on the different media (whith solid wheat bran and its extract as substrate)  in a batch culture. So, 10 mm discs were placed in 250-ml Erlenmeyer flasks for 7 days at 28°C. The cultures were filtered using Whatman No.1 filter paper. The mycelial mats were collected and dried in oven at 80°C till constant weight.

3. RESULTS

 Laccase activity

The results in Fig.1 revealed that laccase activity varied according to the medium used. When solid wheat bran was used as substrate in P. ostreatus media, the highest laccase activity was observed in medium no. 7 (7.8 U/mL). The lowest laccase activity (0.2 U/mL) was attained in medium no. 14. When wheat bran was replaced by wheat bran extract, the laccase activity profile declined . However, hight laccase activity, was observed in medium no. 15 (3.5 U/mL), whereas, media no. 1 and no. 6 were coupled with very low laccase activity (0.1 U/mL).
Figure 1. Effect of media composition on laccase activity [A] (U/mL), protein content in mycelial mats [B] (mg/g) and culture filtrates [C] (mg/mL) of Pleurotus ostreatus after 8 days incubation at 28°C.

Protein content

Protein content was measured in both dry biomass and culture filtrate of P. ostreatus. Solid wheat bran supplementation induced high biomass protein content (250 mg/g in medium no.18), whereas with wheat bran extract, 3 times more protein was reached  (777.0 mg/g in medium no.16). In the case of bran extract, the mycelial protein content was higher than that of solid bran in all tested media (Fig.1). The lowest biomass protein content was observed in medium no. 14 (93.3 mg/g) with solid wheat bran and in medium no. 2 (~195mg/g) with bran extract (Fig.1).

High protein content in culture filtrates of P. ostreatus growing on solid wheat bran supplementation was measured in the case of no. 7 (802.5 mg/mL). The lowest protein content in the culture filtrate was obtained in medium no. 9 (239.8 mg/mL). In the case of wheat bran extract, the best medium was no. 16 (1772.8 mg/mL)  and the worst medium no. 12 (815.3 mg/mL).

Carbohydrate content

Level of reducing sugar in filtrate is the consequence of laccase activity, higher amount indicated higher enzyme activity. Results on Fig. 2 indicated that the highest amount of total reducing sugars (TRS) in P. ostreatus biomass (9.5 mg/g)  was in the medium no.7 (supplemented with wheat solid bran), while the lowest biomass TRS content was recorded in media no.1 and no. 8 (2.8 and 2.7 mg/g , respectively). The highest value of direct reducing sugars (DRS) was detected in medium no. 18 (7.3 mg/g), while it was significantly declined in media no.: 13, 14, 11, 2, 9, 17, 10, 4 and 5 ( 1.5-3.3 mg/g). The lowest biomass DRS were detected on media no.8 (0.7 mg/g). The highest non-reducing sugar (NRS) was obtained in medium no.7 (4.0 mg/g), significant decrease was detected in media no. 14, 10, 9, 4 and 13. The lowest amount of NRS (0.6 mg/g-1) was recorded for media no.15 and no.18.

Figure 2. Effect of media composition on the total reducing sugars (TRS), direct reducing sugars (DRS) and non-reducing sugars (NRS) in dry biomass of Pleurotus ostreatus supplemented with wheat solid bran [A] and bran extract [B] after 8 days incubation at 28°C.

Data in Fig. 2 revealed that higher values of carbohydrate contents in biomass of P. ostreatus were measured when wheat bran extract was used as substrate in the media. In dry biomass obtained with bran extract the highest amount of TRS was produced in medium no.9 (28.0mg/g) and the lowest in medium no. 12 (2.7 mg/g). As shown on Fig. 2 the highest DRS was in medium no. 5 (3.4mg/g) and the lowest in medium no. 1 (0.5mg/g). The highest non-reducing sugars (NRS) were when medium no. 9 was used (24.7mg/g). Medium no.7 was coupled with the lowest amount of NRS (0.4mg/g).

When solid bran was added as substrate , the best value of TRS content (354.8 mg/mL) in culture filtrates of P. ostreatus  was in medium no. 8 (Fig. 3). TRS amount decreased in media no.: 11,16,1 and 9 (216.5, 186.2, 137.8 and 100.6 mg/mLrespectively). However, the smallest TRS content in culture filtrate was attained in medium no.10 (69.8 mg/mL). The DRS content in culture filtrate was the highest in medium no.3 (203.4 mg/mL) and the lowest in medium no.10 (41.4 mg/mL). The NRS content in the culture filtrate of P. ostreatus, was the highest in medium no.4 (239mg/mL) and the lowest in medium no. 9 (40.1mg/mL) .

Results on Fig. 3 indicated that in bran extract containing media TRS in the culture filtrate was high in medium no. 9 (427.7 mg/mL) and low in medium no. 18 (167.8 mg/mL). The composition of medium no.7 was the best for DRS content (302.5 mg/mL). The lowest DRS values were recorded for media no. 11 and no. 17 (~101.5 mg/mL ). The highest NRS content was detected in medium no.12 (222.2 mg/mL) while the lowest value in medium no.7 (45.3 mg/mL).

Figure 3. Effect of media composition on the total reducing sugars (TRS), direct reducing sugars (DRS) and non-reducing sugars (NRS) in culture filtrates of Pleurotus ostreatus supplemented with wheat solid bran [A] and bran extract [B] after 8 days incubation at 28°C.

 Mycelial growth rate

The best medium for mycelial growth rate was medium no. 9 (5.0 mm.day-1) in the case of solid wheat bran and no. 11 (5.3 mm/day) when bran extract was used (Table 3). Significant decrease in growth rate of fungus (0.2-0.9 mm/day) was measured  in media no.: 1, 8, 14 (with solid wheat bran)  and in media no:  10, 13, 6 and 16 9 (with extract)   

Table 3. Effect of media composition on the mycelial growth rate (mm/day-1) of P. ostreatus after daily records for 12 days incubation in case of wheat bran and wheat bran extract.

Media no.

Growth rate (mm/day)

Solid bran

Bran extract

1

0.1

2.7

2

1.7

1.9

3

1.7

1.3

4

1.6

2.8

5

2.2

2.8

6

1.2

0.9

7

1.9

2.3

8

0.8

1.1

9

5.0

3.1

10

0.6

0.2

11

2.5

5.3

12

1.4

1.7

13

1.1

0.6

14

0.8

1.6

15

1.9

1.8

16

1.4

0.9

17

4.2

4.4

18

1.2

1.1

Dry biomass yield and pH

The dry biomass of P. ostreatus varied according to the medium composition (Table 4). The maximal dry biomass was gained in medium no. 6 (8.9 g/L) when the fungus grown on solid wheat bran. However, on wheat bran extract dry biomass in medium no. 3 was also high (5.8 g/L).

The final pH increased moderately when solid wheat bran was used as substrate,  to reach its maximum value in medium no.2 (pH 7.3). Considering wheat bran extract as substrate final pH shift significantly either to the alkaline value ( in media no.: 1, 5, 11, and 14) or to the acidic value ( in media no. 3, 6, 7, 8, 9, 17 and 18). Medium no.7 and no.15 , in which higher laccase activity was recorded were characterized by acidic final pH value.

Table 4. Effect of media composition on dry biomass (g/L) and final pH of P.ostreatus after 8 days incubation at 28°C.

Media no.

Wheat bran

Wheat bran extract

Dry biomass
(g/L)

Initial pH

Final pH

Dry biomass
 (g/L)

Initial pH

Final pH

 1

3.8±0.14

5.0
6.2
3.3±0.25
5.0
8.1

 2

7.8±0.11

5.0
7.3
3.2±0.13

5.0

7.6

 3

3.7±0.12

5.0
4.6
5.8±0.09
5.0
4.2

 4

3.7±0.21

5.0

5.5

3.5±0.14
5.0

6.7

 5

6.5±0.14

5.0
6.0
5.3±0.15
5.0
8.2

 6

8.9±1.45

5.0
6.2
1.5±0.30
5.0
4.2

 7

4.5±0.16

5.0
5.8
4.6±0.17
5.0
4.2

 8

7.2±0.24

5.0
7.1
5.1±0.14
5.0
4.0

 9

3.0±0.21

5.0
6.3
4.5±0.21
5.0
4.0

10

2.2±0.28

5.5
6.8
3.0±0.08
5.5
7.3

11

1.3±0.16

5.5
6.1
5.1±0.13
5.5
8.0

12

3.3±0.21

5.5
6.5
2.5±0.16
5.5
7.1

13

7.7±0.28

5.5
6.1
5.2±0.04
5.5
6.9

14

3.6±0.18

5.5
6.8
4.5±0.16
5.5
8.4

15

5.3±0.28

5.5
6.0
4.1±0.13
5.5
5.6

16

2.3±0.17

5.5
6.7
5.6±0.14
5.5
5.7

17

7.2±0.17

5.5
5.1
3.3±0.15
5.5
4.4

18

2.8±0.21

5.5
6.0
3.5±0.14
5.5
4.4

4. DISCUSSION

In this study, the factorial design L18 (21×37) was used to optimize the growth parameters for maximal laccase production. Similarly, several group of researchers attempted to optimize the production of laccase by either conventional or statistical method. Medeiros et al. [36] found that low pH and high yeast extract concentration without the use of an inducer and buffer system had positive effects on laccase production. Vasconceelos et al. [69] utilized factorial design and surface response method to determine optimum concentrations of veratryl alcohol , yeast extract, optimal time of cultivation and agitation speed for the production of laccase by Botryosphaeria spp. MAMB-5. Several investigators found that 30.4 mM veratryl alcohol, 4.5 day at 28°C with an agitation speed of 180 rpm were the optimal conditions to maximize production of one of the laccase [18, 27, 37]. Galhaup et al. [15] studied the effects of culture composition and various conditions on the production of laccase by T. pubescence and found the following optimized medium composition (l-1): 40g glucose, 10g meat peptone, 1.0g MgSO4.7H2O and 2.0 mM Cu (II). Medeiros et al.[36] and Gursharan et al. [18] found pH 5.5 as an optimum pH value to obtain maximal yield of enzyme from batch culture of P. ostreatus 1804. Leonowicz et al. [24],  Galhaup et al.[15] and Prasad et al.[52], reported effective laccase yield in the submerged culture of P. ostreatus 1804 at pH range of 5.0-5.5. There are many surveys for enzymes involved in lignin degradation by fungi, mainly white-rot basidiomycetes [12,18,31,47,64,70]. The ligninolytic enzymes, lignin peroxidase, manganese peroxidase and laccase are widespread throughout the white rot fungi and different levels of these proteins were found in various strains of the same species. Culture conditions and medium composition also play a major role in the level of enzyme biosynthesis [47,56].

In this study, among 18 tested media, laccase induction and activity reached the maximal value when P. ostreatus wascultivated in medium no.7. While when bran extract was added as substrate the optimum was reached in medium no.15. The addition of syringic acid as inducer (10 mM)  stimulated production of laccase (the highest in medium no.7). It is known that the most efficient elicitors of laccases are lignin hydrolysates and aromatic compounds such as acids, alcohol and aldehydes [3]. As reported by several investigators, laccase activity in fungal cultures can be increased by the addition of different aromatic compounds as inducers to the media [5,17,30]. The positive effect of aromatic inducers such as guaiacol, veratryl alcohol, p-hydroxybenzoic acid, and 2,5-xylidine on the laccase production was confirmed in cultures of white-rot fungi:   Phlebia radiata MJL-1198-spp., Dichomitus flavida MTCC145 [1] and T. versicolor [54] as well as non-white-rot fungi such as Agaricus sp. [56] and an ascomycete Botryosphaeria spp. [7]. Inducers effect is mainly depending on the type, concentration and time of its addition to enzyme production  [15, 39].

The present results indicated that the use of wheat bran as solid substrate was better than the usage of wheat bran extract for induction of laccase produced by P. ostreatus. Results also showed that the addition of wheat bran at concentration of 10 g/L induced the highest activity of laccase. The use of natural solid substrate, especially lignocellulosic agricultural residues, as substrates for fungal growth has been enthusiastically studied for laccase production in recent years. Morais et al. [39] and Erika et al. [12] demonstrated that wheat straw was a better substrate than wheat extract for laccase production using P.ostreatus. Similar effect was observed in cultures of Lentinula edodes 610 [20]. Pickard et al. [49] and Vikineswary et al. [71] demonstrated that the cereal bran liquid medium allowed a high production of laccase by Coriolopsis gallica UAMH8260. Lorenzo et al. [28] reported that barley bran was a better substrate for laccase production by T. versicolor CBS100.29 than grape seed and grape stalks. Fenice et al. [14] demonstrated the successful use of olive mill wastewater-based medium, contained large amounts of recalcitrant aromatic compounds for production of laccase and Mn-peroxidase in batch and solid-state cultures of Panus tigrinus. Ullah et al. [68] investigated wood chips, cereal grain, wheat husk, and wheat bran as substrate of Coriolus (Trametes) versicolor FRRL-28A and found that wheat husk and wheat bran allowed the growth and laccase production. Wheat bran was also successfully used as solid substrate medium for the laccase production by Pleurotus pulmonarius CCB-19 [33] and Fomes sclerodermeus BAFS2752 [46].

In the present study, high laccase production was obtained  in medium no.7 with high glucose concentration (20 g/L) and low in media no. 1 and no.6 with smaller glucose amount.  The highest laccase production was observed at median concentration of urea (7.5 g/l) and lower in both low and high concentration of urea (5 and 10 g/L). In P. ostreatus a high concentration of nitrogen in the medium (34 mM, glutamate as N) did not repress but rather slightly stimulated mineralization of lignin compared to the N-limited medium [23]. Laccase activity in Cerrena unicolor IBB62 depend on the nitrogen source in the culture medium, and the highest levels of laccase activity was observed in the medium with (NH4)2SO4 [10]. Kaal et al. [23], Mirjana et al. [38] and Membrillo et al. [37] showed that N-sufficient peptone medium stimulated laccase activity in Lentinus edodes and P. ostreatus. Buswell et al. [4] using L. edodes, and Srinivasan et al. [61] using Phanerochaete chrysosporium, also observed the highest levels of laccase activity when these fungi were grown under high nitrogen conditions. Stajiæ et al. [62] showed the highest laccase activity produced by both P. eryngii and P.ostreatus  with (NH4)2SO4 as a nitrogen source (20 and 30 mM, respectively). Mansur et al. [32] showed that the use of fructose instead of glucose resulted in a 100-fold increase in the specific laccase activity of basidiomycetes. Elisashvili et al. [11] showed that the highest laccase activity in P. ostreatus was revealed in cultivation on the mannitol-containing medium.

In an attempt to find any correlation between media composition, laccase activity and fungal metabolism, the protein and carbohydrate content of biomass and culture filtrate as well as dry biomass gain and mycelial growth rate of P. ostreatus were estimated. It was found that solid wheat bran containing medium no.7 in which maximum laccase activity has been recorded, was also characterized by the highest content of protein and carbohydrate in mycelium and culture filtrate. Median biomass gain, mycelial growth rate and slight shift to acidic pH value were also detected in medium no.7. In medium containing wheat bran extract, the highest laccase activity was recorded in medium no.15 and was coupled with low biomass but high filtrate protein content. The carbohydrate content revealed low reducing sugar and high non-reducing sugar, the dry biomass and mycelial growth rate are significantly high although the pH was acidic.

From the present results, it can be concluded that high mycelial protein and non-reducing sugars is coupled to high laccase biosynthesis by P. ostreatus. Solid wheat bran added as substrate induces higher laccase activity than when its extract was added to the growth medium. The medium optimum for laccase activity is not necessary optimal for fungal growth. In this respect, the biodegrability of four Pleurotus species; namely P. flabellatus, P. florida, P. sajor-caju and P. ostreatus were evaluated for their yield and nutritional state [48,66]. P. ostreatus found to give maximum harvested yield on wheat straw substrate showing 64% biological efficiency and biomass have high protein content and low carbohydrate content  [8, 9, 53, 60]. P. ostreatus biodegraded rice straw and increased protein content from 1.7 to 9.4% [65]. The compositional profile of cotton seed hull was changed greatly by cultivation of P. ostreatus. The increase in protein content and decrease in carbohydrate content contributed to the increase in dry matter digestibility of the substrate [26]. Effect of glucose on metabolism of Phanerochaete chrysosporium and ligninolytic enzyme activities were observed in culture medium supplemented with glucose [60]. Authors  reported that glucose was required for degradation of polymeric molecules. Biomass production by one of inter-sterility groups of the root pathogen, Heterobasidion annosum was significantly lower in strain P than in strain S. By contrast, laccase activity measured in relation to growth rate, was significantly higher for the P strain compared with S strain [22]. Slight repression of laccase biosynthesis by Pycnosporus sanguineas was observed in high nitrogen culture medium [50]. Laccase levels observed in high nitrogen batch cultures were higher than those in low nitrogen. The N-source experiments seemed to reveal that NH4 plays an important role in inducing laccase of white rot fungus Trametes gallica [63]. These results are in agreement with the investigation performed by Collins and Dobson [5]. Since the addition of these compounds greatly enhance the laccase production and seems a promising approach for large scale production of this enzyme for industrial and biotechnological applications.

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Accepted for print: 30.09.2011

Sorial Adly Moharib
Biochemistry Department, National Research Center, Dokki, Cairo, Egypt

email: smoharib@yahoo.com

Tahany Mohammed Abdel-Rahman
Botany Department,
Faculty of Science, Cairo University Egypt


Tarek Abdel-Maugod Moussa
Botany Department,
Faculty of Science, Cairo University Egypt


Ramy Sayed Yehia
Botany Department,
Faculty of Science, Cairo University Egypt

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