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Effect of mannan oligosaccharide elicitor and ferulic acid on enhancement of laccases production in liquid cultures of basidiomycetes ARTICLE in ENZYME AND MICROBIAL TECHNOLOGY · JUNE 2007 Impact Factor: 2.32 · DOI: 10.1016/j.enzmictec.2006.10.002
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Effect of mannan oligosaccharide elicitor and ferulic acid on enhancement of laccases production in liquid cultures of basidiomycetes Sophie Vanhulle a , Romeo Radman b , Roberto Parra b , Tingting Cui b , Christian-Marie Bols c , Thierry Tron d , Giovanni Sannia e , Tajalli Keshavarz b,∗ b
a Catholic University of Louvain, Louvain-la-Neuve, Belgium Department of Applied and Molecular Biosciences, University of Westminster, London, United Kingdom c Wetlands Engineering SPRL, Louvain-la-Neuve, Belgium d Laboratoire de Bioinorganique Structurale Marseille, France e Department of Organic Chemistry and Biochemistry, University of Naples, Naples, Italy
Abstract The effects of mannan oligosaccharides preparation (MO), as elicitor, and ferulic acid inducer for enhancement in laccases production in liquid cultures of three strains of basidiomycetes, Pycnoporus sanguineus, Coriolopsis polyzona and Pleurotus ostreatus was studied using a full factorial 32 experimental design. MO, either individually or combined with ferulic acid, enhanced laccases levels in the three different strains of the white-rot fungi. The enhancement of laccases production was species specific with the highest increase in liquid cultures of P. sanguineus (88-fold) followed by P. ostreatus (3-fold) and C. polyzona (2-fold). Separate additions of 75 mg/l of MO to the cultures of P. sanguineus and P. ostreatus caused the increase while a combined addition of 150 mg/l of MO and 1 mM ferulic acid resulted in the optimal production of laccases in the cultures of C. polyzona. © 2006 Elsevier Inc. All rights reserved. Keywords: Basidiomycetes; Mannan oligosaccharides; Ferulic acid; Laccases; Response surface methodology
1. Introduction White-rot fungi, a heterogeneous group of lignin-degrading basidiomycetes, have received considerable attention for their bioremediation potential [1–3]. They have the capability of degrading lignin due to their extracellular non-specific and non-stereoselective enzyme system composed by laccases (EC 1.10.3.2), lignin peroxidases (LiP, EC 1.11.1.14) and manganese peroxidases (MnP, EC 1.11.1.13), which function together with peroxide-producing oxidases and secondary metabolites [4,5]. The same unique non-specific mechanisms that give these fungi the ability to degrade lignin, also allows them to degrade a wide range of pollutants, among them polycyclic aromatic hydrocarbons, chlorinated phenols, polychlorinated biphenyls, dioxins, pesticides, explosives and dyes [1,3,6]. Because the key enzymes are extracellular, white-rot fungi can degrade many
∗
Corresponding author. Tel.: +44 20 79115000x3562; fax: +44 20 79115087. E-mail address:
[email protected] (T. Keshavarz).
hazardous environmental pollutants, as they do not require preconditioning to a particular pollutant. This makes them useful for biotechnological applications [4,5]. Recently however, there has been growing interest in studying laccases enzymes of a wider array of white-rot fungi, not only from the standpoint of comparative biology but also with the expectation of finding better systems for use in various biotechnological applications [7]. Potential exploitation of enzymes to replace or reduce the use of hazardous chemicals in industrial processes such as pulp and paper, textile and leather industry has attracted attention in recent years due to the public concern and governmental regulations. In the pulp and paper industry, for example, enzymatic applications at different parts of the manufacturing process have proved successful. The use of xylanases has helped in reduction of the need for chlorine and lipases have proved effective in controlling the accumulation of pitch. Enzymes have great potential in degrading contaminants in industrial waste streams and laccases with their extensive properties can play important role in reducing the toxicity of industrial wastewaters. However,
laccases production on an industrial scale presents a problem due to low titres and high costs. Optimisation of media and culture conditions has been one approach for improvement in laccases production. The small amounts of laccases produced constitutively in basidiomycetes, however, can be considerably enhanced by the presence of a wide variety of inducing substances, mainly aromatic or phenolic compounds related to lignin or lignin derivatives such as ferulic acid, 2,5-xylidine, p-anisidine and veratryl alcohol. The successful biotechnological application of laccases requires production of high amounts of the enzyme at low operation cost; so far only obtained in flask cultures [8,9]. The application of statistical methodologies is helpful in finding the effect and interactions between the physiological factors that play a role in biotechnological process such as microbial enzyme production. The use of different statistical designs for medium optimisation has been recently employed for lysozyme, xylanase, amylase and laccases production by fungal cultures [10–16]. Statistical optimisation methods can take into account the interactions of variables in generating a process response [17,18]. Thayer et al. [19] used response surface designs to optimise media and process conditions rapidly. Parra et al. [10] used an orthogonal design to optimise 13 medium components to enhance squalestatin production. Bull et al. [20] used a Plackett–Burman design to screen 20 different culture media components to identify those important for cell growth and protein production. An inexpensive and economically viable approach for enhancement of secondary metabolites production in fungi has been through the use of oligosaccharides as elicitors [21]. Oligosaccharides such as OG (oligoguluronate), OM (oligomannuronate), MO (mannan oligosaccharides) and pectin oligosaccharide, as elicitors, enhance the production of penicillin G in Penicillium chrysogenum P2 cultures [21] and increase the levels of chrysogenin in P. chrysogenum NRRL 1951 [22] and P. chrysogenum MUCL 30168 [23]. Locust bean gumderived mannan oligosaccharides were shown to be the most generic and potent elicitors investigated in filamentous fungi [21]. Although the effects of several medium ingredients have been reported, there are no published work on the synergistic effect of elicitors and inducers in the production of laccases. Therefore, the aim of this study was to apply a full factorial 32 design and response surface methodology to investigate the interaction between the levels of mannan oligosaccharides (elicitor) and ferulic acid (inducer) on laccases production by three strains of white-rot fungi, Pycnoporus sanguineus (P. sanguineus), Corylopsis polyzona (C. polyzona) and Pleurotus ostreatus (P. ostreatus) in submerged fermentation. 2. Materials and methods 2.1. Organism and their maintenance P. sanguineus MUCL 38531 and C. polyzona MUCL 38443, and P. ostreatus IT01 strains were used in this study. The master cultures were kept at −80 ◦ C. The cultures were activated on potato dextrose agar plates and grown for 2 weeks at 28 ◦ C.
Table 1 Medium composition designed for each strain from based on previous studies
Pycnoporus sanguineusa Coriolopsis polyzonab Pleurotus ostreatusc a b c
Potato dextrose (g/l)
Glucose (g/l)
Lactose (g/l)
Yeast extract (g/l)
5 5 25
0 0 7
15 15 15
1 5 5
CuSO4 75 M and pH 5.0. CuSO4 225 M and pH 4.0. CuSO4 225 M and pH 5.0.
2.2. Media preparation Based on previous studies (data not included) the best medium for laccases production designed for each strain was used to evaluate the effect of ferulic acid and mannan oligosaccharide additions on laccases production. Table 1 shows the optimal medium designed for high laccases activity for each strain.
2.3. Mannan oligosaccharide preparation The preparation of mannan oligosaccharide elicitors from locust bean gum (Ceratonia siliqua) was performed as described previously [24]. The preparation contained oligosaccharides with the degree of polymerisation of 5–8.
2.4. Fermentation conditions Actively growing 8-day-old colonies of P. sanguineus, C. polyzona and P. ostreatus on potato yeast agar (PDA) were used to inoculate 250-ml Erlenmeyer flasks with 50 ml of the medium designed for each strain. The flasks were inoculated with three 10 mm plugs and incubated in an orbital shaker (2 cm throw) at 150 rpm and 28 ◦ C for 26 days.
2.5. Addition of ferulic acid and MO Different additions corresponding to different MO and ferulic acid combinations were prepared according to a full factorial experimental design. MO was added at 24 h and ferulic acid at 72 h. The factors and levels are coded as positive (+1), zero (0) and negative (−1) for the higher, middle and lower levels of the variables used in the experiment (Table 2).
2.6. Laccases assay Samples were taken from the flasks every 48 h and centrifuged at 13,000 rpm and 4 ◦ C for 15 min. Laccases activity of the cell-free broth was determined using ABTS as substrate. The assay mixture contained 200 l of 2.5 mM ABTS (dissolved in 100 mM sodium tartrate buffer pH 3), 50 l of supernatant and 950 l 100 mM sodium tartrate buffer at pH 3. The product formation rate from enzymatic oxidation of ABTS was measured spectrophotometrically at 415 nm with an extinction coefficient ε = 3.6 × 104 M−1 cm−1 . The unit activity of laccases was expressed as 1 M of product formed per min. The enzymatic reactions were carried out at room temperature.
Table 2 Concentrations and levels for different combinations of factors to be investigated Factor
MO (mg/l) Ferulic acid (mM)
Levels −1
0
+1
0 0
75 0.5
150 1
The oligosaccharide MO was added at 24 h and the inducer at 72 h.
Table 3 Factors and levels used for the full factorial design Key
X1 X2
Factor
Levels
MO (mg/l) Ferulic acid (mM)
Low (−1)
Medium (0)
High (+1)
0 0
75 0.5
150 1.0
2.7. Cell dry weight Biomass was estimated at the end of the run in all cases. The whole culture volume of each flask was filtered through pre-weighed Whatman no. 1 filter paper and washed with 20 ml of distilled water. The filters were dried to a constant weight in an 80 ◦ C oven, weighed and the dry cell weight calculated.
2.8. Experimental design and evaluation The effects of MO and ferulic acid on laccase production were tested to determine the best combination this factor for laccases produced by three strains of bacidiomisetes. The optimisation was based on a full factorial experimental design 32 . Table 3 shows the factor codes and natural values used in this experiment. The factors are arranged into three levels, coded −1, 0, and + 1 for low, middle and high concentration (or value), respectively. For predicting the optimal point, a second-order polynomial function was fitted to correlate the relationship between variables and response (laccases activity).
2.9. Statistical analysis To investigate the effect of the MO and ferulic acid on the production of laccases a full factorial design was used for each strain studied in this work. This design is important especially when the investigator is interested in finding all the interaction effects of the factors under investigation. A mathematical model describing the relationships between laccases produced and the medium component in a second-order equation was developed. The laccases activity produced by the three strains of bacidiomycetes were multiply regressed with respect to MO and ferulic acid concentrations by the least squares method as follows Y = A0 +
Ai Xi +
Aii Xi2 +
Aij Xi Xj
(1)
where Y is the predicted response variable (laccase produced), A0 , Ai , Aii , Aij are constant regression coefficients of the model, and Xi , Xj (i = 1 and 2; j = 1 and 2). The coefficients represent the independent variables (medium composition) in the form of coded values. The accuracy and general ability of the above polynomial model could be evaluated by the coefficient of determination R2 . All experimental designs were randomised to exclude any bias. The analysis of regression and variance (ANOVA) was carried out using the experimental design Statistica 7 (StatSoft Inc., USA).
Fig. 1. Time course of laccases produced by Pycnoporus sanguineus. () 0.5 mM ferulic acid and 75 mg/l MO; (♦) 1.0 mM ferulic acid and 75 mg/l MO; () 0.5 mM ferulic acid and 150 mg/l MO; () 1.0 mM ferulic acid and 150 mg/l MO; () 150 mg/l MO; () 1.0 mM ferulic acid; () control. Figure represents mean of triplicate experiments and the S.D. is added as error bars.
acid at a concentration of 1 mM was inhibitory to laccases productions. However, mannan oligosaccharides at a concentration of 150 mg/l significantly increased the levels of laccases produced by P. sanguineus. There was 88-fold increase in laccases levels when compared to the control. Biomass concentration obtained from triplicate samples was similar for the control and supplemented cultures (8.74 g/l with a S.D. of 0.42). For P. sanguineus the analysis of variance shows that mannan oligosaccharides and ferulic acid play a significant role in the enhancement of laccases as individual components rather than having synergistic mode of action (Table 4). It appears to be little synergy between the two factors for enhancement of laccases by P. sanguineus (Table 4). Based on the experimental data a response surface model was constructed to estimate the optimal levels of both mannan oligosaccharides and ferulic acid (Fig. 2). The parameterised model is presented below laccase (U/l) = 25376.5 − 328.2X1 − 17532.6X12 1402.2X2 −5745.9X22 917.1X1 X2
(2)
3. Results
where X1 is the coded value of MO concentrations and X2 is the coded value of ferulic acid concentration. The optimal con-
3.1. Evaluation of the effect of mannan oligosaccharides and ferulic acid on laccases production in liquid cultures of P. sanguineus
Table 4 Analysis of variance of the full factorial design for the laccases produced by P. sanguineus
Laccases levels increased significantly in the presence of both ferulic acid and mannan oligosaccharides for P. sanguineus. All experimental combinations tested showed an increase compared to the control cultures where no ferulic acid and mannan oligosaccharides were added. The most effective combination was of 0.5 mM ferulic acid and 75 mg/l mannan oligosaccharides with a maximum of approximately 30,000 U/l (Fig. 1). Ferulic
MO (L) MO (Q) Ferulic acid (L) Ferulic acid (Q) MO × ferulic acid
MS
F
P
7.51E + 05 6.76E + 08 1.37E + 07 7.26E + 07 5.40E + 06
0.044 39.163 0.794 4.206 0.313
0.841 0.000 0.402 0.079 0.593
MS = mean square, F = Fisher value, P = level of significance, L = linear term of the polynomial, Q = quadratic term of the polynomial.
Table 5 Analysis of variance of the central composed experimental design for the laccases produced by C. polyzona
MO (L) MO (Q) Ferulic acid (L) Ferulic acid (Q) MO × ferulic acid
MS
F
P
577,244 2,628,168 26,895,303 667,678 8,431,840
0.8722 3.9711 40.6380 1.0088 12.7402
0.3747 0.0775 0.0001 0.3414 0.0060
MS = mean square, F = Fisher value, P = level of significance, L = linear term of the polynomial, Q = quadratic term of the polynomial.
Fig. 2. Response surface model of laccases activity produced by P. sanguineus under the experimental conditions.
centrations for the elicitor and inducer are 75 mg/l and 0.5 mM, respectively (Fig. 2). 3.2. Evaluation of the effect of mannan oligosaccharides and ferulic acid on laccases production in liquid cultures of C. polyzona For C. polyzona the interactions were more complex. Fig. 3 shows that ferulic acid at 1 mM and a combination of 0.5 mM ferulic acid and 150 mg/l of mannan oligosaccharides are inhibitory to laccases production when compared to control. However, it is important to notice that the synergistic effect of 1 mM ferulic acid and 150 mg/l mannan oligosaccharides was significant (the highest production level for this strain with more than two-fold increase in the production of laccases in cultures of C. polyzona. The effect was not limited to the titres only but the laccases pro-
Fig. 3. Time course of laccases produced by Coriolopsis polyzona. () 0.5 mM ferulic acid and 75 mg/l (♦); 1.0 mM ferulic acid and 75 mg/l MO; () 0.5 mM ferulic acid and 150 mg/l MO; (䊉) 1.0 mM ferulic acid and 150 mg/l MO; () 150 mg/l MO; () 1.0 mM ferulic acid; () control. Figure represent mean of triplicate experiments and the S.D. is added as error bars.
duction started earlier too. The onset of laccases production in C. polyzona supplemented with 1 mM ferulic acid and 150 mg/l mannan oligosaccharides was 4 days earlier than the control without the elicitor and inducer (Fig. 3). Biomass concentration obtained from triplicate samples was similar for the control and supplemented cultures (6.12 g/l with a S.D. of 0.46). The analysis of variance of the results show that both ferulic acid and mannan oligosaccharides have a significant effect on laccases production with P values of 0.0001 and 0.0775, respectively (Table 5). The combined effect of the elicitor and the inducer is significant in enhancing levels of laccases, with a P value of 0.0060. A response surface model was designed (Fig. 4) based on the experimental data. The regressed model is presented next: laccase (U/l) = 2067.2 − 285.8X1 1038.5X12 +1950.8X2 523.4X22 + 1145.6X1 X2
(3)
where X1 is the coded value of MO concentrations and X2 is the coded value of ferulic acid concentration. It shows strong synergistic effect of both factors investigated. Fig. 4 shows the contribution of mannan oligosaccharides to enhancement of laccases. The model shows that mannan oligosaccharide concen-
Fig. 4. Response surface model of laccases activity produced by C. polyzona under the experimental conditions.
Fig. 5. Time course of laccases produced by Pleurotus ostreatus. () 0.5 mM ferulic acid and 75 mg/l; (♦) 1.0 mM ferulic acid and 75 mg/l MO; () 0.5 mM ferulic acid and 150 mg/l MO; (䊉) 1.0 mM ferulic acid and 150 mg/l MO; () 150 mg/l MO; () 1.0 mM ferulic acid; () control. Figure represent mean of triplicate experiments and the S.D. is added as error bars.
tration has limited effect on laccases production in C. polyzona cultures while ferulic acid has a stimulatory effect (Fig. 4).
Fig. 6. Response surface model of laccases produced by P. ostreatus under the experimental conditions.
3.3. Evaluation of the effect of mannan oligosaccharides and ferulic acid on laccases production in liquid cultures of P. ostreatus
used for higher laccases production (Fig. 6). The response surface was generated based on the next mathematical model:
The onset of laccases production was considerably earlier for P. ostreatus when compared with the other strains. The pattern of laccases production was similar for all factors investigated with the exception of 0.5 mM ferulic acid and 75 mg/l manna oligosaccharide addition (Fig. 5). Under this condition at day 15 there was three-fold increase in laccases levels when compared to the control cultures. Biomass concentration obtained from triplicate samples was similar for the control and supplemented cultures (4.14 g/l with a S.D. of 0.64). The analysis of variance show that both ferulic acid and mannan oligosaccharides were significant factors contributing to laccases production in P. ostreatus cultures with P-values of 0.014 and 0.059, respectively (Table 6). However, there was no appreciable synergistic effect in the over-production of laccases. The response surface model constructed based on the experimental data shows the relationship of the elicitor and the inducer Table 6 Analysis of variance of the central composed experimental design for the laccases produced by P. ostreatus
MO (L) (mg/l) MO (Q) (mg/l) Ferulic acid (L) (mM) Ferulic acid (Q) (mM) MO × ferulic acid
MS
F
P
345,631 4,638,254 2,266,861 10,667,230 759,334
0.441 5.917 2.892 13.608 0.969
0.536 0.059 0.150 0.014 0.370
MS = mean square, F = Fisher value, P = level of significance, L = linear term of the polynomial, Q = quadratic term of the polynomial.
laccase (U/l) = 6989.5 − 238.2X1 − 1594.2X12 − 610.1X2 −2542.7X22 − 375.8X1 X2
(4)
where X1 is the coded value of MO concentrations and X2 is the coded value of ferulic acid concentration. The optimal concentrations of mannan oligosaccharides and ferulic acid were 75 mg/l and 0.5 mM (Fig. 6), respectively. 4. Discussion In this work the use of a full factorial experimental design was successfully applied to mannan oligosaccharides as elicitors in liquid cultures of white-rot fungi with the scope for enhancing laccases production. The use of a full factorial design to optimise the selected factors for maximal production was shown to be a good method for the identification of statistically significant effects and interactions. Although mannan oligosaccharides enhanced laccases production in all the strains in this study, the effect was not generic. The elicitor as a singular factor had significant effect when it was added to the liquid cultures of P. ostreatus and P. sanguineus. However, in the case of C. polyzona the synergistic effect of the combined mannan oligosaccharides and ferulic acid was statistically significant for the increase in laccases production. The optimal concentration obtained form the response surface models for the mannan oligosaccharides was 75 mg/l for P. ostreatus and P. sanguineus. Variety of oligosaccharides derived from alginate and locust bean gum have been reported to act as elicitors in liquid cultures of filamentous fungi resulting in increase in the levels of secondary metabo-
lites [21,25,26]. These studies showed that, while there was no generic effect in terms of the type, size and concentration of the elicitor, mannan oligosaccharides elicited the highest effect for enhancement of fungal metabolites in most cases. The choice of mannan oligosaccharides for this study was on this basis. This study showed that mannan oligosaccharides, either individually or combined with ferulic acid, enhance laccases levels in three different strains of white-rot fungi. The increase in laccases production was species specific with the highest increase in liquid cultures of P. sanguineus followed by P. ostreatus and C. polyzona. The reasons why mannan oligosaccharides induce higher titres of laccases in white-rot fungi is to be speculated at this stage. In filamentous fungi as well as in plants elicitor induction of metabolites is seen as an activation of cellular defence mechanisms. In the cultures of P. chrysogenum, mannan oligosaccharides not only enhance penicillin G levels, but also its intermediates ␦-(l-␣-aminoadipyl)-l-cysteinyl-d-valine (tripeptide ACV), isopenicillin N and 6-aminopencillanic acid [21,24,27] Other physiological changes elicited by mannan oligosaccharide in P. chrysogenum include increased germination rates, increase of hyphal tip numbers and clump size, as well as increase in the concentration of spores and pigmentation [22,24] and changes in the levels of reactive oxygen species [28]. Although laccases are involved in lignin degradation, they are also involved in conidial pigmentation and in overcoming host organism reactions during phytopathogenesis [29] and are also known to play a role in melanin production effecting fungal virulence. Laccases are involved in the microbial morphogenesis such as fungal spore development and fungal differentiation [30] and extracellular laccases have been implicated in the infectivity of pathogenic fungi [31]. This inherent multifunctional aspect of laccases and particularly their involvement in defence mechanism of the fungi makes them good targets for increased production, in response to the addition to the cultures, of elicitors such as mannan oligosaccharides. Acknowledgement The authors thank the European Commission for financial support of this work under the Framework VI Integrated Project, SOPHIED. References [1] Higson FK. Degradation of xenobiotics by white rot fungi. Rev Environ Contam Toxicol 1991;122:111–52. [2] Smith M, Thurnston CF. Fungal laccases: role in delignification and possible industrial application. In: Messerschmidt A, editor. Multi-copper oxidases. Singapore: World Scientific; 1997. p. 253–9. [3] Pointing SB. Feasibility of bioremediation by white-rot fungi. Appl Microbiol Biotechnol 2001;57:20–33. [4] Field AJ, De Jong E, Feijoo-Costa FE, de Bont JAM. Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics. TIBTECH 1993;11:44–8. [5] Kuhad RC, Singh A, Eriksson KE. Microorganisms and enzymes involved in the degradation of plant cell walls. Adv Biochem Eng Biotechnol 1997;57:45–125.
[6] Kirk TK, Schultz E, Connors WJ, Lorenz LF, Zeikus JG. Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch Microbiol 1978;117:277–85. [7] D’Souza TM, Merritt CS, Reddy CA. Lignin-modifying enzymes of the white rot basidiomycete Ganoderma lucidum. Mycologia 1999;65: 5307–13. [8] Collins PJ, Dobson ADW. Regulation of laccases gene transcription in Trametes versicolor. Appl Environ Microbiol 1997;63:3444–50. [9] Kahraman S, Yesilada O. Industrial and agricultural wastes for laccases production by white-rot fungi. Folia Microbiol 2001;46:133–6. [10] Parra R, Aldred D, Magan N. Medium optimization for the production of the secondary metabolite squalestatin S1 by a Phoma sp. combining orthogonal design and response surface methodology. Enzyme Microb Technol 2005;37:704–11. [11] Parra R, Aldred D, Magan N. A novel immobilised design for the production of the heterologous protein lysozyme by a genetically engineered Aspergillus niger strain. Appl Microbiol Biotechnol 2005;67:336–44. [12] Francis F, Sabu A, Madhavan NK, Ramachandran S, Ghosh S, Szakacs G, et al. Use of response surface methodology for optimizing process parameters for the production of a-amilase by Aspergillus oryzae. Biochem Eng J 2003;15:107–15. [13] Nyanhongo GS, Gomes J, G¨ubitz GM, Zvauya R, Read J, Steiner W. Decolourisation of textile dyes by laccases from a newly isolated strain of Trametes modesta. Water Res 2002;36:1449–56. [14] Dey G, Mitra A, Banerjee R, Maiti BR. Enhanced production of amylase optimization of nutritional constituents using response surface methodology. Biochem Eng J 2001;7:227–31. [15] Ghanem NB, Yusef HH, Mahrouse HK. Production of Aspergillus terreus xylanase in solid state cultures: application of the Plackett–Burman experimental design to evaluate nutritional requirements. Biores Technol 2000;73:113–21. [16] Vasconcelos AFD, Barbosa AM, Dekker RFH, Scarminio IS, Rezende MI. Optimization of laccase production by Botryosphaeria sp. in the presence of veratryl alcohol by the response surface method. Proc Biochem 2000;35:1131–8. [17] Khuri AI, Cornell JA. Determining optimum condition. In: Response surface design and analysis. New York: Marcel Dekker; 1987. p. 149–205. [18] Haaland PD. Experimental design in biotechnology. New York: Dekker; 1989; Higson FK. Degradation of xenobiotics by white rot fungi. Rev Environ Contam Toxicol 1991;122:111–52. [19] Thayer DW, Muller WS, Buchanan RL, Phillips JG. Effect of NaCl, pH, temperature, and atmosphere on growth of Salmonella typhimurium in glucose–mineral salts medium. Appl Environ Microbiol 1987;53: 1311–5. [20] Bull AT, Castro PM, Hayter PM, Ison AP. Application of a statistical design to the optimization of culture medium for recombinant interferong production by Chinese hamster ovary cells. Appl Microbiol Biotechnol 1992;38:84–90. [21] Radman R, Saez T, Bucke C, Keshavarz T. Review: elicitation of plants and microbial cell systems. Biotechnol Appl Biochem 2003;37:91–102. [22] Asilonu E, Bucke C, Keshavarz T. Enhancement of chrysogenin production in cultures of Penicillium chrysogenum by uronic acid oligosaccharides. Biotechnol Lett 2000;22:931–6. [23] Nair R, Radman R, Roy I, Bucke C, Keshavarz T. Towards unravelling the elicitation mechanism in cultures of Penicillium chrysogenum Chrysogenin elicitation. Chem Eng Trans 2005;6:899–994. [24] Radman R, Bucke C, Keshavarz T. Elicitor effects on Penicillium chrysogenum morphology in submerged cultures. Biotechnol Appl Biochem 2004;40:229–33. [25] Ariyo TA, Bucke C, Keshavarz T. Alginate oligosaccharides as enhancers of penicillin production in cultures of Penicillium chrysogenum. Biotech Bioeng 1997;53:17–20. [26] Petruccioli M, Federici F, Bucke C, Keshavarz T. Enhancement of glucose oxidase production by Penicillium variabile P16. Enzyme Microb Technol 1999;24:397–401. [27] Tamerler C, Ariyo B, Bucke C, Keshavarz T. Effect of mannan and alginate oligosaccharides on production in bioreactors of peni-
cillin G and its biosynthetic intermediates. Ann Microbiol 2001;51:53– 60. [28] Radman R, Bucke C, Keshavarz T. Elicitor effects on reactive oxygen species in liquid cultures of Penicillium chrysogenum. Biotechnol Lett 2004;26:147–52. [29] Mayer AM. Polyphenol oxidases in plant: recent progress. Phytochem 1987;26:11–20.
[30] Timonen S, Sen R. Heterogeneity of fungal and plant enzyme expression in intact Scots pine – Suillus bovimus and – Paxillus involutus mycorrhizospheres developed in natural forest humus. The New Phytologist 1998;138:355–66. [31] Rigling D, van Alfen NK. Regulation of laccases biosynthesis in plantpathogenic fungus Cryphonectria parasitica by double stranded RNA. J Bacteriol 1991;173:8000–3.
