The effect of pH on the production of chitinolytic enzymes of Verticillium fungicola in submerged cultures

Bioresource Technology 101 (2010) 9236–9240

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The effect of pH on the production of chitinolytic enzymes of Verticillium fungicola in submerged cultures Laura Ramirez-Coutiño a, Jesús Espinosa-Marquez a, Martin G. Peter b, Keiko Shirai a,* a b

Universidad Autónoma Metropolitana, Biotechnology Department, Laboratory of Biopolymers, Av. San Rafael Atlixco, No. 186, 09340 Mexico City, Mexico Universität Potsdam, Institut für Chemie, P.O. Box 601553, D-14415 Potsdam, Germany

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Article history: Received 14 May 2010 Received in revised form 29 June 2010 Accepted 30 June 2010 Available online 24 July 2010 Keywords: Chitooligosaccharides Chitinases N-Acetylhexosaminidase Verticillium fungicola Chitosan

a b s t r a c t Chitinase and N-acetylhexosaminidase activities in submerged cultures of Verticillium fungicola increased up to 5-fold and 2.5-fold, respectively when the pH of the culture medium was raised from 5 to 8. SDS– PAGE and zymograms of the freeze-dried crude enzyme obtained from the cultures indicated four chitin degrading proteins of Mw 24, 40, 55 and 63 kDa, whereas isoelectric focusing displayed the separation of three chitin degrading enzymes with isoelectric points of 4.7, 6.8 and 10, as well as two N-acetylhexosaminidases having isoelectric points of 3.2 and 13. Freeze-dried crude enzyme was characterized for its ability to produce chito-oligosaccharides from chitosans. Matrix-assisted laser desorption ionization time of flight mass spectrometry analyses revealed that monomers as well as hetero-oligomers with degree of polymerization 4 were initially the main products, whereas oligomers with degree of polymerization 2– 11 were detected after extended reaction times. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction The mycoparasitic fungus Verticillium fungicola causes dry bubble disease in the mushroom Agaricus bisporus (Amey et al., 2003). Infection of A. bisporus by V. fungicola is mediated by fungal cell wall degrading enzymes, including glucanases, proteases and chitinases (Matsumoto et al., 2004; Shirai, 2006), which cause necrosis and browning, thereby leading to important economical losses in A. bisporus production (Ramirez-Coutiño et al., 2006). Fungal chitinases are produced constitutively and they can be induced. These chitinases are essential to cell wall destruction and formation during growth and morphogenesis. They are also required for antagonism and when chitin is assimilated as carbon source (Matsumoto et al., 2004; Shirai, 2006). Furthermore, chitinolytic enzymes promote the synthesis of chito-oligosaccharides, which are at present intensively studied as plant growth regulators, food additives, as well as potential drugs (Peter, 2002; Shirai, 2006). Synthesis and secretion of fungal cell wall degrading enzymes are regulated by several environmental factors, most importantly by extracellular pH which acts as a key signal for growth, differentiation and virulence of fungal pathogens of humans, plants, insects and other fungi (Caracuel et al., 2003; Maccheroni et al., 2004). Thus, fungi have developed efficient mechanisms for sensing and responding to changes in the ambient pH (St. Leger et al., 1998). Despite these reports, the influence of pH of the culture * Corresponding author. Tel.: +52 5 5804 49 21; fax: +52 5 5804 47 12. E-mail address: [email protected] (K. Shirai). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.160

medium on the production of chitinases of V. fungicola in submerged culture (SmC) has not yet been investigated. The aim of this study was to evaluate the role of the pH of the culture medium on the production of chitinolytic enzymes, i.e. chitinases and b-N-acetylhexosaminidases (HexNase), by growing V. fungicola in the presence of chitin as the sole carbon source. Furthermore, we report the characteristics of the enzymes on molecular weights (Mw), chitinases activities in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and isoelectric points (pI) determinations. As well, the freeze-dried crude enzyme (FDCE) was applied in the production of oligomers from chitosan and the products were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analyses. 2. Methods 2.1. Materials

a-Chitin was prepared from shrimp (Litopenaeus spp.) waste by lactic acid fermentation and purified according to Cira et al., (2002). Chitosans were prepared by partial homogeneous deacetylation of a-chitin (Kurita et al., 1993). The mole fraction of N-acetylglucosamine (FA) value was determined by elemental analysis (Perkin Elmer–Series II, Connecticut, USA) (Ramírez-Coutiño et al., 2006). Mw was estimated by intrinsic viscosity (Rocha-Pino et al., 2008). All other chemicals were analytical grade and used without further purification.

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2.2. Fungal strain and culture conditions V. fungicola var. flavidum was obtained from the Entomopathogenic Fungal Culture Collection (Ithaca, New York USA). Fungal strain was maintained on potato dextrose agar (PDA) slants at 4 °C until needed. Spore suspension was prepared by agitation of PDA cultures with a solution of Tween 80 0.1 v/v% up to a concentration of 107 spores/mL. 2.3. Submerged culture (SmC) SmC were carried out in a 3-L instrumented bioreactor (Applikon B.V, The Netherlands), using Czapeck medium supplemented with chitin 10 g/L. The operating conditions were 1 L of air per L of medium per minute of aeration rate at 25 °C and an agitation rate of 250 rpm. The reactor was inoculated with 107 spores/mL. pH was kept constant at 5 for 144 h by the addition of either acid (0.1 N HCl) or alkali (0.1 N NaOH). In another series of experiments, pH was kept constant for 72 h and then it was increased every 24 h up to pH 8 until 144 h. Fifty millilitre samples were taken every 12 h and centrifuged at 4 °C and 12,700 g (Beckman J2-MI, USA). Protein contents in the supernatants were determined by Bradford (1976). 2.4. Preparation of crude enzyme from culture media with shifts of pH Fungal biomass was collected by centrifugation of the SmC at 4 °C and 12,700 g during 20 min (Beckman J2-MI, USA). The supernatant (1550 mL) was concentrated to approximately 150 mL by ultrafiltration with a molecular weight cut-off membrane of 10 kDa (Millipore Pellicon XL equipment, Bedford, Massachusetts). The retentate was freeze-dried (Lyph-Lock 6 Labconco 195, Kansas City, Missouri) prior to further characterization and chitosan hydrolysis experiments. 2.5. SDS–PAGE Proteins were separated by SDS–PAGE using 5 wt/v% stacking and 12 wt/v% resolving gels (mini Protean II Bio-Rad, Hercules California USA) and visualized by staining with Coomassie brilliant blue R-250. A broad range of molecular weight (Mw) protein standard (Bio-Rad) was used as a reference. 2.6. Detection of chitinase activity in SDS–PAGE Gels for SDS–PAGE were prepared including 0.01 wt/v% of chitosan (FA 0.64) in the resolving gel. After electrophoresis, a strip of the gel was stained with Coomassie Brilliant Blue R-250 to visualize protein bands. Another strip of the gel was incubated in a 50 mM sodium phosphate renaturing buffer (pH 5) containing Triton X-100 0.1 wt/v%, for 2 h at room temperature. The strip was then rinsed with distilled water and incubated in deionized water for 1 h at 37 °C. Further, gel was immersed for 5 min into a freshly prepared solution Calcofluor white M2R 0.01 wt/v% in 0.5 M tris– HCl buffer (pH 9). Chitinolytic activities appeared as dark zones within homogenously fluorescent background upon illumination with an UV transilluminator (Gel Doc Bio-Rad) (Trudel and Asselin 1989). The experiments were carried out by duplicate. 2.7. Isoelectric focusing FDCE was dissolved in 45 mL of deionized water, containing 1 v/ v% of ampholytes with a pH range of 3 to 10 (Bio-Rad), 5 v/v% of glycerol and 0.5 v/v% of Triton-X100. Then it was subjected to isoelectric focusing in a Rotofor electrofocusing cell (Bio-Rad). A constant current of 15 W was applied for 5 h at 4 °C. pH, enzyme

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activities and protein concentrations were determined in 20 fractions using deionized water and ampholytes as blanks. 2.8. Enzymatic reactions Chitinase and HexNase activities were determined using colloidal chitin and p-nitrophenyl N-acetylglucosamine (GlcNAc-pNP) as substrates with an UV/VIS spectrophotometer (JENWAY 6305 Essex, U.K.) at k = 510 and 400 nm, respectively (Tronsmo and Harman 1993). One unit of chitinase was defined as the amount of the crude enzyme preparation required to reduce 5% of the initial turbidity of a 1 wt/v% colloidal chitin solution. One unit of HexNase was defined as the amount of crude enzyme required to release 1 lmol of p-nitrophenol (PNP) in a 2.92 mM solution of GlcNAcPNP in 0.2 M of buffer citrate–phosphates (pH 5.6). 2.9. Enzymatic hydrolysis of chitosan and analysis by MALDI-TOF MS of the reaction products Enzymatic hydrolyses were carried out in 100 mL reactors using 5 mg of FDCE/ mL of 50 mM citrate–phosphate buffer (pH 5.0), containing 0.02 wt/v% of sodium azide. Chitosan samples were added at a concentration of 5 mg/mL and the reaction was carried out at 40 °C under continuous stirring (Ramírez-Coutiño et al., 2006). Aliquots of the reaction mixture were removed and ultrafiltrated through a cut-off membrane of 5 kDa, the permeates were freeze-dried and the residue was dissolved in methanol/water 1:1 to give a concentration of oligosaccharides of ca. 1 mg/mL. Then, 0.35 lL was mixed on the target with 0.7 lL of the matrix solution (25 g/L of 2,5-dihydroxybenzoic acid in acetonitrile, containing 0.1 v/v% of trifluoroacetic acid). The samples were dried at room temperature under a stream of air. Mass spectra were recorded with a Bruker Reflex II (Bruker Daltonik, Bremen Germany) instrument in the positive ion mode, using a nitrogen laser (337 nm, 3 ns pulse width, 3 Hz). Ca. 120 single shots were accumulated and averaged (Bahrke et al., 2002; Letzel et al., 2000). 3. Results and discussion 3.1. Effect of pH on the production of chitinolytic enzymes Chitinases were initially detected in the SmC of V. fungicola medium kept at constant pH of 5.0 for 36 h. The activity was ca.100 U/g in the initial dry substrate (IDS) and afterwards it remained between 50 and 100 U/g IDS throughout the experiment for up to 120 h. Interestingly, the activity showed a remarkable increase to 550 U/g IDS when the pH of the culture medium was raised stepwise to 8.0 between 72 and 120 h (Fig. 1). Similarly, HexNase activity was positively affected by the increasing pH, since a maximum of 400 mU/g IDS at constant pH 5.0 (120 h) rise to 950 mU/g IDS (ca. 2.5-fold increase) when the pH increased, as shown in Fig. 2. Relatively high pH of the culture medium triggers an important signal for the expression of chitinolytic enzymes in V. fungicola, which is in agreement with earlier reports on the effects of the pH on extracellullar enzymatic activities, including transcription, translation, protein processing, enzyme stabilities and the toxic effects of non-optimal pH conditions on protein synthesis (St. Leger et al., 1998; Mach et al., 1999; Caracuel et al., 2003). St. Leger et al., (1998) reported that the genes encoding cuticle degrading enzymes, proteases and chitinases of Metarhizium anisopliae are regulated by the combination of substrate induction and carbon catabolite repression. Our sequence of enzymatic activities expression, suggests that the chitinases and HexNases were expressed in relation to certain chemical signals, such as chitin

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Fig. 3. SDS–PAGE of: (a) Mw standards, stained with Coomassie blue; (b) extracellular proteins from V. fungicola, stained with Coomasie blue; (c) extracellular protein, zymogram with chitin (FA 0.64) and Calcofluor white.

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as inducer and oligosaccharides as products of hydrolysis. The experimental results shown in Figs. 1 and 2 indicate that HexNases were expressed at the early stages, followed by the production of chitinases. The action of chitinases and HexNases were essential to fulfill the fungal cell requirements from the chitin used as sole carbon source, owing to the production of the mixture of chitobiose, chitotriose and chitotetraose as substrates for the HexNases. Chitinases appear at 36 h in the SmC carried out at pH 5. At this time chitin was first split into oligos which further could be broken down by HexNases (Fig. 2). In a related work, Mach et al., (1999) reported that chitin degradation products (DP 1–3) acted as inducers of HexNases expression of Trichoderma harzianum, whereas chitinases were triggered by the exhaustion of carbon, low temperature and high osmotic pressure. Herein, it is remarkable that chitinases were more influenced by the shifts of pH in the culture than HexNases, which was more likely induced by chitin and its degradation products (Figs. 1 and 2). Likewise the production of proteases and chitinases by Metarhizium anisopliae were expressed as a response to the ambient pH (St. Leger et al., 1998). Mw of proteins present in the crude enzyme after 144 h of SmC of V. fungicola cultured with the shift of pH from 5 to 8 were estimated by SDS–PAGE (Fig. 3). Four bands, i.e. 24, 40, 55 and 63 kDa (Fig. 3b), displayed chitinolytic activity, as revealed by the zymogram shown in Fig. 3c. Most of the chitinolytic activity appears

associated with the protein band at 24 kDa. However, a diffusion leading to band broadening cannot be excluded with certainty. The multiple bands of the crude enzyme observed on SDS–PAGE (Fig. 3C) also suggest the presence of isoenzymes which might explain the capability of V. fungicola strain to hydrolyze chitin at different pHs. This is not surprising since several fungi are known to produce multiple proteins for chitin cleavage by chitinases or chitosanases, such as the mycoparasitic strain of T. harzianum (De la Cruz et al.,1992). The chitinolytic activity and the pH of fractions collected after preparative isoelectrofocusing are shown in Fig. 4. Two HexNase and three chitinase/chitosanase activities were detected. The pIs of the HexNases were 3.2 and 13, whereas the pI values of the chitinolytic activities were 4.7, 6.8 and 10, respectively. Acid and basic chitinolytic enzymes were isolated from SmC of Lecanicillium lecanii with chitin as substrate and identified as CHI1 with Mw of 40.9 and CHI2 with 45.9 kDa. The pIs for these chitinases were 4.5 and 7.61 for CHI1 and CHI2, respectively. According to other authors, CHI1 may play a protective role within the fungal cell, whereas CHI2 was secreted outside the cell for chitin digestion (Lu et al., 2005). In other related fungi, Verticillium chlamydosporium and V. suchlasporium, endochitinases having 43 kDa (CHI43) were purified from SmC with colloidal chitin and main bands with pI of 7.6 and 7.9 for V. suchlasporium and V. chlamydosporium, respectively, were detected after isoelectrofocusing analysis. Additionally, two isoforms with pIs of 7.1 and 7.4 were determined in V. suchlasporium as well as another two, with pIs of 7.3 and 7.6, for V. chlamydosporium (Tikhonov et al., 2002). 3.2. Enzymatic hydrolysis of chitosan Mw of 345 kDa and FA of 0.43 were determined in the chitosan sample employed in the hydrolysis with the FDCE prepared after 144 h of SmC of V. fungicola with the 5 to 8 pH increase. The 5 kDa-permeates of the enzymatic hydrolysis were analyzed by MALDI-TOF MS. Glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) monomers were formed and assigned to peaks with m/z 179.08 and 260.18, respectively (see Supplementary Data 1 in the online version of this article for the MALDI-TOF MS of the end products from hydrolysis experiments with chitosan using FDCE of V. fungicola). At an earlier reaction time (1.5 h), chito-oligo-

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tion depends not only on the substrate but also on product concentrations as it was reported by Dvorakova et al., (2001) when the accumulation of A repressed the hydrolysis thus favoring transglycosylation. Herein, all the oligomers present in the reaction media could act either as donors or acceptors during the transglycosylation reaction, in addition, water acts as acceptor thus promoting hydrolysis. In the earlier stages of the reaction (1.5 h), the hydrolysis was certainly the main mechanism involved, which produced oligomers with DP from 1 to 7. These oligosaccharides could act as acceptors or even donors towards production of higher DP oligomers. Experimental observations show that FA of the oligomers with the same DP decreases as the reaction proceeds, which clearly points out to the activity of chitin deacetylases.

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The stepwise increase of pH from 5 to 8 during SmC of V. fungicola supplemented with chitin displayed a remarkable influence on chitinases production. The detection of enzyme activity in SDS–PAGE showed the presence of four chitinases in the FDCE with protein bands of Mw of 24, 40, 55 and 63 kDa. Crude enzyme produced by V. fungicola was able to produce oligomers with a wide range of DP, which are important due to their biological activities. Further work is underway on enzyme purification and their application on chitin/chitosan towards biological production of oligosaccharides. Acknowledgements

Fraction number Fig. 4. Chitinases activities detected in fractions collected after isoelectric focusing of crude enzyme of V. fungicola.

saccharides of DP 6 7, being monomers, dimers (A2, D1A1), trimers (D3, D1A2) and tetramers (D1A3) were detected with the highest intensities. Whereas, at the ending of the reaction (120 h), monomers, dimers (D1A1), trimers (D1A2), tetramers (D2A2, D1A3), pentamers (D2A3) and hexamers (D3A3) were identified as the most abundant. In addition, oligomers with DP10 and DP11, absent at the beginning of the reaction, were present at 120 h (see Supplementary Data 1 in the online version of this article for the MALDITOF MS of the end products from hydrolysis experiments with chitosan using FDCE of V. fungicola). Hydrolysis of the b-1,4-glycosidic linkage of chitin can be carried out by a chitinolytic system: endochitinases (EC 3.2.1.1.4), exochitinases (EC 3.2.1.14), b-N-acetyl hexosaminidases (EC 3.2.1.52) and chitosanases (EC 3.2.1.1.32). The production of low molecular weight oligomers of GLcNAc, such as chitotetraose, chitotriose, and diacetylchitobiose might be attributed to random cleavage by endochitinases. Later on, the action of HexNases released GLcNAc from these oligomers. The arrangement of hydrolysis products showed that HexNase was significantly high since monomers were detected during the whole experiment (see Supplementary Data 1 in the online version of this article for the MALDI-TOF MS of the end products from hydrolysis experiments with chitosan using FDCE of V. fungicola). Reaction is rather complex considering that the FDCE contains a wide variety of chitinases, which did not drive only to hydrolysis but also to other side reactions such as transglycosylation and deacetylation. Several chitinases of family 18, which includes fungal chitinases, are reported to catalyze transglycosylation (Dvorakova et al., 2001; Boer et al., 2004). For instance, transglycosylation was observed during the hydrolysis reaction of b-1,4-galactosylated and a-1,3-fucosylated chito-oligosaccharides by chitinases with 33 and 42 kDa of T. harzianum (Boer et al., 2004). Transglycosyla-

Financial support was provided by the National Council for Science and Technology of Mexico (CONACyT 105628). L.R. and J.E. thank the European Commission for scholarships in the Alfa Programme, Project Polylife. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2010.06.160. References Amey, R.C., Mills, P.R., Bailey, A., Foster, G.D., 2003. Investigating the role of a Verticillium fungicola b-1, 6-glucanase during infection of Agaricus bisporus using targeted gene disruption. Fungal Genet. Biol. 39, 264–275. Bahrke, S., Einarsson, J.M., Gislason, J., Haebel, S., Letzel, M.C., Peter-Katalinic, J., Peter, M.G., 2002. Sequence analysis of chitooligosaccharides by matrix-assisted laser desorption ionization postsource decay mass spectrometry. Biomacromolecules 3, 696–704. Boer, H., Munck, N., Natunen, J., Wohlfahrt, G., Söderlund, H., Renkonen, O., Koivula, A., 2004. Differential recognition of animal type b4-galactosylated and a3fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum. Glycobiology 14 (12), 1303–1313. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Caracuel, Z., Roncero, I.G., Espeso, E.A., González-Verdejo, C.I., García-Maceira, F.I., Di Pietro, A., 2003. The pH signalling transcription factor PacC controls virulence in the plant pathogen Fusarium oxysporum. Mol. Microbiol. 48, 765–779. Cira, L.A., Huerta, S., Hall, G.M., Shirai, K., 2002. Pilot scale lactic acid fermentation of shrimp wastes for chitin recovery. Process Biochem. 37, 1359–1366. De la Cruz, J., Hidalgo-Gallego, A., Lora, J.M., Benitez, T., Pintor-Toro, J.A., Llobel, A., 1992. Isolation and chracterization of 3 chitinases from Trichoderma-harzianum. Eur. J. Biochem. 206, 859–867. Dvorakova, J., Schmidt, D., Hunkova, Z., Thiem, J., Kren, V., 2001. Enzymatic rearrangement of chitine hydrolysates with b-N-acetylhexosaminidase from Aspergillus oryzae. J. Mol. Catal. B Enzym. 11, 225–232. Kurita, K., Tomita, K., Tada, T., Shigeru, I., Nishimura, S.I., Shimoda, K., 1993. Squid chitin as a potential alternative chitin source. deacetylation behaviour and characteristic properties. J. Polym .Sci. Pol. Chem. 31, 485–491. Letzel, M.C., Synstad, B., Eijsink, V.G.H., Peter-Katalinic, J., Peter, M.G., 2000. In: Peter, M.G., Domard, A., Muzzarelli, R.A.A. (Eds.), Advances of Chitin Science, vol. 4. University of Potsdam, Germany, pp. 545–552.

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Lu, Z.X., Laroche, A., Huang, H.C., 2005. Isolation and characterization of chitinases from Verticillium lecanii. Can. J. Microbiol. 51, 1045–1055. Maccheroni, W., Araújo, W.L., Azevedo, J.L., 2004. Ambient pH-regulated enzyme secretion in endophytic and pathogenic isolates of the fungal genus Colletotrichum. Sci. Agri. 61, 298–302. Mach, R.L., Peterbauer, C.K., Payer, K., Jaksits, S., Woo, S.L., Zeilinger, S., Kullnig, C.M., Lorito, M., Kubicek, C.P., 1999. Expression of two major chitinase genes of Trichoderma atroviride (T. harzianum P1) is triggered by diferent regulatry signals. Appl. Environ Microbial. 65, 1858–1863. Matsumoto, Y., Saucedo-Castañeda, G., Revah, S., Shirai, K., 2004. Production of b-Nacetylhexosamidase for Verticillium lecanii by solid state fermentation using shrimp waste silage as substrate and inducer. Process Biochem. 39, 665–671. Peter, M.G., 2002. Chitin and Chitosan from Fungi. In: Steinbüchel, A. (Ed.), Biopolymers, vol. 6 Polysaccharides II. Wiley-VCH, Weinheim, pp. 123–157. Ramírez-Coutiño, L., Marín-Cervantes, M., Huerta, S., Revah, S., Shirai, K., 2006. Enzymatic hydrolysis of chitin for production of N-acetylglucosamine using Lecanicillium fungicola chitinases. Process Biochem. 41, 1106–1110.

Rocha-Pino, Z., Martinez Piña, M., Arias, L., Vazquez, H., Shirai, K., 2008. Effect of water quality and particle size on the production of chitosan from b-chitin isolated from jumbo squid processing wastes (Dosidicus gigas). Revista Mexicana de Ingenieria Quimica 7 (3), 299–307. Shirai, K., 2006. Advances in Agricultural and Food Biotechnology. In: Guevara, R.G., Torres, I. (Eds.). Research Signpost, Kerala, India, pp. 289–304. St. Leger, R.J., Joshi, L., Roberts, D., 1998. Ambient pH is a major determinant in the expression of cuticle-degrading enzymes and hydrophobin by Metarhizium anisopliae. Appl. Environ. Microbial. 64, 709–713. Tikhonov, V.E., Lopez-Llorca, L.V., Salinas, J., Jansson, H.B., 2002. Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V Suchlasporium. Fungal Genet. Biol. 35, 67–78. Tronsmo, A., Harman, G.E., 1993. Detection and quantification of N-acetyl-b-Dglucosaminidase, chitobiosidase, and endochitinase in solutions and on gels. Anal. Biochem. 208, 74–79. Trudel, J., Asselin, A., 1989. Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal. Biochem 178, 362–366.