Chitinolytic enzyme activity of Penicillium janthinellum P9 in bench-top bioreactor

JOWKWLOF FERMENTATION ANDBLOENGWEERWG Vol. 86, No. 5.520-523. 1998

Chitinolytic Enzyme Activity of Penicillium jan thinellum P9 in Bench-Top Bioreactor MASSIMILIANO

FENICE,‘*

JEAN-LOUIS

LEUBA,2 AND FEDERICO

FEDERICI’

Dipartimento Agrobiologia e Agrochimica, University o/ Tuscia, Viterbo-01100, Italy’ and Nest16 Research Center, Nestec Ltd., CH-1000, Lausanne 26, Switzerlandz Received 8 June 199WAccepted 18 September 1998 The chitinolytic activity of Penicillium janthinellum P9 was studied in shaken cultures and in a 3-1 benchtop bioreactor by varying culture conditions such as initial medium PI-I, growth temperature, stirrer speed and aeration rate. In shaken flasks, the highest levels of enzyme activity (468 and 483 U-Z-*) were obtained at a growth temperature of 24°C and at an initial medium pH of 4.0, respectively. In the bioreactor, both agitation and aeration significantly influenced the enzyme production: the highest level of enzyme activity (497 U-Z-l) was obtained at an impeller speed of 500 rpm and an aeration rate of 1.5 wm. Culturing P. janthinellum P9 under opthnised conditions led to an increase in the enzyme activity of ca. 65% (686 U-l-’ as compared to the 415 U.Z-l obtained under the initial culture conditions). [Key words:

chitinolytic activity, Penicillium junfhinelium, culture conditions, optimization]

In recent years, the use of chitinolytic enzymes in applicative fields such as biological pest control (l-3), the degradation of chitin-rich wastes (4) and the production of chitin hydrolysates for pharmaceutical or chemical purposes and for the food/feed industry (5, 6) has become of increasing interest. Unfortunately, large-scale production of chitinases is too expensive and uneconomical to make these enzymes available in sufficient quantities. Besides the traditional use of selected strains of Serratia murcescens and Streptomyces griseus for the production of chitinolytic enzymes, fungi have recently been attracting increasing attention (7-9). However, the only fungal chitinase preparation of any commercial value comes from Trichoderma harzianum cultivated by submerged fermentation. The optimization of fermentation parameters very often leads to markedly increased enzyme production (IO, 11); in this respect, however, very little has been done to optimise the production of fungal chitinase by submerged processes (12). In this paper we report a study of the fermentation conditions for optima1 production of chitinolytic enzymes in a bench-top bioreactor by a strain of Penicillium janthineflum (P9) previously selected in a preliminary screening (unpublished results) for the high levels of its extracellular chitinase activity. The time course for growth and enzyme activity under optimal fermentation conditions is reported. Strain P9 of P. janthinellum is stocked in the culture collection of the Dipartimento di Agrobiologia e Agrochimica, University of Tuscia, Viterbo, Italy. During the study, the culture was maintained on malt extract agar (MEA) at 4-6°C and subcultured every month. The standard medium (SM) contained: colloidal chitin and corn steep liquor (CSL) 0.5 g.l-‘. The 15g.l-1, medium was sterilized at 121°C for 20min. Initial conditions for shaken cultures (orbital shaker, BBraun, Mesulgen, Germany) were: inoculum size, ca. 2.0 x lo6 conidia ml-‘; agitation, 200 rpm; growth temperature, 28°C; * Corresponding author.

and initial medium pH, 5.0. Colloidal chitin was prepared as reported by Hankin and Anagnostakis (13): 50 g of chitin from crab shells were dissolved in 500 ml of H2S04 (50%) and the mixture was quickly poured into 10 I of distilled water, After swelling, the colloidal chitin was repeatedly (five times) washed with distilled water (10 1 each time) until the pH was approximately 5.5. Samples were withdrawn every 12-24 h of fermentation and, after centrifugation (10,OOOxg for IOmin), the supernatants were used as enzyme solutions for the enzyme assay. The fermentors used were 3-1 (total volume) jacketed bench-top stirrer tank reactors (STR) (Applikon Dependable Instruments, Schiedam, The Netherlands) filled with 2.01 of medium. The bioreactors were equipped with control instrumentation for temperature, dissolved oxygen, pH, aeration rate, and stirrer speed. Both stirrers were equipped with 2 standard rushton-type turbines (6 flat blades, 4.5 cm). The initial conditions of fermentation were as follows: inoculum size, ea. 2.0 X IO6 conidia ml-‘; stirrer speed, 5OOrpm; aeration rate, l.Ovvm; growth temperature, 28°C; initial dissolved oxygen concentration, 100% of saturation (non-controlled); and initial medium pH, 5.0 (non-controlled). The culture medium contained: colloidal chitin, 15 g. f-l plus CSL, 0.5 g-1-l. To prevent foam formation, siliconic antifoam, 0.5 g . I-‘, was added before sterilization (121°C for 30 min). The fermentation process was controlled by means of a microcomputer controller (ADI 1020, Applikon Dependable Instruments, Schiedam, The Netherlands). Bioprocesses were performed in duplicate using two identical bioreactors. Mycelial growth was measured indirectly as total DNA content of the biomass. DNA extraction was performed as described by Raeder and Broda (14). The amount of extracted DNA was evaluated spectrophotometrically at 260nm. A calibration curve (correlation: r=0.97) was plotted between the mycelial dry weights of the fungus grown on glucose (10 g. 1-l) and the corresponding DNA content. The total chitinolytic activity present in the culture broth, was determined as follows: a mixture of 0.5 ml of

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citratephosphate buffer, pH 5.5 and 0.5 ml of enzyme solution was incubated at 50°C for 10 min. The amount of reducing sugars released was measured by the dinitro-salicylic acid method of Miller (15) using N-acetyl-o-glucosamine (NAG) for the standard curve. Under the assay conditions, one unit (U) of enzyme activity was defined as the amount of enzyme which released 1 ,nmol of N-acetyl-Dglucosamine per milliliter per minute. The effects of growth temperature (22, 24, 26, 28, 30°C) and initial pH of the medium (3.5, 4.0, 4.5, 5.0, 5.5 and 6.0) were tested in shaken cultures. Experiments were performed in triplicate. The influence of aeration rate and stirrer speed on the enzyme production in the bioreactor was evaluated at 0.5, 1.0 and 1.5 vvm under 300, 400, 500, 600 and 700rpm. Fermentation at constant pH (pH 4.0) was obtained by automatic addition of HzS04 3.0 M. In all cases, the experiments were performed in duplicate. One way analysis of variance (ANOVA) and pairwise multiple comparison procedures (Tukey test) were carried out using the statistical software SigmaStat, version 2.0 (Jandel Corp., San Rafael, CA, USA). P. janthinellum P9 grew well in the temperature range 22-30°C with an optimum at 24°C (Fig. l), thus confirming previous results of Rapper and Thomm (16) on the thermal preferences of this species. The chitinase activity followed the same time course as the growth which appeared related in some way (Fig. 1). In fact, at 22°C the enzyme activity was low (210 lJ.l-I), most likely due to the reduced mycelial concentration colloidal

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TABLE 1. Maximum growth and chitinase activities of

Biomass (g. I- ‘) Chitinase (U I- ‘) Activity (A%) Sp. act. (U.g- ‘)

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I -.-

3,5

4,0

4,5

5,O

5,5

6,0

6,5

PH

FIG. 1. Effect of incubation temperature on growth (0) and chitinolytic enzyme prodution (0) by P. janthinellum P9 in shaken flasks. Biomass and enzyme activities are given at the time of maximal enzyme production (168 h of fermentation at 24, 26, 28, 30°C and 192 h at 22°C). a 1% (w/v)

621

FIG. 2. Effect of initial medium pH on growth (0) and chitinolytic enzyme production ( l) by P. janthinelllum P9 in shaken flasks. Biomass and enzyme activities are given at the time of maximal enzyme production (168 h of fermentation).

(5.35 g. I-r); peaked at 24°C (478 U. I-~‘; mycelial growth 6.39g.I-I), ca. 13% higher than that at 28°C (415 U.l-r) and was significantly reduced (95 lJ.l-l) at 30°C. The very limited enzyme activity at this last temperature (6°C above the optimum growth temperature) might depend not only on the low mycelial concentration (5.01 g. 1-l) but also on increased cell turnover of proteins and nucleic acids leading to less energy being available for growth-associated functions (17). The differences recorded between cultures grown at 24 and 28”C, both in enzyme activity and mycelial concentration, were not statistically significant. Thus, and in view of a possible scale-up, all other fermentations were carried out at 28°C. On the industrial scale, cooling is more expensive than heating; thus, the slightly lower enzyme production at 28°C could be compensated by the lower energy required for temperature control; large energy savings, however, can only be achieved at temperatures of or over 40°C (18). The effect of the initial medium pH was studied in shaken cultures in the pH range 3.5-6.0 (steps of 0.5 pH); results are shown in Fig. 2. An initial pH of 4.0 appeared to be optimal for chitinase production. At this pH, the enzyme activity (483 U.l-‘) was ca. 16% higher than that (415 IJ.l-l) obtained at pH 5.0 (initial conditions) (Table 1). This is important since pH 4.0 is considered to be sufficiently low to limit bacterial contamination (19). The pH of the medium increased progressively during fermentation from the initial value of pH 4.0 to 6.5 or even higher (data not shown), probably due to the catabolism of NAG liberated by chitin hydrolysis (17, 20); on glucose the growth of P. janthinellum P9 did P.

janfhinelkm

P9 under different culture conditions

Initial

Best temp.

Best pH

Best aer/rpm

Optimized

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6.39kO.308 468k 19” 13 73

6.38?0.30a 483 t 19ab 16 18

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Sp. act. =Specific enzyme activity expressed as U of chitinase per g of biomass formed; Initial=data obtained at standard conditions (istandard deviation); Best temp. =data obtained at the best temperature (*standard deviation); Best pH=data obtained at the best pH (istandard deviation); Best aer/rpm=data obtained at the best combination of aeration rate and stirrer speed (*standard deviation); Optimized=data obtained under optimized fermentation parameters (*standard deviation). Mean values in the same row followed by the same superscript letter were not significantly different (P=O.O5) as determined by the Tukey test.

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Time (h) FIG. 3. Time course of chitinolytic enzyme activity (-_) and pH (---) of P. janthinellum P9 grown in a bioreactor at pH 4 controlled (0) and uncontrolled (0).

FIG. 4. Effect of stirrer speed and aeration rate on the maximum chitinolytic activity of P. janthineilum P9 in the bioreactor.

not lead to any pH increase (data not shown). When the fungus was grown in the bioreactor at the constant pH of 4.0, the enzyme activity decreased (Fig. 3). Varying the initial medium pH by OS-l.0 pH units from the optimum value (pH 4.0) affected the enzyme activity but not the biomass formed, thus confirming earlier observations of Forage et al. (17). Figure 4 shows the combined effects of aeration and agitation on the chitinolytic activity of P. janthinellum P9. Experiments were carried out at various agitation rates (300, 400, 500, 600 and 700 rpm) in combination with three different aeration rates: 0.5, 1.0 and 1.5 vvm. The enzyme production was strongly influenced by both stirring and aeration: chitinase activity was highest (497 U .I-‘) at a stirrer speed of 500 rpm and an aeration rate of 1.5 vvm, at 168 h of cultivation. The microorganism probably suffered a certain degree of shear stress: stirrer speeds above 500rpm resulted in lower levels of enzyme activity (around 345 U .1-r at 600 and 700 rpm with 1.0 or 1.5 wm; around 300 USIP1 at 600 and 700 with 0.5 vvm). Nevertheless, stirrer speeds lower than 400rpm resulted in lower enzyme production (168, 305, 358 U. I- l at 300 rpm with 0.5, 1.O and 1.5 vvm, respectively), probably due to insufficient oxygenation that

could be partially compensated by increasing tion rate (Fig. 4). Low enzyme activity (ca. accompanied by poor mycelial growth (data was recorded when the fungus was grown speed of 300 rpm and an aeration rate of 0.5

the

aera-

150 U-l-I), not shown), at a stirrer

vvm. Figure 5 shows the time course of growth and chitinase activity of P. janthinellum P9 cultivated in a bench-top reactor under the following optimised conditions: initial medium pH, 4.0 (uncontrolled); initial DO, 100% (uncontrolled); aeration rate, 1.5 vvm; stirrer speed, 500rpm; and temperature, 28°C. Evolution of dissolved oxygen and pH is also shown. The enzyme activity appeared related to the mycehal growth. For the first 48 h of cultivation (likely, the time necessary for enzyme induction), activity was practically absent (23 U-l-r at 48 h). It began to increase after 72 h (66, 258, 432 U. 1.k’ at 72, 96 and 120 h, respectively) and reached its maximum (648 U. 1-l) after 168 h of incubation. Afterwards, the enzyme activity remained almost constant for another 24 h period but decreased rapidly thereafter. After 120 h of fermentation, the DO dropped to ca. 10% and remained below that value for a further 48 h without, however, falling below 5%. During this period,

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of P. janthinellum

P9 grown in a bioreactor under optimised conditions;

VOL. 86, 1998

NOTES

both the enzyme production and the mycelial growth reached their maximum. Under the above optimised conditions of fermentation, fungal growth* and specific enzyme activity were also maximal (7.06 g . l- ’ and 97 U . g - I, respectively) (Table 1). The optimization of fermentation parameters led to an increase in the enzyme activity of about 65%, from 415 to 686 U. 1-l (initial and optimised conditions, respectively). However, the increased chitinase production of P. janthinelhm P9 appeared to be more dependent on appropriate combination of different fermentation conditions than on the optimization of any single parameter which did not lead to enzyme activity increases of more than 13-20%. This work was partially supported by a grant from NESTEC Ltd., Nestle Research Center, Lausanne, Switzerland. REFERENCES 1. Di Pietro, A., Lorito, M., Hayes, C. K., Broadway, R. M., and Harmaa, G. E.: Endochitinase from Gliocladium virens: isolation, characterization and synergistic antifungal activity in combination with gliotoxin. Phitopathology, 83, 308-313 (1993). 2. Lorito, M., Hayes, C. K., Di Pietro, A., Woo, S. L., and Harman, G. E.: Purification, characterization, and synergistic activity of a glucan 1,3+glucosidase and an N-acetyl-b-glucoaminidase from Trichoderma harzianum. Phytopathology, 84, 398-405 (1994). 3. Lorito, M., Woo, S. L., DonaeBi, B., and Scala, F.: Synergistic antifungal interactions of chitinolytic enzymes from fungi, bacteria and plants, p. 157-164. In Muzzarelli, R. A. A. (ed.), Chitin enzimology. Edizioni Atec, Grottammare, Italy (1996). 4. Chen, J. P. and Chang, K. C.: Immobilization of chitinase on a reversibly soluble-insoluble polymer for chitin hydrolysis. .I. Chem. Tech. Biotechnol., 60, 133-140 (1994). 5. Gyorgy, P. and Rose, C. S.: Microbiological studies on growth factor for L. bifidus var. pennsylvanicus. Proc. Sot. Exp. Biol. Med., 90, 219-223 (1955). 6. SakaI, K., Nanjo, F., and Usui, T.: Production and utilization of oligosaccharides from chitin and chitosan. Denpun Kagaku, 37, 79-86 (1990). 7. Deshpande, M. V.: Enzymatic degradation of chitin and its biological applications. J. Scient. Ind. Res., 45, 273-281 (1986).

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8. Chet, I.: Trichoderma: application, mode of action, and potential as biocontrol agent of soilborne plant pathogenic fungi, p. 137-160. In Chet, I. (ed.), Innovative approaches to plant disease control, John Wiley & Sons, New York (1987). 9. Vyas, P. and Deshpande, M.: Enzymatic hydrolysis of chitin by Myrothecium verrucaria chitinase complex and its utilization to produce SCP. J. Gen. Appl. Microbial., 37, 267-275 (1991). 10. Petruccioli, M., Fenice, M., Piccioni, P., and FederIci, F.: Effect of stirrer speed and buffering agents on the production of glucose oxidase and catalase by Penicillium variabile (P16) in benchtop bioreactor. Enzyme Microb. Technol., 17, 336-339 (1995). 11. Park, Y., Uehara, H., Teruya, R., and Okabe, M.: Effect of culture temperature and dissolved oxygen concentration on expression of a-amylase gene in batch culture of spore-forming host, Bacillus subtilis lA289. J. Ferment. Bioeng., 84, 53-58 (1997). 12. Kapat, A., R&hit, S. K., and Panda, T.: Optimization of carbon and nitrogen sources in the medium and environmental factors for enhanced production of chitinase by Trichoderma harzianum. Bioproc. Eng., 15, 13-20 (1996). 13. Hankin, L. and Anagnostakis, S.L.: The use of solid media for detection of enzyme production by fungi. Mycologia, 67, 597-607 (1975). 14. Raeder, U. and Broda, P.: Rapid preparation of DNA from filamentous fungi. Lett. Appl. Microbial., 1, 17,-20 (1985). 15. Miier, G. L.: Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal. Chem., 31, 426-430 (1959). 16. Rapper, K. R. and Thomm, C.: Manual of Pennicillia. Afner Publication Co., NY and London (1968). 17. Forage, R. G., Harrison, D. E. F., and Pitt, D. E.: Effect of environment on microbial activity, p. 251-280. In Moo-Young, M. (ed.), Comprehensive biotechnology, vol. 1. Pergamon Press, Oxford, New York, Toronto, Sydney, Frankfurt (1985). 18. Stanbury, P. F., Whitaker, A., and Hall, S. J.: The isolation, preservation and improvement of industrially important microorganisms, p, 35-91. In Stanbury, P. F., Whitaker, A., and Hall, S. J. (ed.), Principles of fermentation technology, 2nd ed. Pergamon Press, Elsevier Sciences, Oxford (1995). 19. Cooney, C. L.: Growth of microorganisms, p. 73-112. In Rhem, H-J. and Reed, G. (ed.), Biotechnology, vol. 1. Verlag Chemie, Weinheim, Deerfield Beach, Base1 (1981). 20. Wang, D. I. C., Cooney, C. L., Demain, A. L., Dunnil, P., Humphrey, A., and Lilly, M. D.: Fermentation kinetics, p. 5797. In Wang, D. I. C., Cooney, C. L., Demain, A. L., Dunnil, P., Humphrey, A., and Lilly, M. D. (ed.), Fermentation and enzyme technology. John Wiley & Sons, New York (1979).