World Journal of Microbiology & Biotechnology 12, 413-414
Short Note: Exocellular hydrolases from Penicillium ulaiense
V.B. Rajal, L. Carrillo* and C.M. Cuevas Penicillium ulaiense exhibited carboxymethylcellulase, pectinase, protease, amylase and phenolase activities, while no xylanase, cellulase, lipase or ligninase activities were found. Pectinolytic action was studied in liquid medium, showing low levels of pectinesterase and pectinase production. No mycotoxins were detected by thin-layerchromatography. Keywords: Hydrolase, pectinase, Penicillium ulaiense.
Penicillium ulaiense Hsieh, Su & Tzean is, together with Penicillium digitatum (Pers.:Fr) Sacc and Penicillium italicum Wehmer, a citrus post-harvest pathogenic fungus. It was described in 1987 (Holmes et aI. 1994). Since only taxonomic or phyto-pathological information about it has been published, studies of its exocellular hydrolase activities were carried out. Three strains of P. ulaiense (1640, i610 and 1653), four
The authors are with INIQUI, Universidad Nacional de Salta, Buenos Aires 177, 4400 Salta, Argentina; fax: 0054-87-251006. * Corresponding author.
of P. italicum and one of P. digitatum were maintained in agar slants using Czapek and Pumpkin Agar. Hydrolase assays were performed in Petri dishes at 28°C using different substrates. After nine days the plates were developed showing carboxymethylcellulase, pectinase, amylase, protease on skim milk (two strains) and phenolase (two strains) activities. They did not show xylanase, cellulase, lipase, protease on gelatin or ligninase activities. The strains were cultured in liquid medium with citrus pectin as sole carbon source, shaken at 120 rev/min, at 28°C. Pectinesterase activity (Figure 1A) was measured by continuous titration and results were corrected for pH
0,1
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0,3
0,06 0,2
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< 0,04 tU a. []
0,1
O
0,02
0
1
2
3
4
5
Culture time (days)
8
7
0
1
2
3
4
5
6
7
Culture time (days)
Figure 1. Pectinolytic action by P. ulaiense in liquid medium. Strains /X--1640; O--1610; [Z]--1653. (A) Pectinesterase production, expressed as pectinesterase units (PEU) per ml of supernatant (SN). (B) Pectinase activities expressed as the fluidity variation. @ 1996 Rapid Science Publishers World Journal of Microbiology & Biotechnology, Vol 12, 1996
4 ~3
V.B. Rajal, L. Carillo and C.M. Cuevas deviation (Whitaker 1992). One pectinesterase unit was defined as the amount of enzyme that releases 1/lmol of methoxyl group/rain at 35°C and pH 4.5. Total pectinase activity was determined by fluidity variation (Yamasaki et al. 1967). The assays with pectic enzymes showed that P. ulaiense strains, in general, produce low concentrations of pectinases (Figure 1B). Pectinesterase activity is high in P. digitatum and low in P. italicum. A search for secondary toxic exocellular metabolites using YES Agar cultures was made by thin-layer chromatography (Paterson & Bridge 1994). The P. ulaiense strains studied did not produce fluorescent nor visible spots before or after spraying with the developer. These results indicate the potential use of P. ulaiense for enzyme production in the food industry.
414
Wor~a]ou~l of Microbiology& Biotechnology,Vo112,1996
References Holmes, G.J., Eckert, J.W. & Pitt, J.I. 1994 Revised description of Penicillium ulaiense and its role as a pathogen of citrus fruit. Phytopathology 84, 719-727. Paterson, R.R.M. & Bridge, P.D. 1994 Biochemical techniques for filamentous fungi, Volume 1, IMI Technical Handbooks. p. 21. Wallingford: CAB International. Whitaker, J.R. 1992 Microbial pectolytic enzymes. In Microbial Enzymes and Biotechnology, 2nd Edition. eds Fogarty, W.M. & Kelly, C.T. pp. 133-176. New York: Elsevier Applied Science. Yamasaki, M., Kato, A., Chu, S.Y. & Arima, K. I967 Pectic enzymes in the clarification of apple juice. Part II. The mechanism of clarification. Agricultural and Biological Chemistry 31, 552-560.
(Received in revised form 5 March 1996; accepted 22 March I996)
Short Note: Exocellular hydrolases from Penicillium ulaiense
V.B. Rajal, L. Carrillo* and C.M. Cuevas Penicillium ulaiense exhibited carboxymethylcellulase, pectinase, protease, amylase and phenolase activities, while no xylanase, cellulase, lipase or ligninase activities were found. Pectinolytic action was studied in liquid medium, showing low levels of pectinesterase and pectinase production. No mycotoxins were detected by thin-layerchromatography. Keywords: Hydrolase, pectinase, Penicillium ulaiense.
Penicillium ulaiense Hsieh, Su & Tzean is, together with Penicillium digitatum (Pers.:Fr) Sacc and Penicillium italicum Wehmer, a citrus post-harvest pathogenic fungus. It was described in 1987 (Holmes et aI. 1994). Since only taxonomic or phyto-pathological information about it has been published, studies of its exocellular hydrolase activities were carried out. Three strains of P. ulaiense (1640, i610 and 1653), four
The authors are with INIQUI, Universidad Nacional de Salta, Buenos Aires 177, 4400 Salta, Argentina; fax: 0054-87-251006. * Corresponding author.
of P. italicum and one of P. digitatum were maintained in agar slants using Czapek and Pumpkin Agar. Hydrolase assays were performed in Petri dishes at 28°C using different substrates. After nine days the plates were developed showing carboxymethylcellulase, pectinase, amylase, protease on skim milk (two strains) and phenolase (two strains) activities. They did not show xylanase, cellulase, lipase, protease on gelatin or ligninase activities. The strains were cultured in liquid medium with citrus pectin as sole carbon source, shaken at 120 rev/min, at 28°C. Pectinesterase activity (Figure 1A) was measured by continuous titration and results were corrected for pH
0,1
0,4 E
Z &
0,08 . _
/k
E ,a. v
0,3
0,06 0,2
>
< 0,04 tU a. []
0,1
O
0,02
0
1
2
3
4
5
Culture time (days)
8
7
0
1
2
3
4
5
6
7
Culture time (days)
Figure 1. Pectinolytic action by P. ulaiense in liquid medium. Strains /X--1640; O--1610; [Z]--1653. (A) Pectinesterase production, expressed as pectinesterase units (PEU) per ml of supernatant (SN). (B) Pectinase activities expressed as the fluidity variation. @ 1996 Rapid Science Publishers World Journal of Microbiology & Biotechnology, Vol 12, 1996
4 ~3
V.B. Rajal, L. Carillo and C.M. Cuevas deviation (Whitaker 1992). One pectinesterase unit was defined as the amount of enzyme that releases 1/lmol of methoxyl group/rain at 35°C and pH 4.5. Total pectinase activity was determined by fluidity variation (Yamasaki et al. 1967). The assays with pectic enzymes showed that P. ulaiense strains, in general, produce low concentrations of pectinases (Figure 1B). Pectinesterase activity is high in P. digitatum and low in P. italicum. A search for secondary toxic exocellular metabolites using YES Agar cultures was made by thin-layer chromatography (Paterson & Bridge 1994). The P. ulaiense strains studied did not produce fluorescent nor visible spots before or after spraying with the developer. These results indicate the potential use of P. ulaiense for enzyme production in the food industry.
414
Wor~a]ou~l of Microbiology& Biotechnology,Vo112,1996
References Holmes, G.J., Eckert, J.W. & Pitt, J.I. 1994 Revised description of Penicillium ulaiense and its role as a pathogen of citrus fruit. Phytopathology 84, 719-727. Paterson, R.R.M. & Bridge, P.D. 1994 Biochemical techniques for filamentous fungi, Volume 1, IMI Technical Handbooks. p. 21. Wallingford: CAB International. Whitaker, J.R. 1992 Microbial pectolytic enzymes. In Microbial Enzymes and Biotechnology, 2nd Edition. eds Fogarty, W.M. & Kelly, C.T. pp. 133-176. New York: Elsevier Applied Science. Yamasaki, M., Kato, A., Chu, S.Y. & Arima, K. I967 Pectic enzymes in the clarification of apple juice. Part II. The mechanism of clarification. Agricultural and Biological Chemistry 31, 552-560.
(Received in revised form 5 March 1996; accepted 22 March I996)
