World Journal
of Microbiology
and Biotechnology,
9. 300-302
Production and preliminary characterization of RNA-depolymerase from Trichoderma harzianum E.S. Vasileva-Tonkova Trichoderma harzianum produced RNA-depolymerase with maximum activity after 72 to 120 h of growth. Addition of K,HPO, repressed enzyme production by the fungus. The optimal activity was at pH 7.8 and 40 to 50°C. The enzyme was stable at pH 3.2 to 9.0 and 80% of activity remained after 60 min at 4O“C. EDTA and pchloromercuribenzoate had no effect on the enzyme activity. Key words: Production,
RNA-depolymerase,
Trichoderma
There have been many reports on microbial RNA-degrading enzymes which play an important role in the metabolism of nucleic acids. They are also important for structural studies of RNA (Takahashi & Moore 1982). RNAases are good material for analysis of protein from the viewpoint of comparative biochemistry (Beintema et al. 1977; Hill et al. 1983). Various species of Trichoderma produce xylanases, chitinases, chitobiases, cellulases, pectinases and a number of products of commercial value @lanes et al. 1988; Ujiie et al. 1991; Ulhoa & Peberdy 1992) but very little information exists on the degradation of RNA by extracellular enzymes of these fungi (Harada & Irie 1974; Vasileva-Tonkova & Bezborodova 1986). This paper reports the production, precipitation and some preliminary characteristics of RNA-depolymerase in the culture filtrate of Trichoderma harziantrm.
Materials
and Methods
Microorganism and Growth Conditions Trichoderma harzianum 01 was maintained on Czapek’s agar slants at 4’C. The growth medium contained (g/l): glucose, 50.0; peptone, 10.0; soybean meal, 5.0; MgSOJH,O, 0.5; CaCl,.ZH,O, 0.1; KNO,, 0.2. Erlenmeyer flasks (750 ml), each containing 150 ml of the medium, were each inoculated with 1.0 ml of spore
suspension and incubated at 25°C with agitation. ES. Vasileva-Tonkova Academy of Sciences, fax: 359-2-700109.
World
mycelium
is with the Institute of Microbiology, Bulgarian Acad. G. Bonchev str., Bl. 26, 1113 Sofia, Bulgaria;
@ 1993 Rapid Communications
300
The
of Oxford
Joumdof Microbiologyand
Ltd Biotechnology, Vol 9, 1993
was removed by filtration activity, soluble protein,
and the filtrate pH and residual
Precipitation of Enzyme After 96 h cultivation, RNA-depolymerase (pH 7.6) was precipitated by adding
saturation) with stirring at 4’C. After collected
10
by centrifugation,
dissolved
sodium phosphate buffer, pH same buffer for 24 h. mM
was assayed for enzyme glucose.
in the culture filtrate (NH,),SO, (30% to 90% 30 min, the precipitate was with the minimal amount of 6.4, and dialysed against the
Enzyme Assay RNA-depolymerase activity was determined, according to the modified method of Anfinsen et al. (1954), at pH 8.0, using 0.8% (w/v) RNA (sodium salt) as substrate. In determination of the pH optimum, 0.1 M sodium acetate buffer (pH 4.0 to 6.0) and 0.1 M Tris/HCl buffer (pH 7.0 to 9.5) were used. Protein
determination
was carried
out
Granum (1980) or using the absorbance determined by the method of Somogyi the fungus was determined by filtering, water and drying at 105°C overnight.
according
to Whitaker
&
at 280 run. Glucose was (1952). The dry weight of washing with distilled
Results Growth and RNA-depolymerase Production Trichoderma harzianum produced RNA-depolymerase in Ogata’s medium containing 5% (w/v) glucose but without added phosphate (Figure 1). Maximal growth was obtained after 72 h, when the glucose in the medium had been completely consumed. RNA-depolymerase first appeared in
RNA-depolymerase
from T. harzianum
90 80
Time (h)
30
Figure 1. Time course of growth, pH, glucose and protein levels and RNA-depolymerase production in T. harzianum cultures. O-Activity at pH 8.0; n-dry weight; A-glucose; O-protein; W-PH.
the medium after the growth reached the stationary phase; maximum activities were attained after 72 to 120 h. After 3 days of growth in media with 0.1, 1.0 and 10.0 mM K,HPO,, relative activities in culture filtrates were 52%, 29% and 4%, of the activity in the absence of phosphate, respectively. Production of the enzyme on 3% (w/v) glucose was a third of that seen on 5% (w/v). The addition of RNA (0.4% w/v) to the basic medium did not affect RNA-depolymerase production. Precipitation and Characterization of RNA-depolymerase Precipitation with (NH&O, at 50% and 70% saturation gave recoveries of 38% and 69%, respectively, with a two-fold purification. Precipitation at 90% saturation resulted in four-fold purification and 88% recovery. This fraction was dialysed and used as a crude RNAdepolymerase for preliminary characterization. The pH optimum for enzyme activity was 7.8. There was a small increase in activity at pH 4.5 to 5.0 which suggested the presence of at least two RNA-depolymerases in the culture filtrate of T. harziantrm (Figure 2). The optimal temperature for enzyme activity was 40 to 50°C. The crude enzyme was 100% stable in the range pH 7.6 to 9.0, about 80% in the range pH 3.2 to 6.3 and completely unstable at pH 1.2. Thermostability was determined by pre-incubation of the crude enzyme precipitate at different temperatures (at pH 6.4) for 10 and 60 min, followed by assay for activity at pH 8.0 and 25°C. After 10 min at 40, 50 and 6O”C, 94%, SO%, and 20%, respectively, of the initial enzyme activity remained. Preincubation of the enzyme with 0.1 or 10 mM EDTA for 15 min at pH 6.4 had no effect whereas preincubation with 10 mM p-chloromercuribenzoate inhibited enzyme activity, determined at pH 8.0, by 20%.
20
9
345678
10
ph Figure 2. Effect (NH&SO,-precipitate
of
pH on RNA polymerase activity from culture filtrate of T. harzianum.
in
Discussion The fungus T. harziantlm produces RNA-depolymerase when grown in liquid medium with low content of ortho-phosphate. This medium was used as it appeared to be the most suitable for RNA-depolymerase production from other fungal sources (Bezborodova & Bezborodov 1979). The fungus produced mainly alkaline RNAdepolymerase, with a pH optimum (7.8) similar to that of the RNAase AC, from Acrocylindrium sp. (Suhara et al. 1972); Mg, from A4ucor genevensis (Rushizky et al. 1964); and Fs, from Fusarimm semifecfum (Bezborodova & Markauskaite 1972). The enzyme was stable over a broad range of pH values (3.2 to 9.0) and up to 50°C. It was presumed that, at 60 to SO”C, protease activity in the culture filtrate inactivated the RNA-depolymerase, as was observed with the RNAase from Aspergilltis candidus (Mohammad Kunhi and Singh 1981). As divalent cations were not required for its catalytic activity, the isolated RNAdepolymerase is probably an RNAase.
References Anfinsen, C.B., Rodfied, R.R., Choate, W.L., Page, J.T. & Caroll, W.R. 1954 Studies of the structure, cross linkages and terminal sequences in ribonucleases. ]ouma\ of Biobgicd Chemistry 207, 201-210.
Beintema, J.J., Gaastra, W., Len&a, J.A., Welling, G.W. & Fitch, W.M. 1977 The molecular evolution of pancreatic ribonuclease. ]ownal
of A4oleculur
Euolufion
10, 4(r71.
ES.
Vu&vu-Tonkova
Bezborodova, S.I. & Bezborodov, A.M. 1979 Extracellular ribonucleases in comparative study. In Biosynthesis of Ntlcleuses and Proteases from Microorgunisms, ed Imshenetzky, A.A. pp. 92-130. Moscow: Science. Bezborodova, S.I. & Markauskaite, R.B. 1972 Purification of extracellular ribonuclease from Fusuriwn semifectwn. Prikludnuyu Biochimiu and Microbiologiu 8, 107-111. Harada, M. & Irie, M. 1974 Partial purification of ribonuclease from Trichoderm koningii. Chemical and Pharmaceutical Bulletin 22, 1850-1856. Hill, C., Dodson, G., Heinemann, U., Saenger, W., Mitsui, Y., Nakamura, K., Borisov, S., Tischenko, G., Polyakov, K. & Pavlovsky, S. 1983 The structure and sequence homology of a family of microbial ribonucleases. Trends in Biochemicul Sciences 8, 364-369. Illanes, A., Gentina, J.C. & Marchase, M.P. 1988 Production and stabilization of cellulases from Trichodermu reesei. ]oarnul of Applied Microbiology and Biotechnology 4, 407-417. Mohammad Kunhi, A.A. & Singh, R. 1981 Standardization of assay produce and some properties of ribonuclease from Aspergillw cundidw. Foliu Microbiologica 26, 328-333. Rushizky, G.W., Greco, A.E., Hartley, R.W. & Sober, H.A. 1964 Studies on the characterization of ribonucleases. Journal of Biological Chemistry 239, 2165-2169.
302
World ~oumal
of Minobiology and Biotechnology.
Vol 9, 1993
Somogyi, M. 1952 Determination of reducing sugar. Journal of Biological Chemistry 195, 19-23. Suhara, I., Kawashima-Kusaba, F., Nakao, Y., Yoneda, M. & Ohmura, E. 1972 The nucleases from Acrocylindriwn sp, I. Purification and properties of ribonuclease. )ownul af Biochemistry
71, 941-950. Takahashi, K. & Moore, S. 1982 Ribonuclease T,. In The Enzymes, Nucleic Acids, Vol. 15, ed Boyer, P.D. pp. 435-468. New York and London: Academic Press. Ujiie, M., Roy, C. & Yaguchi, M. 1991 Low-molecular weight xylanase from Trichoderm viride. Applied and Environmenful Micrabiology 57, 1860-1862. Ulhoa, CJ. & Peberdy, J.F. 1992 Purification and some properties of the extracellular chitinase produced by Trichodermu hurziunum. Enzyme and Microbiul Technology 14, 23&240. Vasileva-Tonkova, E.S. & Bezborodova, S.I. 1986 Production of exocellular RNA-depolymerases by Trichodermu fungi, Micrabiologiu 55, 401-406. Whitaker, J.R. & Granum, P.E. 1980 An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Analytical Biochemistry 109, 156-159. (Received
in
November
1992)
revised
form
9 November
1992;
accepted
12
of Microbiology
and Biotechnology,
9. 300-302
Production and preliminary characterization of RNA-depolymerase from Trichoderma harzianum E.S. Vasileva-Tonkova Trichoderma harzianum produced RNA-depolymerase with maximum activity after 72 to 120 h of growth. Addition of K,HPO, repressed enzyme production by the fungus. The optimal activity was at pH 7.8 and 40 to 50°C. The enzyme was stable at pH 3.2 to 9.0 and 80% of activity remained after 60 min at 4O“C. EDTA and pchloromercuribenzoate had no effect on the enzyme activity. Key words: Production,
RNA-depolymerase,
Trichoderma
There have been many reports on microbial RNA-degrading enzymes which play an important role in the metabolism of nucleic acids. They are also important for structural studies of RNA (Takahashi & Moore 1982). RNAases are good material for analysis of protein from the viewpoint of comparative biochemistry (Beintema et al. 1977; Hill et al. 1983). Various species of Trichoderma produce xylanases, chitinases, chitobiases, cellulases, pectinases and a number of products of commercial value @lanes et al. 1988; Ujiie et al. 1991; Ulhoa & Peberdy 1992) but very little information exists on the degradation of RNA by extracellular enzymes of these fungi (Harada & Irie 1974; Vasileva-Tonkova & Bezborodova 1986). This paper reports the production, precipitation and some preliminary characteristics of RNA-depolymerase in the culture filtrate of Trichoderma harziantrm.
Materials
and Methods
Microorganism and Growth Conditions Trichoderma harzianum 01 was maintained on Czapek’s agar slants at 4’C. The growth medium contained (g/l): glucose, 50.0; peptone, 10.0; soybean meal, 5.0; MgSOJH,O, 0.5; CaCl,.ZH,O, 0.1; KNO,, 0.2. Erlenmeyer flasks (750 ml), each containing 150 ml of the medium, were each inoculated with 1.0 ml of spore
suspension and incubated at 25°C with agitation. ES. Vasileva-Tonkova Academy of Sciences, fax: 359-2-700109.
World
mycelium
is with the Institute of Microbiology, Bulgarian Acad. G. Bonchev str., Bl. 26, 1113 Sofia, Bulgaria;
@ 1993 Rapid Communications
300
The
of Oxford
Joumdof Microbiologyand
Ltd Biotechnology, Vol 9, 1993
was removed by filtration activity, soluble protein,
and the filtrate pH and residual
Precipitation of Enzyme After 96 h cultivation, RNA-depolymerase (pH 7.6) was precipitated by adding
saturation) with stirring at 4’C. After collected
10
by centrifugation,
dissolved
sodium phosphate buffer, pH same buffer for 24 h. mM
was assayed for enzyme glucose.
in the culture filtrate (NH,),SO, (30% to 90% 30 min, the precipitate was with the minimal amount of 6.4, and dialysed against the
Enzyme Assay RNA-depolymerase activity was determined, according to the modified method of Anfinsen et al. (1954), at pH 8.0, using 0.8% (w/v) RNA (sodium salt) as substrate. In determination of the pH optimum, 0.1 M sodium acetate buffer (pH 4.0 to 6.0) and 0.1 M Tris/HCl buffer (pH 7.0 to 9.5) were used. Protein
determination
was carried
out
Granum (1980) or using the absorbance determined by the method of Somogyi the fungus was determined by filtering, water and drying at 105°C overnight.
according
to Whitaker
&
at 280 run. Glucose was (1952). The dry weight of washing with distilled
Results Growth and RNA-depolymerase Production Trichoderma harzianum produced RNA-depolymerase in Ogata’s medium containing 5% (w/v) glucose but without added phosphate (Figure 1). Maximal growth was obtained after 72 h, when the glucose in the medium had been completely consumed. RNA-depolymerase first appeared in
RNA-depolymerase
from T. harzianum
90 80
Time (h)
30
Figure 1. Time course of growth, pH, glucose and protein levels and RNA-depolymerase production in T. harzianum cultures. O-Activity at pH 8.0; n-dry weight; A-glucose; O-protein; W-PH.
the medium after the growth reached the stationary phase; maximum activities were attained after 72 to 120 h. After 3 days of growth in media with 0.1, 1.0 and 10.0 mM K,HPO,, relative activities in culture filtrates were 52%, 29% and 4%, of the activity in the absence of phosphate, respectively. Production of the enzyme on 3% (w/v) glucose was a third of that seen on 5% (w/v). The addition of RNA (0.4% w/v) to the basic medium did not affect RNA-depolymerase production. Precipitation and Characterization of RNA-depolymerase Precipitation with (NH&O, at 50% and 70% saturation gave recoveries of 38% and 69%, respectively, with a two-fold purification. Precipitation at 90% saturation resulted in four-fold purification and 88% recovery. This fraction was dialysed and used as a crude RNAdepolymerase for preliminary characterization. The pH optimum for enzyme activity was 7.8. There was a small increase in activity at pH 4.5 to 5.0 which suggested the presence of at least two RNA-depolymerases in the culture filtrate of T. harziantrm (Figure 2). The optimal temperature for enzyme activity was 40 to 50°C. The crude enzyme was 100% stable in the range pH 7.6 to 9.0, about 80% in the range pH 3.2 to 6.3 and completely unstable at pH 1.2. Thermostability was determined by pre-incubation of the crude enzyme precipitate at different temperatures (at pH 6.4) for 10 and 60 min, followed by assay for activity at pH 8.0 and 25°C. After 10 min at 40, 50 and 6O”C, 94%, SO%, and 20%, respectively, of the initial enzyme activity remained. Preincubation of the enzyme with 0.1 or 10 mM EDTA for 15 min at pH 6.4 had no effect whereas preincubation with 10 mM p-chloromercuribenzoate inhibited enzyme activity, determined at pH 8.0, by 20%.
20
9
345678
10
ph Figure 2. Effect (NH&SO,-precipitate
of
pH on RNA polymerase activity from culture filtrate of T. harzianum.
in
Discussion The fungus T. harziantlm produces RNA-depolymerase when grown in liquid medium with low content of ortho-phosphate. This medium was used as it appeared to be the most suitable for RNA-depolymerase production from other fungal sources (Bezborodova & Bezborodov 1979). The fungus produced mainly alkaline RNAdepolymerase, with a pH optimum (7.8) similar to that of the RNAase AC, from Acrocylindrium sp. (Suhara et al. 1972); Mg, from A4ucor genevensis (Rushizky et al. 1964); and Fs, from Fusarimm semifecfum (Bezborodova & Markauskaite 1972). The enzyme was stable over a broad range of pH values (3.2 to 9.0) and up to 50°C. It was presumed that, at 60 to SO”C, protease activity in the culture filtrate inactivated the RNA-depolymerase, as was observed with the RNAase from Aspergilltis candidus (Mohammad Kunhi and Singh 1981). As divalent cations were not required for its catalytic activity, the isolated RNAdepolymerase is probably an RNAase.
References Anfinsen, C.B., Rodfied, R.R., Choate, W.L., Page, J.T. & Caroll, W.R. 1954 Studies of the structure, cross linkages and terminal sequences in ribonucleases. ]ouma\ of Biobgicd Chemistry 207, 201-210.
Beintema, J.J., Gaastra, W., Len&a, J.A., Welling, G.W. & Fitch, W.M. 1977 The molecular evolution of pancreatic ribonuclease. ]ownal
of A4oleculur
Euolufion
10, 4(r71.
ES.
Vu&vu-Tonkova
Bezborodova, S.I. & Bezborodov, A.M. 1979 Extracellular ribonucleases in comparative study. In Biosynthesis of Ntlcleuses and Proteases from Microorgunisms, ed Imshenetzky, A.A. pp. 92-130. Moscow: Science. Bezborodova, S.I. & Markauskaite, R.B. 1972 Purification of extracellular ribonuclease from Fusuriwn semifectwn. Prikludnuyu Biochimiu and Microbiologiu 8, 107-111. Harada, M. & Irie, M. 1974 Partial purification of ribonuclease from Trichoderm koningii. Chemical and Pharmaceutical Bulletin 22, 1850-1856. Hill, C., Dodson, G., Heinemann, U., Saenger, W., Mitsui, Y., Nakamura, K., Borisov, S., Tischenko, G., Polyakov, K. & Pavlovsky, S. 1983 The structure and sequence homology of a family of microbial ribonucleases. Trends in Biochemicul Sciences 8, 364-369. Illanes, A., Gentina, J.C. & Marchase, M.P. 1988 Production and stabilization of cellulases from Trichodermu reesei. ]oarnul of Applied Microbiology and Biotechnology 4, 407-417. Mohammad Kunhi, A.A. & Singh, R. 1981 Standardization of assay produce and some properties of ribonuclease from Aspergillw cundidw. Foliu Microbiologica 26, 328-333. Rushizky, G.W., Greco, A.E., Hartley, R.W. & Sober, H.A. 1964 Studies on the characterization of ribonucleases. Journal of Biological Chemistry 239, 2165-2169.
302
World ~oumal
of Minobiology and Biotechnology.
Vol 9, 1993
Somogyi, M. 1952 Determination of reducing sugar. Journal of Biological Chemistry 195, 19-23. Suhara, I., Kawashima-Kusaba, F., Nakao, Y., Yoneda, M. & Ohmura, E. 1972 The nucleases from Acrocylindriwn sp, I. Purification and properties of ribonuclease. )ownul af Biochemistry
71, 941-950. Takahashi, K. & Moore, S. 1982 Ribonuclease T,. In The Enzymes, Nucleic Acids, Vol. 15, ed Boyer, P.D. pp. 435-468. New York and London: Academic Press. Ujiie, M., Roy, C. & Yaguchi, M. 1991 Low-molecular weight xylanase from Trichoderm viride. Applied and Environmenful Micrabiology 57, 1860-1862. Ulhoa, CJ. & Peberdy, J.F. 1992 Purification and some properties of the extracellular chitinase produced by Trichodermu hurziunum. Enzyme and Microbiul Technology 14, 23&240. Vasileva-Tonkova, E.S. & Bezborodova, S.I. 1986 Production of exocellular RNA-depolymerases by Trichodermu fungi, Micrabiologiu 55, 401-406. Whitaker, J.R. & Granum, P.E. 1980 An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Analytical Biochemistry 109, 156-159. (Received
in
November
1992)
revised
form
9 November
1992;
accepted
12
