Purification, physicochemical properties, and specificity of a ribonuclease produced by Trichoderma harzianum

ELSEVIER

Purification, physicochemical properties, and specificity of a ribonuclease produced by Trichoderma harzianum Evgenia S. Vasileva-Tonkova Institute of Biochemistry,

and Svetlana I. Bezborodova

Russian Academy

of Sciences,

Moscow, Russia

An extracellular ribonuclease of Trichoderrna harzianum, named RNase Th, was purified to homogeneity by CM-cellulose chromatography followed by DEAE-cellulose and Sephadex G-50 chromatography. The enzyme consisted of one polypeptide chain of 102 residues. The molecular weight was found to be 11,000 by SDS/PAGE and 10,342 from the amino acid composition. The pH optimum was at 8.0 with yeast RNA and 6.0 with GpC. RNase Th had a unique high pI of 9.5. The enzyme degraded only poly(I), GpN, G > p, and I > p {2’,3’cyclophosphate) substrates. Products were 3’-IMP (with poly(I)) and 3’-GMP (with GpN) via guanosine 2’,3’cyclophosphate. It was concluded that RNase Th is a guanyl-specific cyclizing RNase (EC 3.1.27.3). The kinetic parameters of reactions with GpN were measured and shown to be similar to those of other fungal guanyl RNases. Keywords:

Ribonuclease;

Trichodernza;

purification;

specificity

Introduction Trichodermo species can be effective as biological control agents against fungal pathogens.‘*2 The main mechanism involved in the antagonism of Trichoderma sp. and pathogenic fungi appears to be the release of hydrolytic enzymes including cellulases, chitinases, chitobiases, xylanases, pectinases, and proteases. Species of the genus Trichoderma also produce ribonucleases (RNases).7-9 These enzymes are produced by numerous microorganisms among which the fungi are the most potent producers. RNases are used in various industries, particularly in the food industry,‘O for study of the structure and function of RNA,” and for synthesis of various oligoribonucleotides.‘2 RNases with angiogenic, immunosuppressive, antitumor, neurotoxic, and other actions have been reported.‘3*14 These enzymes may be good material for analysis of protein from the viewpoint of comparative biochemistry. i5, *6 It has been reported previously that T. harzianum OZ is a

good producer of a low molecular alkaline RNase when grown in liquid medium with a low content of phosphate. l7 Partial isolation and some basic characteristics of the RNase were described. 18 In the present paper, the extracellular RNase of T. harzianum was purified to homogeneity and some physicochemical properties and the specificity of the enzyme were investigated.

Materials and methods Organism

Enzyme and Microbial Technology 18:147-152, 1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

conditions

The fungus T. harzianum strain 01 was maintained on Czapek’s agar slants at 4°C. For a deep cultivation, a modified medium on the basis of Ogata’s medium was used.” Incubation was carried out in 750-ml Erlenmeyer flasks with 150 ml medium at 2YC on a rotary shaker for 72 h. The mycelium was removed by filtration and the filtrate was used as a source of enzyme. Enzyme

Address reprint requests to Dr. Evgenia S. Vasileva-Tonkova, Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev str., Bl. 26, BG-1113 Sofia, Bulgaria Received 13 October 1994; revised 26 April 1995; accepted 4 May 1995

and growth

and protein

assays

RNase, phosphatase, and phosphodiesterase activities were determined as described previously. ” Deoxyribonucleolytic activity was measured the same as RNase activity except that RNA was substituted by DNA in the reaction mixture. Protein concentrations were determined by using the method of

0141-0229/96/$15.00 SSDI 0141-0229(95)00095-M

Papers Whitaker and Granum19 or by measuring umn eluates at 280 nm.

the absorbance

of col-

Enzyme purification All purification

steps were performed

at 4°C.

Concentration of crude enzyme. The culture filtrate (5 I), after adjusting to pH 7.6, was brought to 90% (NH&SO, saturation. The suspension was retained for three days at 4°C and centrifuged at 5,000 rpm for 30 min. The precipitate was dissolved in 10 mM sodium phosphate buffer (pH 6.5) and dialyzed overnight against the same buffer. CM-cellulose chromatography. The dialyzed enzyme solution (660 ml) was applied to a CM-cellulose column (6.0 x 15 cm) equilibrated with 10 mM sodium phosphate buffer (pH 6.5). The column was washed with a 600 ml linear gradient of 0.014.5 M equilibration buffer. Fractions (10 ml) were collected and assayed for activity. Active fractions were combined and dialyzed overnight against the starting buffer. DEAEI-cellulose chromatography. The CM-cellulose pool (232 ml containing 10,556 U enzyme) was applied to a DEAE
Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) was carried out as described by LaemmliZo on a 12% (w/v) polyacrylamide slab gel. The proteins were stained using Coomassie Brilliant Blue R-250. Isoelectric focusing (IEF) was carried out in 7.5% polyacrylamide gels in the pH range of ampholytes 3.0-10.0 according to the method of Wrigley.2’ The gel (7.4 cm in length) was sliced into 2-mm slices. Protein was extracted from each band by incubating overnight with 1 ml of distilled water. The fractions were assayed for RNase activity; pH measurements of each fraction were made with a combination electrode.

Amino acid analysis The enzyme protein (1.8 mg) was dialyzed against distilled water, freeze-dried, and hydrolyzed with 6 M HCI in evacuated tubes for 24 h at 110°C. The amino acids in the hydrolysate were determined with a Biotronik LC-5001 amino acid analyzer.

148

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Microb. Technol.,

1996, vol. 18, February

Determination

of substrate specificity

The reaction mixture for degradation of synthetic polyribonucleotides (poly(N)) and the poly (I) poly(C) duplex by purified RNase (weight correlation E/S for poly(1) = l/560; for other poly(N) = 11125) was incubated at pH 8.0 and 37°C. Aliquots (0.1 ml) were taken at intervals, diluted with 0.1 ml water, and 0.1 ml of 0.75% uranyl acetate in 25% perchloric acid was added to precipitate unhydrolyzed substrate. The supematant fluids were diluted with distilled water and the absorbance at 260 nm was measured. To determine the ability of RNase to hydrolyze the 16 dinucleoside monophosphates (NpN’), a 0.6 x 10“ M concentration of NpN’ (where N is A, G, C, or U) was used. Kinetic measurements for GpN substrates were made using a Beckman 35 spectrophotometer at 25°C in 0.1 M sodium acetate buffer containing 0.2 M NaCl pH 6.0. Kinetic parameters were calculated by means of the Lineweaver-Burk plot. The enzyme concentration for all GpN substrates was 4.7 x lo-* M. The initial rates of substrate cleavage were determined as the increase in absorbance over time using changes in the molar coefficients of absorbance22s23 at pH 6.2 and 25°C. Thin-layer chromatography (TLC) of the reaction products was carried out on a Silufol plate (50 x 150 mm) using a solvent system of isopropanol, NH,OH, and water (7: 1:2, v/v/v) by the ascendent method24 for 3-4 h at 25°C. After the plates were air-dried, the spots were visualized with a UV lamp.

Chemicals DEAE-cellulose, poly(A), poly(C), poly(U), and poly(1) were from Reanal (Budapest, Hungary). NpN’ and CM-cellulose were from Serva (Heidelberg, Germany). 2’,3’-cyclophosphates (N > p) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Poly(1) poly(C) was from Boehringer Mannheim (Mannheim, Germany). Sephadex G-50 was from Pharmacia LKB Biotechnology (Uppsala, Sweden). All other chemicals used were reagent grade.

Results Purification RNase was purified from a 5 1 culture filtrate by using the purification procedure described in MATERIALSAND METHODS. The results obtained are presented in Table 1. During chromatography on CM-cellulose (Figure I), the bound RNase was eluted over a narrow range of buffer gradient (0.24-0.38 M). This step resulted in a high purification (506-fold) with a yield of 35%. After chromatography on DEAE-cellulose, a small fraction of the proteins with high RNase activity (84% of the enzyme units applied) was eluted in the flow-through. Purification was 2,750-fold and recovery of RNase activity was 30%. The final purification step was performed by gel filtration on Sephadex G-50. The RNase activity was found in the third peak of proteins eluted from the column (Figure 2). The final yield of enzyme activity was 18% with a specific activity of 4,230 representing 4,700-fold purification over the culture filtrate. The final preparation was free of acid and alkaline phosphatases, phosphodiesterase, and deoxyribonuclease activities. Analysis of the purified enzyme (designated as RNase The T. harziunum

ribonuclease:

Trichoderma Table 1

Purification

of T. harzianum

RNase

Step Culture filtrate Ammonium sulfate precipitate First CM-cellulose (gradient fraction) DEAE-cellulose (breakthrough fraction) Second CM-cellulose (gradient fraction) Sephadex G-50 The RNase from T. harzianum

E. Vasileva-Tonkova and S. Bezborodova

Protein O-ng)

RNase activity (U)

Specific activity (U mg-‘)

Purification (-fold)

Yield (%I

33.000.00 15,770.oo 23.20 3.60 3.10 1.26

30,000 24,000 10,556 8,910 8,091 5,330

0.9 1.5 455.0 2,475.0 2,610.O 4,230.O

1.0 1.7 506.0 2.750.0 2,900.o 4,700.o

100 80 35 30 27 18

was purified and assayed for protein concentration

77~)by SDS/PAGE revealed a single protein band (Figure 3), with molecular mass of about 11,000. The pH optimum for RNase Th activity was 8.0 with RNA as substrate and 6.0 with GpC as substrate using sodium acetate and Tris/HCl buffers. Isoelectric point and amino acid composition The p1 value of the purified enzyme was estimated by isoelectric focusing. The protein showed homogeneity giving a single peak of RNase activity with a p1 of 9.5. The ultraviolet absorption spectrum of the purified enzyme showed a maximum at 278 nm and a small shoulder at 285 nm, indicating a lack of tryptophan (not shown). The amino acid composition is reported in Table 2. It is characterized by the absence of methionine, the low content of tyrosine, and the high contents of glycine and alanine. The molecular mass calculated from this composition is 10,342. Substrate specificity

and activity as described in MATERIALS AND METHODS

side monophosphates (NpN’). Among the poly(N), RNase Th hydrolyzes only poly(1) and double-stranded polymer poly(1) f poly(C) (Figure 4). Poly(1) as the closest structural analog of poly(G) was chosen as substrate because of the tendency of poly(G) to aggregate in solutions. RNase Th do not split poly(A), poly(U), and poly(C). Among the 16 NpN’, RNase Th (E/S = 1:1,240) hydrolyzed only the series GpN. At concentrations of RNase 50fold higher than for GpN, cleavage of the other NpN’ substrates was not observed. The rate of degradation of GpC was fourfold higher than the rate for GpU (Table 3). The reaction products were analyzed by TLC on Silufol plates using standards for reference. Hydrolyzates of GpN (E/S = 1:30) by RNase Th were chromatographed after 20 mm, 35 min, 1 h, 2 h, 6 h, and 24 h incubations. Only two spots were observed from the start of the degradation of each GpN, corresponding to the 3’-GMP nucleotide and G > p. The spots corresponding to G > p decreased with time and disappeared within 24 h; only spots of 3’--GMP were observed at the end of the reactions which were more intensive than at the initial stage. Figure 5 shows TLC only

The base specificity of RNase Th was determined using three types of substrates: synthetic polyribonucleotides (poly (N)),2’,3’-cyclophosphates (N > p), and 16 dinucleo-

0

:

50

3 0.1 60

100 Fraction

140

180

number

Figure 1 Ion-exchange chromatography on a CM-cellulose column (6 x 15 cm) of T. harzianom RNase. The column was washed with 10 mM sodium phosphate buffer pH 6.5 (arrow 1) and was eluted with a 6OO-ml gradient of the same buffer O.Ol0.5 M pH 6.5 (arrow 2). Fraction size, 10 ml; l, absorbance at 280 nm of fractions; o, RNase activity with RNA as substrate

t

I R 30

I

I

60

Fraction

b

I 100

I

h 140

Jo

number

Figure 2 Gel filtration on a Sephadex G-50 column (2 x 95 cm) of T. harzianum RNase. For elution, 10 mM sodium phosphate buffer containing 20 mM NaCl pH 6.5 was used. Fraction size, 2.15 ml. Symbols same as in Figure 7

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Papers

0.5

0

1.0

Time

1.5

24

(h)

Figure 4 Rate of release of acid-soluble nucleotides from poly(N) substrates by RNase Th. RNase was incubated under optimal conditions (see MATERIALSAND METHODS)with the indicated polynucleotides. At the indicated time intervals, the reactions were stopped by an equal volume of 0.75% uranyl acetate in 25% HCIO,. Substrates: poly(l), l; polyfl) . PolyfC), o PO/~(A), poly(C), and poly(U) are not hydrolyzed by the enzyme

for GpC. In the parallel reaction of RNase Th with G > p and I > p, only one spot representing 3’-GMP (for G > p) and 3’-IMP (for I > p) was observed, indicating that RNase Th hydrolyzes G > p to 3’-GMP and I > p to 3’-IMP. A > p, U > p, and C > p were not hydrolyzed by RNase Th.

Figure 3 SDS/PAGE of T. harzianum RNase. Tracks 1 and 2 contained molecular weight markers: a-chymotrypsinogen mw = 24,000 (1) and cytochrome C, mw = 12,400 (2). Track 3 contained the purified RNase Th

Discussion Alkaline RNase (pH optimum 8.0) from the culture filtrate of the phytopathogenic fungus T. harzianum (RNase Th) Table 2

Amino acid composition

of some fungal guanyl-specific

RNases RNase

Amino acid

Th

Ch26

F,27

Arg LYs His Asp Glu Ser Thr GUY Ala Val Leu Ile Pro Met Half-cystine Phe Tyr Trp

3 4 3 8 4 12 8 15 14 6 2 4 4 0 4 5 6 ND

1 4 3 8 6 15 8 14 9 5 5 3 5 1 4 6 70 3

2 1 2 15 8 10 11 14 12 6 0 3 5 0 4 4 9 0

Total

102

110

106

T,**

C,29

AP,~’

Pb,3’

1 1 3 15 9 15 6 12 7 8 3 2 4 0 4 4 9 1

4 0 3 13 8 13 4 15 9 6 4 2 4 0 4 4 10 1

4 2 3 12 7 16 4 15 10 5 4 2 4 0 4 4 IO 1

1 2 3 14 5 11 9 10 11 7 3 4 4 0 4 5 9 0

104

104

107

102

All data are presented as residues molecule protein-’ ND, not determined

150

Enzyme Microb. Technol.,

1996, vol. 18, February

N,32

tJ,=

3 0 2 15 6 11 5 11 13 8 3 3 4 0 4 5 9 0

3 3 3 14 4 14 4 13 10 4 4 5 5 2 4 5 9 1

2 3 2 15 6 14 8 14 5 6 1 2 4 0 4 4 11 0

102

107

101

Pch,29

Trichoderma Table 3 Kinetic parameters of the cleavage cleoside phosphates by RNase Th

(M)

Vmax x lOA (M min-‘)

K,,, x lo3 (min-‘)

2.00 3.57 2.86 5.00

10.00 5.56 4.54 2.50

21.2 11.8 9.6 5.3

K,,, x ‘IO4 Substrate GPC GPA GPG GPU

reactions of dinu-

RNase Th was incubated with the indicated substrates (E/S = 1:1,240) at pH 6.0. I = 0.3 M, T = 25°C. and kinetic measurements were carried out as described in MATERIALSAND METHODS.The molecular rate constant (Kc,,) is the initial rate divided by the concentration of the enzyme (4.7 x 10” M)

purified to homogeneity with a yield of 18%. It was observed by SDS/PAGE that RNase Th is a monomer with a MW of 11,000. By amino acid analysis, a MW of 10,342 was obtained. All other extracellular fungal RNases have low MWs (in the range 10,000-14,000) and a pH optimum in the range 6.0-8.8.25 The p1 of T. harzianum RNase indicates that this enzyme is an alkaline protein. This unique high p1 of RNase Th contrasts sharply with the strongly acidic p1 of the other fungal RNases and is similar to p1 values of pancreatic RNases.15.25The p1 reflect the amino acid composition of the enzyme. Table 2 shows the amino acid composition of some guanyl-specific RNases. These enzymes have many similarities. They have two disulfide bridges, few basic amino acwas

Fro
of

the

solvent

ribonuclease:

E. Vasileva-Tonkova

and S. Bezborodova

and many aspartic acid, serine, and glycine residues. The tyrosine level is also relatively high among these RNases. RNase Th lacks methionine and tryptophan (from the shoulder of the UV spectrum) residues and belongs to RNases from Penicillium SP.,~~RNase F, from Fusarium moniliforme,27 and RNase U, from Ustilago sphaerogena.32 The protein has more basic amino acids (10 residues) and fewer acidic amino acids (12 residues) than other extracellular fungal RNases. In many RNases from various microbial sources, the cleavage of single-stranded RNA occurs as a two-step mechanism yielding 3’-nucleotides via 2’,3’-cyclic nucleotide intermediates. These RNases are “cyclizing RNases.” In view of base specificity, they are classified into three groups: guanine-specific, purine-specific, and nonspecific. RNase Th completely degraded only poly(I), slightly denatured poly(1) . poly(C), but did not affect the other poly(N) tested. Among the 16 NpN’, the enzyme hydrolyzed only the series GpN. As demonstrated by TLC with standards, the degradation product of GpN nucleotides was G > p which was converted to 3’-GMP. RNase Th hydrolyzes only G > p and I > p but not the other N > p. Thus, on the basis of the results obtained, RNase Th belongs to the group of guanyl-specific cyclizing RNases (EC 3.1.27.3, ribonucleate 3’-guanylo-oligonucleotido-hydrolase). They depolymerize single-stranded RNA through the cleavage of the P-5’0 ester bonds of GpN sequences in two separate stages: transphosphorylation and hydrolysis. The first step in the reaction consists of a transesterification and results in a terminal G > p. In a second step, this cyclic intermediate is hydrolyzed by RNase to give 3’-GMP. It was shown earlier that double-stranded polyribonucleotides are split by cyclizing RNases including guanylRNases because the enzyme binds opened nucleotides which appear spontaneously at the double helix.33 Comparison of the kinetic parameters for GpN substrates of RNase Th with those for the other guanylic RNases studied so far shows that they have similar values and depend on the nature of the nucleoside at its S’oxygen end: V,,,,, is highest for GpC because its K, is lowest (Table 3). The molecular rate constant (KC,) of RNase Th for GpC (21 x lo4 min-‘) was very similar to those of RNases N, and U,.25 ids,

References 1.

2.

t

start 0.3 Time

of

0.6

1

incllbation

2

55

24

3.

4.

(hours)

Figure 5 Thin-layer chromatography of products of the reaction of RNase Th with GpC. Standards used were 3’-GMP (1) and G > p (2)

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