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Production of 5′-ribonucleotides by enzymatic hydrolysis of RNA
Production of 5'-ribonucleotides by enzymatic hydrolysis of RNA M. D. Benaiges, J. Lopez-Santin and C. Sola Unitat d'Enginyeria Quimica, Facultat de Ci~ncies, Universitat AutOnoma de Barcelona, Bellaterra, (Barcelona), Spain
Study of optimal operational conditions for RNA enzymatic hydrolysis to obtain 5'-ribonucleotides has been carried out. RNA from brewer's yeasts, obtained by ammonium extraction, was hydrolysed by a partially purified 5'-phosphodiesterase from barley rootlets. Temperature of 60°C and pH 7 have been determined as the best operational conditions. Low RNA initial concentration (-0.1%) and reaction time (~1 h) have been identified as necessary to obtain a good yield of 5'-ribonucleotides.
Keywords: Enzymatichydrolysis;5'-phosphodiesterase; RNA; 5'-ribonucleotides
Introduction 5'-Ribonucleotides are high added value products used as flavor enhancers in food industries (5'-IMP, 5'-GMP) or having important applications in the pharmaceutical industry (5'-CMP, 5'-UMP, 5'-AMP). ~,2 These compounds can be obtained in several ways, but the aim of this work was to study the production of 5'-ribonucleotides by enzymatic hydrolysis of ribonucleic acid, selecting the operational conditions that give a high yield of 5'-ribonucleotides. ~ Enzymatic hydrolysis of RNA can be carried out by different enzymatic activities; however, in order to obtain 5'-ribonucleotides, it is necessary to work with a 5'-phosphodiesterase activity. The feasibility of a process of RNA hydrolysis is directly related to the availability of economical sources for both substrate and enzyme used. In this work, a partially purified 5'-phosphodiesterase activity from barley rootlets was used to hydrolyse RNA obtained from brewer's yeasts, because both raw materials are economical breweffs by-products. Although 5'-phosphodiesterase activity can be found in different sources, it is always found together with other enzymatic activities that become contaminating activities 4 Operational conditions for hydrolysis have to be selected depending on the degree of purifi-
Address reprint requests to Dr. Lopezat the Chemical Engineering Dept., UniversitatAut6nomade Barcelona,08193 Bellaterra, Barcelona, Spain Received 26 June 1988; revised I May 1989 86
Enzyme Microb. Technol., 1990, vol. 12, February
cation achieved in the enzymatic preparation and also on the intrinsic properties of the enzymes. Thus, RNA hydrolysis by enzymatic broth of Strept o m y c e s aureus (which is carried out by the cooperative action of an endo- and an exonucleolytic enzyme) is performed at two consecutive different conditions of the two enzymatic activities involved (i.e. temperature 42-65°C and pH 7-8). 5 Concerning the nuclease Pj from Penicillium citrinum (exonucleolytic enzyme), working conditions are constant and the temperature is 65°C and pH 5. 6 With 5'-phosphodiesterase activity from barley rootlets, depending on the purification degree of the final enzymatic solution, optimal temperature is considered to be between 55°C and 65°C and optimal pH about 6. 7 Referring to initial RNA concern tration, it is advised to hold these between 0.5% and 2% in all cases. The main purpose of this work is to develop an operational strategy yielding a product of high purity using a partially purified 5'-phosphodiesterase activity by minimizing the consecutive action of phosphomonoesterase, the main contaminating activity of the obtained enzymatic preparation.
Materials and methods Substrate, enzyme, and reagents
RNA was obtained by ammonia extraction from brewer's yeasts, as previously reported. 8 The source of 5'phosphodiesterase was barley rootlets, and the partial purification procedure, including extraction, acetone precipitation and DEAE-Sepharose chromatography, is reported in a previous paperfl © 1990 Butterworth Publishers
5'-Ribonucleotides by enzymatic hydrolysis of RNA: M. D. Benaiges et al. 5'-Phosphodiesterase and phosphomonoesterase activities were measured by using thymidine 5'-monophosphate p-nitrophenyl ester and p-nitrophenylphosphate as substrates. One unit of activity was defined as the amount of enzyme that produces 1 p,mol p-nitrophenol per minute under the assay conditions. 9 Nucleotides and nucleosides used as standards were from Sigma, 8-bromoguanosine from Chemical Dynamics Corporation, PIC A (tetrabutylammonium phosphate) for HPLC was from Waters S.A., and all other chemicals were reagent grade quality.
0.20
A
0.16
u
0.12
o
0.08
0.04
/ /
Optimal conditions study
2
4
6
8 Time
Optimal conditions for temperature and pH have been determined by initial rate of reaction tests, working with l ml RNA solution (0.5%), 1 ml enzymatic solution, 1 ml bromoguanosine (0.8 g 1- a) (as internal standard for HPLC), and 2 ml of the suitable buffer solution in each case (glycine-HC1, citrate-citric, Tris-HCl, glycine-OH). After 1 h of reaction, a sample was injected into the HPLC system.
Study of hydrolysis reaction Batch experiments were carried out in a stirred flask containing 4 ml of RNA solution, 4 ml of enzymatic solution, and 8 ml of buffer solution, working at 100 rev min-a. The initial RNA concentration was from 0.05% up to 1%. The flask was submerged in a constant temperature bath at the desired temperature. The development of the reaction was followed by HPLC analysis of 200-/~1 samples, adding 50 ~1 of bromoguanosine (0.8 g I a) as an internal standard.
Analysis of nucleotides and nucleosides The HPLC analysis was made with an HP-1090 chromatograph working with a Ca80DS-Hypersyl 5/., column by ion pairing chromatographic technique using 200/~M tetrabutylammonium phosphate as a PIC A reagent in 0.25% KH2PO 4 solution, a° Conditions were: flow rate 0.6 ml min- ', temperature 37°C, and pressure 220 atm. After about 10 analyses, the column was washed with distilled water and afterwards with ethanol-water (50 : 50) at the same flow rate for 1 h in order to avoid losses in column resolution. The detector used was a DAD (diode array detector) UV at a wavelength of 260 nm. In order to evaluate nucleotide and nuceloside concentrations, 8-bromoguanosine was used as an internal standard.
Results and discussion In order to determine optimal experimental conditions for the RNA hydrolysis reaction, the RNA used as substrate was extracted from brewer's yeasts by a procedure already described (see Materials and methods). The enzymatic preparation was a partially purified solution from barley rootlets (see Materials and methods), containing about 15 units of 5'-phosphodiesterase activity ml i and about 2 units of phosphomonoester-
,
14
•
,
16
•
•
•
18
(hours)
Figure 1 Evolution of 5'-ribonucleotide concentration over time at 1 g I 1 of initial substrate concentration at 60°C. (A) 5'-AMP; ([3) 5'-CMP; (A) 5'-GMP; (O) 5'-UMP
ase activity ml - a, as a main contaminating activity (this last activity is responsible for hydrolysis of 5'-ribonucleotide to nucleosides). The use of such a partially purified enzymatic preparation requires optimum reaction conditions to maximize the yield of 5'-ribonucleotides, by minimizing the concentration of nucleosides in the final product.
Optimal conditions study RNA hydrolysis tests were performed at different operational pHs between 3 and 10, although at pH 3, 4, and I0 the rate of reaction is extremely slow and the enzymatic activity can be considered as null. A value of about 7 was determined as the optimum pH in order to achieve a high yield in 5'-nucleotides. Temperature influence was studied at the optimum pH. The maximum activity was found at 60°C. At this temperature, the enzymatic activity exhibited a relatively low thermal deactivation and the selectivity obtained was higher than at lower temperatures.
RNA hydrolysis: study of the distribution curves RNA hydrolysis experiments were carried out at pH 7 and 60°C, working at initial substrate (RNA) concentrations in a range from 0.5 to 10g i a. The analysis of the initial rates of reaction showed a substrate inhibitory effect at initial substrate concentrations higher than 1.5
gl -l.
From the different RNA hydrolysis experiments, the results obtained with l g l-a of initial substrate concentration are shown in Figures I and 2, as an example. The behavior observed is similar to that seen at other initial substrate concentrations. From Figures I and 2, the following conclusions can be drawn from the distribution curves: Purine 5'-nucleotides are produced faster than pyrimidine ones, following the order 5'-AMP, 5'-GMP, 5'-UMP, 5'-CMP. No 2'- or 3'-nucleotides were detected in the analysis of hydrolysis products. Enzyme Microb. Technol., 1990, vol. 12, February
87
Papers tide deaminase able to degrade the adenosine (produced by 5'-phosphodiesterase) to inosine. The expected mechanism for the R N A hydrolysis that was studied consisted of two consecutive reactions: R N A 5'-phosphodiesterase ~ 5'-ribonucleotides phosphomonoesterase nucleosides 2
4
6
8 Time
16
18
20
(hours)
Figure 2 Evolution of nucleoside concentration over time with 1 g I 1 of initial substrate concentration at 60°C. (A) Ado; (1~) Cyd; (A) Guo; (©) Urd
80
This mechanism would lead to an initial rate ofnucleoside formation equal to zero. In fact, the experimental results exhibited a nonzero initial rate of reaction for the nucleosides (with the exception only of cytidine), suggesting a possible small contribution of a direct nucleoside formation from R N A to the overall reaction rate. The different behavior of cytidine can be due to a reduced number of cytosine terminals in the RNA
70
60
1.1
50
1.0
40
0.9
30
~z
0.8
0.7
20
0.6
10 >,
0.5
2
4
6
8
i0
Initial s u b s t r a t e c o n c e n t r a t i o n (g.l-I ) 0.4
Figure 3 Yield evolution with time and also with initial substrate concentration. Reaction times: (m) 0.5 h; (A) 1 h; (O) 1.5 h; (¢) 2 h (E~) 3 h
Nucleosides follow the same order of appearance as nucleotides, except adenosine (Ado), whose concentration decreases after reaching a maximum. The behavior of the concentration of Ado was checked by carrying out additional hydrolysis experiments using 5'-AMP as substrate. The results (not shown) confirmed the described pattern of change, suggesting the presence of an enzymatic activity nucleo-
88
Enzyme Microb. Technol., 1990, vol. 12, February
0 . 3"
0. 2'
2
4
6
8
i0
Initial s u b s t r a t e c o n c e n t r a t i o n (g.l-ll
Figure 4 Evolution of productivity with time and also with initial substrate concentration. Reaction times: ( , ) 0.5 h; (A) 1 h; (~) 1.5 h; (U) 2 h; (C]) 3 h
5'-Ribonucleotides by enzymatic hydrolysis of RNA: M. D. Benaiges et al. chain, which is in accordance with the lowest yield of 5'-CMP. Nevertheless, a principal mechanism could be described by the following scheme: ,.~ 5 ' - U M P RNA --'* 5'-CMP --~ 5'-GMP 5'-AMP
~ ~ ~ ~
Urd Cyd Guo Ado ~ Ino
RNA hydrolysis: selection of operational conditions F r o m different R N A hydrolysis batch reactions, the best operational conditions, such as reaction time and initial substrate concentration, should be selected. Several criteria can be used: concentration of products obtained, selectivity (grams 5'-nucleotides/grams R N A degraded), yield grams 5'-nucleotides/grams R N A initial), and also productivity (grams 5'-nucleotides/l h). T w o of these criteria were not suitable to select the operational conditions. Product concentrations obtained in different RNA hydrolysis were in all cases of the same order of magnitude, and selectivity is rather high working at any initial substrate concentration, about 90% at low reaction times. Concerning the yield and productivity, great differences have been observed, but as their trends are opposite, an intermediate position should be taken. Thus, while yield increases with time, productivity decreases, as can be seen from Figures 3 and 4. Taking into account the productivity, the maximum is moved decreasing from 1 g I-1 to 3 g 1 J of initial substrate concentration when the reaction time is increased. H o w e v e r , the yield increases with the time, and the maximum is observed near I g 1 ~ of initial substrate concentration, although it is necessary to work at high reaction times to get yields about 70%. Both criteria indicate an initial substrate concentration about 1 g 1-1, which corresponds to the absence of substrate inhibition effect, and fix the reaction time between 0.5 h and 1.5 h. The final accurate selection of reaction time should be made taking into account an economic criterion, that is, to maximize the overall benefit of the process, after its optimization.
Conclusions 5'-Ribonucleotides can be obtained, as a main product, from yeast R N A by a partially purified 5'-phosphodiesterase from barley rootlets working at optimal operational conditions: 60°C and p H 7. Substrate inhibition is observed above 1.5 g 1-1 o f the initial substrate concentration. High yields and productivity are obtained working at low R N A concentration (1 g 1-1) and reaction time about 1 h. Selectivity of 5'-ribonucleotides at these conditions is about 90%, which is high enough to consider this process suitable for producing 5'-ribonucleotides.
Nomenclature Ado Cyd Guo Ino RNA Urd 5'-AMP 5'-CMP 5'-GMP 5'-UMP
adenosine cytidine guanosine inosine ribonucleic acid uridine adenosine 5'-monophosphate cytidine 5'-monophosphate guanosine 5'-monophosphate uridine 5'-monophosphate
References 1
Sinsky, A. J. in Organic" Chemicals from Biomass (Wise, D. L., ed.) Benjamin Cummings, Menlo Park, CA, 1983, pp.
2
Nakao Y. in Microbial Technology 1 (Peppier, H. J. and Perlman, D., eds.) Academic Press, New York, 1979, pp. 311-354 Gutcho, S. in Nucleotides and Nucleosides (Noys Data Corporation, Park Ridge, NJ, 1970), 84-90 Walsh, C. in Enzymatic Reaction Mechanisms 1 W. H. Freeman and Company, San Francisco, 1978, 184-209 Crueger, W. and Crueger, A. in Biotechnology: A Textbook o f Industrial Microbiology (Brock, T. D., ed.) Sinauer Associates, Sunderland, MA, 1984 Linn, S. M. in Nucleases (Roberts, R. J., ed.) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982 Ikenaga, H. and Horie, Y. Dept. Res. Lab. Kirin Brewery Co. Ltd. 1978, 21, 11 Andreu, G., Benaiges, M. D., L6pez-Santin, J. and Sol~t, C. Biotechnol. Bioeng. 1988 32, 927 Benaiges, M. D., L6pez-Santin, J. and Sol,t, C. Enzyme Microb. Technol. 1989, 11, 444-451 Zakaria, M. and Brown, P. R. J. Chromatogr 1981, 226, 267
1-68
3 4 5 6 7 8 9 10
Enzyme Microb. Technol., 1990, vol. 12, February
89
Study of optimal operational conditions for RNA enzymatic hydrolysis to obtain 5'-ribonucleotides has been carried out. RNA from brewer's yeasts, obtained by ammonium extraction, was hydrolysed by a partially purified 5'-phosphodiesterase from barley rootlets. Temperature of 60°C and pH 7 have been determined as the best operational conditions. Low RNA initial concentration (-0.1%) and reaction time (~1 h) have been identified as necessary to obtain a good yield of 5'-ribonucleotides.
Keywords: Enzymatichydrolysis;5'-phosphodiesterase; RNA; 5'-ribonucleotides
Introduction 5'-Ribonucleotides are high added value products used as flavor enhancers in food industries (5'-IMP, 5'-GMP) or having important applications in the pharmaceutical industry (5'-CMP, 5'-UMP, 5'-AMP). ~,2 These compounds can be obtained in several ways, but the aim of this work was to study the production of 5'-ribonucleotides by enzymatic hydrolysis of ribonucleic acid, selecting the operational conditions that give a high yield of 5'-ribonucleotides. ~ Enzymatic hydrolysis of RNA can be carried out by different enzymatic activities; however, in order to obtain 5'-ribonucleotides, it is necessary to work with a 5'-phosphodiesterase activity. The feasibility of a process of RNA hydrolysis is directly related to the availability of economical sources for both substrate and enzyme used. In this work, a partially purified 5'-phosphodiesterase activity from barley rootlets was used to hydrolyse RNA obtained from brewer's yeasts, because both raw materials are economical breweffs by-products. Although 5'-phosphodiesterase activity can be found in different sources, it is always found together with other enzymatic activities that become contaminating activities 4 Operational conditions for hydrolysis have to be selected depending on the degree of purifi-
Address reprint requests to Dr. Lopezat the Chemical Engineering Dept., UniversitatAut6nomade Barcelona,08193 Bellaterra, Barcelona, Spain Received 26 June 1988; revised I May 1989 86
Enzyme Microb. Technol., 1990, vol. 12, February
cation achieved in the enzymatic preparation and also on the intrinsic properties of the enzymes. Thus, RNA hydrolysis by enzymatic broth of Strept o m y c e s aureus (which is carried out by the cooperative action of an endo- and an exonucleolytic enzyme) is performed at two consecutive different conditions of the two enzymatic activities involved (i.e. temperature 42-65°C and pH 7-8). 5 Concerning the nuclease Pj from Penicillium citrinum (exonucleolytic enzyme), working conditions are constant and the temperature is 65°C and pH 5. 6 With 5'-phosphodiesterase activity from barley rootlets, depending on the purification degree of the final enzymatic solution, optimal temperature is considered to be between 55°C and 65°C and optimal pH about 6. 7 Referring to initial RNA concern tration, it is advised to hold these between 0.5% and 2% in all cases. The main purpose of this work is to develop an operational strategy yielding a product of high purity using a partially purified 5'-phosphodiesterase activity by minimizing the consecutive action of phosphomonoesterase, the main contaminating activity of the obtained enzymatic preparation.
Materials and methods Substrate, enzyme, and reagents
RNA was obtained by ammonia extraction from brewer's yeasts, as previously reported. 8 The source of 5'phosphodiesterase was barley rootlets, and the partial purification procedure, including extraction, acetone precipitation and DEAE-Sepharose chromatography, is reported in a previous paperfl © 1990 Butterworth Publishers
5'-Ribonucleotides by enzymatic hydrolysis of RNA: M. D. Benaiges et al. 5'-Phosphodiesterase and phosphomonoesterase activities were measured by using thymidine 5'-monophosphate p-nitrophenyl ester and p-nitrophenylphosphate as substrates. One unit of activity was defined as the amount of enzyme that produces 1 p,mol p-nitrophenol per minute under the assay conditions. 9 Nucleotides and nucleosides used as standards were from Sigma, 8-bromoguanosine from Chemical Dynamics Corporation, PIC A (tetrabutylammonium phosphate) for HPLC was from Waters S.A., and all other chemicals were reagent grade quality.
0.20
A
0.16
u
0.12
o
0.08
0.04
/ /
Optimal conditions study
2
4
6
8 Time
Optimal conditions for temperature and pH have been determined by initial rate of reaction tests, working with l ml RNA solution (0.5%), 1 ml enzymatic solution, 1 ml bromoguanosine (0.8 g 1- a) (as internal standard for HPLC), and 2 ml of the suitable buffer solution in each case (glycine-HC1, citrate-citric, Tris-HCl, glycine-OH). After 1 h of reaction, a sample was injected into the HPLC system.
Study of hydrolysis reaction Batch experiments were carried out in a stirred flask containing 4 ml of RNA solution, 4 ml of enzymatic solution, and 8 ml of buffer solution, working at 100 rev min-a. The initial RNA concentration was from 0.05% up to 1%. The flask was submerged in a constant temperature bath at the desired temperature. The development of the reaction was followed by HPLC analysis of 200-/~1 samples, adding 50 ~1 of bromoguanosine (0.8 g I a) as an internal standard.
Analysis of nucleotides and nucleosides The HPLC analysis was made with an HP-1090 chromatograph working with a Ca80DS-Hypersyl 5/., column by ion pairing chromatographic technique using 200/~M tetrabutylammonium phosphate as a PIC A reagent in 0.25% KH2PO 4 solution, a° Conditions were: flow rate 0.6 ml min- ', temperature 37°C, and pressure 220 atm. After about 10 analyses, the column was washed with distilled water and afterwards with ethanol-water (50 : 50) at the same flow rate for 1 h in order to avoid losses in column resolution. The detector used was a DAD (diode array detector) UV at a wavelength of 260 nm. In order to evaluate nucleotide and nuceloside concentrations, 8-bromoguanosine was used as an internal standard.
Results and discussion In order to determine optimal experimental conditions for the RNA hydrolysis reaction, the RNA used as substrate was extracted from brewer's yeasts by a procedure already described (see Materials and methods). The enzymatic preparation was a partially purified solution from barley rootlets (see Materials and methods), containing about 15 units of 5'-phosphodiesterase activity ml i and about 2 units of phosphomonoester-
,
14
•
,
16
•
•
•
18
(hours)
Figure 1 Evolution of 5'-ribonucleotide concentration over time at 1 g I 1 of initial substrate concentration at 60°C. (A) 5'-AMP; ([3) 5'-CMP; (A) 5'-GMP; (O) 5'-UMP
ase activity ml - a, as a main contaminating activity (this last activity is responsible for hydrolysis of 5'-ribonucleotide to nucleosides). The use of such a partially purified enzymatic preparation requires optimum reaction conditions to maximize the yield of 5'-ribonucleotides, by minimizing the concentration of nucleosides in the final product.
Optimal conditions study RNA hydrolysis tests were performed at different operational pHs between 3 and 10, although at pH 3, 4, and I0 the rate of reaction is extremely slow and the enzymatic activity can be considered as null. A value of about 7 was determined as the optimum pH in order to achieve a high yield in 5'-nucleotides. Temperature influence was studied at the optimum pH. The maximum activity was found at 60°C. At this temperature, the enzymatic activity exhibited a relatively low thermal deactivation and the selectivity obtained was higher than at lower temperatures.
RNA hydrolysis: study of the distribution curves RNA hydrolysis experiments were carried out at pH 7 and 60°C, working at initial substrate (RNA) concentrations in a range from 0.5 to 10g i a. The analysis of the initial rates of reaction showed a substrate inhibitory effect at initial substrate concentrations higher than 1.5
gl -l.
From the different RNA hydrolysis experiments, the results obtained with l g l-a of initial substrate concentration are shown in Figures I and 2, as an example. The behavior observed is similar to that seen at other initial substrate concentrations. From Figures I and 2, the following conclusions can be drawn from the distribution curves: Purine 5'-nucleotides are produced faster than pyrimidine ones, following the order 5'-AMP, 5'-GMP, 5'-UMP, 5'-CMP. No 2'- or 3'-nucleotides were detected in the analysis of hydrolysis products. Enzyme Microb. Technol., 1990, vol. 12, February
87
Papers tide deaminase able to degrade the adenosine (produced by 5'-phosphodiesterase) to inosine. The expected mechanism for the R N A hydrolysis that was studied consisted of two consecutive reactions: R N A 5'-phosphodiesterase ~ 5'-ribonucleotides phosphomonoesterase nucleosides 2
4
6
8 Time
16
18
20
(hours)
Figure 2 Evolution of nucleoside concentration over time with 1 g I 1 of initial substrate concentration at 60°C. (A) Ado; (1~) Cyd; (A) Guo; (©) Urd
80
This mechanism would lead to an initial rate ofnucleoside formation equal to zero. In fact, the experimental results exhibited a nonzero initial rate of reaction for the nucleosides (with the exception only of cytidine), suggesting a possible small contribution of a direct nucleoside formation from R N A to the overall reaction rate. The different behavior of cytidine can be due to a reduced number of cytosine terminals in the RNA
70
60
1.1
50
1.0
40
0.9
30
~z
0.8
0.7
20
0.6
10 >,
0.5
2
4
6
8
i0
Initial s u b s t r a t e c o n c e n t r a t i o n (g.l-I ) 0.4
Figure 3 Yield evolution with time and also with initial substrate concentration. Reaction times: (m) 0.5 h; (A) 1 h; (O) 1.5 h; (¢) 2 h (E~) 3 h
Nucleosides follow the same order of appearance as nucleotides, except adenosine (Ado), whose concentration decreases after reaching a maximum. The behavior of the concentration of Ado was checked by carrying out additional hydrolysis experiments using 5'-AMP as substrate. The results (not shown) confirmed the described pattern of change, suggesting the presence of an enzymatic activity nucleo-
88
Enzyme Microb. Technol., 1990, vol. 12, February
0 . 3"
0. 2'
2
4
6
8
i0
Initial s u b s t r a t e c o n c e n t r a t i o n (g.l-ll
Figure 4 Evolution of productivity with time and also with initial substrate concentration. Reaction times: ( , ) 0.5 h; (A) 1 h; (~) 1.5 h; (U) 2 h; (C]) 3 h
5'-Ribonucleotides by enzymatic hydrolysis of RNA: M. D. Benaiges et al. chain, which is in accordance with the lowest yield of 5'-CMP. Nevertheless, a principal mechanism could be described by the following scheme: ,.~ 5 ' - U M P RNA --'* 5'-CMP --~ 5'-GMP 5'-AMP
~ ~ ~ ~
Urd Cyd Guo Ado ~ Ino
RNA hydrolysis: selection of operational conditions F r o m different R N A hydrolysis batch reactions, the best operational conditions, such as reaction time and initial substrate concentration, should be selected. Several criteria can be used: concentration of products obtained, selectivity (grams 5'-nucleotides/grams R N A degraded), yield grams 5'-nucleotides/grams R N A initial), and also productivity (grams 5'-nucleotides/l h). T w o of these criteria were not suitable to select the operational conditions. Product concentrations obtained in different RNA hydrolysis were in all cases of the same order of magnitude, and selectivity is rather high working at any initial substrate concentration, about 90% at low reaction times. Concerning the yield and productivity, great differences have been observed, but as their trends are opposite, an intermediate position should be taken. Thus, while yield increases with time, productivity decreases, as can be seen from Figures 3 and 4. Taking into account the productivity, the maximum is moved decreasing from 1 g I-1 to 3 g 1 J of initial substrate concentration when the reaction time is increased. H o w e v e r , the yield increases with the time, and the maximum is observed near I g 1 ~ of initial substrate concentration, although it is necessary to work at high reaction times to get yields about 70%. Both criteria indicate an initial substrate concentration about 1 g 1-1, which corresponds to the absence of substrate inhibition effect, and fix the reaction time between 0.5 h and 1.5 h. The final accurate selection of reaction time should be made taking into account an economic criterion, that is, to maximize the overall benefit of the process, after its optimization.
Conclusions 5'-Ribonucleotides can be obtained, as a main product, from yeast R N A by a partially purified 5'-phosphodiesterase from barley rootlets working at optimal operational conditions: 60°C and p H 7. Substrate inhibition is observed above 1.5 g 1-1 o f the initial substrate concentration. High yields and productivity are obtained working at low R N A concentration (1 g 1-1) and reaction time about 1 h. Selectivity of 5'-ribonucleotides at these conditions is about 90%, which is high enough to consider this process suitable for producing 5'-ribonucleotides.
Nomenclature Ado Cyd Guo Ino RNA Urd 5'-AMP 5'-CMP 5'-GMP 5'-UMP
adenosine cytidine guanosine inosine ribonucleic acid uridine adenosine 5'-monophosphate cytidine 5'-monophosphate guanosine 5'-monophosphate uridine 5'-monophosphate
References 1
Sinsky, A. J. in Organic" Chemicals from Biomass (Wise, D. L., ed.) Benjamin Cummings, Menlo Park, CA, 1983, pp.
2
Nakao Y. in Microbial Technology 1 (Peppier, H. J. and Perlman, D., eds.) Academic Press, New York, 1979, pp. 311-354 Gutcho, S. in Nucleotides and Nucleosides (Noys Data Corporation, Park Ridge, NJ, 1970), 84-90 Walsh, C. in Enzymatic Reaction Mechanisms 1 W. H. Freeman and Company, San Francisco, 1978, 184-209 Crueger, W. and Crueger, A. in Biotechnology: A Textbook o f Industrial Microbiology (Brock, T. D., ed.) Sinauer Associates, Sunderland, MA, 1984 Linn, S. M. in Nucleases (Roberts, R. J., ed.) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982 Ikenaga, H. and Horie, Y. Dept. Res. Lab. Kirin Brewery Co. Ltd. 1978, 21, 11 Andreu, G., Benaiges, M. D., L6pez-Santin, J. and Sol~t, C. Biotechnol. Bioeng. 1988 32, 927 Benaiges, M. D., L6pez-Santin, J. and Sol,t, C. Enzyme Microb. Technol. 1989, 11, 444-451 Zakaria, M. and Brown, P. R. J. Chromatogr 1981, 226, 267
1-68
3 4 5 6 7 8 9 10
Enzyme Microb. Technol., 1990, vol. 12, February
89