ORIGINAL ARTICLE This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Braz. J. Food Technol., Campinas, v. 24, e2020246, 2021 | https://doi.org/10.1590/1981-6723.24620 1/13 5'-Ribonucleotides production using 5'- phosphodiesterase from spent malt roots Produção de 5'-ribonucleotídeos utilizando 5'-fosfodiesterase da radícula de malte cervejeiro Edson Marcelino Alves1* , Joyce Faria de Souza1 , Szymon Macieja1, Pedro de Oliva Neto1 1Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Faculdade de Ciências e Letras de Assis, Departamento de Biotecnologia, Assis/SP - Brasil *Corresponding author: Edson Marcelino Alves, Universidade Estadual Paulista Júlio de Mesquita Filho, Faculdade de Ciências e Letras de Assis, Departamento de Biotecnologia, Av. Dom Antônio, 2100, CEP: 19806- 900, Assis/SP - Brasil, e-mail: edsonmaralves@hotmail.com Cite as: Alves, E. M., Souza, J. F., Macieja, S., & Oliva Neto, P. (2021). 5'-Ribonucleotides production using 5'- phosphodiesterase from spent malt roots. Brazilian Journal of Food Technology, 24, e2020246. https://doi.org/10.1590/1981-6723.24620 Abstract 5'-ribonucleotides are high value-added molecules widely used in the food and pharmaceutical industries because of their bioactive properties. The present work aims to produce a composition of 5’-ribonucleotides using spent brewer’s yeast as cheap source of RNA, and barley malt rootlets as cheap source of 5'-phosphodiesterase (5'-PDE). This is a very promising and innovative strategy because both spent yeast and malt rootles are residues of the brewing process and are closely linked in a cycle that until now is not yet commercially exploited due to lack of studies. Our results showed that extraction of 5’-PDE was mainly influenced by the fineness of the rootlets and amount of extraction solvent (water). The main molecules formed during RNA hydrolysis were 5’-ribonucleotides, which represented 85.86% of the total hydrolyzed molecules. Finally, the results of the proposed approach can generate a new perspective for the brewing industry regarding the management of its wastes, generating from them products of high added value and with a wide range of applications. Keywords: Brewing industry wastes; Spent brewer’s yeast; Barley malt rootlets; RNA hydrolysis; Enzyme kinetics; Bioprocess optimization. Resumo 5'-ribonucleotídeos são moléculas de alto valor agregado amplamente utilizadas nas indústrias alimentícia e farmacêutica, em razão de suas propriedades bioativas. O presente trabalho visa produzir uma composição de 5'- ribonucleotídeos a partir de levedura residual cervejeira, como fonte de RNA, e radícula de malte cervejeiro, como fonte barata de 5'-fosfodiesterase (5'-PDE). A abordagem proposta mostrou-se promissora e com caráter inovador, pois ambas, levedura e radícula de malte, são resíduos do processo cervejeiro, estando intimamente ligadas em um ciclo que até o momento ainda não foi explorado comercialmente por falta de estudos. Os resultados do presente trabalho mostraram que a extração de 5'-PDE foi influenciada principalmente pelo tamanho das radículas e pela quantidade de solvente de extração (água). As principais moléculas formadas durante a hidrólise de RNA foram 5'- ribonucleotídeos, os quais representaram 85,86% do total de moléculas hidrolisadas. Por fim, os resultados deste https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0002-3633-0824 https://orcid.org/0000-0002-5225-1528 5'-Ribonucleotides production using 5'-phosphodiesterase from spent malt roots Alves, E. M. et al. Braz. J. Food Technol., Campinas, v. 24, e2020246, 2021 | https://doi.org/10.1590/1981-6723.24620 2/13 trabalho podem gerar uma nova perspectiva para a indústria cervejeira no que se refere ao gerenciamento de seus resíduos, a fim de gerar a partir deles produtos de alto valor agregado e com ampla gama de aplicações. Palavras-chave: Resíduos da indústria cervejeira; Levedura residual cervejeira; Radícula de malte de cevada; Hidrólise de RNA; Cinética enzimática; Otimização de bioprocessos. 1 Introduction 5'-ribonucleotides and nucleosides are high value-added molecules and are widely used in the food and pharmaceutical industries because of their bioactive properties (Jiao et al., 2019). Nucleosides are composed of a nitrogenous base (purine or pyrimidine) and a pentose (ribose for RNA or 2'-deoxyribose for DNA). When one or more phosphate groups are present, the compound is known as nucleotide and is the most important class of nucleic acid derivatives (Trelles et al., 2019). Phosphodiester bonds connect the 5' carbon of one nucleotide to the 3' carbon of another nucleotide forming polymers known as polynucleotides or nucleic acids. The selective chemical or enzymatic hydrolysis of ribonucleic acids produces a composition of nucleosides and nucleotides. The process to be chosen for RNA hydrolysis deserves due attention because it directly affects the characteristics of the molecules produced. Chemical RNA hydrolysis can be carried out under alkaline conditions but is considered limited because RNA is degraded to lower molecular weight compounds without any specificity (Smernik et al., 2015). In this process, ribonucleotides with 2'- and 3'-phosphate groups are obtained mostly. These molecules show no flavoring characteristics and are of little commercial interest. 5'-ribonucleotides, on the other hand, are involved in several basic biological processes and are considered high value-added molecules. The production of 5'-ribonucleotides requires the hydrolysis of the crude RNA by means of a very specific enzyme, 5'-phosphodiesterase (5'-PDE). This enzyme hydrolyzes the RNA ester bonds at the 3' position, promoting the release of 5'-ribonucleotides in which the phosphate group is attached to the 5' carbon of the pentose molecule (Luo et al., 2017). Cohn and Volkin (1953) were the first to demonstrate the presence of 5'-PDE activity in snake venom. Despite its high efficiency, it cannot be used in the agri-food industry for obvious reasons. Other important sources of 5'-PDE include certain fungi such Penicillium citrinum (Chen et al., 2019), certain species of Halobacterium (Boonpan et al., 2015) and Aspergillus sp. (Luo et al., 2017). However, this type of enzyme production requires industrial fermentation and quite complex purification steps, which ultimately results in an expensive enzyme. 5'-PDE from Penicillium citrinum, for example, is available commercially at Amano Enzyme among others, under the name Nuclease RP-1G (or EC3, 1.30.1). Because of its very high cost, this enzyme is mainly used on an industrial scale in an immobilized form. Its use in a process for producing yeast extracts enriched in 5'-ribonucleotides induces very large additional enzyme costs. Animal tissues are other sources of 5’-PDE (Morelli et al., 2011), which can also be obtained from germs of some plants, such as rootlets of oats, barley, wheat, corn, rye, millet, sorghum rice and malt rootlets (Xu et al., 2013). Compared with many other sources, barley malt rootlets are by-products of the brewing industry and, therefore, cheaper materials for the preparation of 5’-PDE and additionally, there is no doubt about the safety of the enzyme. According to Zou et al. (2008), 5'-PDE can be obtained by simple mixing malt root powder in an aqueous solution to extract the enzyme. Barley rootlets are good source of this enzyme among others including deoxyribonuclease, ribonuclease and adenosine-5’-phosphomono-esterase. As previously mentioned, this enzyme can be obtained from a variety of other sources such as plant (Beluhan et al., 2020), animal (Cesarini et al., 2020) and microbial (Okado et al., 2016). However, considering economic aspects, preparations of 5'-PDE from a cheap source residue such malt roots is very promising. Besides, it can serve as good raw material for large-scale production of 5’-PDE. 5'-Ribonucleotides production using 5'-phosphodiesterase from spent malt roots Alves, E. M. et al. Braz. J. Food Technol., Campinas, v. 24, e2020246, 2021 | https://doi.org/10.1590/1981-6723.24620 3/13 Based on the explanations above, the present work aims to study the optimal conditions for 5’-PDE extraction from spent malt rootlets as well as its application for hydrolyzing RNA form spent brewer’s yeast. It was also objective to determine the enzyme characteristics, including optimal pH and temperature, heat stability and Michaelis’ constant (Km). 2 Material and methods 2.1 Extraction of 5′-PDE Malt roots were kindly provided by Agrária (Cooperativa Agrária Agroindustrial, Guarapuava, PR, Brazil). Approximately 1 kg of malt roots were sieved and fractioned according to their granulometry (Tamis mesh 16- 100, Bertel, Caieras, Brazil). The influence of granulometry and water proportion for the enzyme extraction was evaluated. Malt rootlets (1 g) of the different granulometry were soaked with 6 ml distilled water at 20 °C for 20 min to extract crude 5’-PDE. For the water-rootlet proportion assay, 1g of malt rootlet were soaked with water in the following proportions (w/w): 4:1, 8:1, 12:1, 16:1 and 20:1. After extraction, the samples were filtrated with gauze and centrifuged for 10 minutes at 956 x G (Heraeus Megafuge 16R, Thermo Scientific, Osterode am Harz, Germany). The supernatant was collected and used for 5’-PDE activity assays. Finally, the total protein content of the 5'-PDE extracts was determined according to the classical Kjeldahl method described by AOAC (Association of Official Agricultural Chemists, 2000), thus evaluating the specific activity of the enzyme. 2.2 5'-PDE activity assay The 5'-PDE activity was determined as described by Fujimoto et al. (1974). It consists of the measure of the absorbance at 260 nm of nucleotides molecules released after RNA hydrolysis by the enzyme. Test tubes (13x100 mm) containing 0.9 ml of RNA solution 1% (w/v) (Sigma-R6625) in acetate buffer solution 0.125 M pH 5.3 and 3 mM Zn2+ ion were incubated in a water bath (MA-093, Marconi, Piracicaba, Brazil) at 69 °C for 10 minutes to equilibrate the temperature. Then, 100 μl of crude enzyme solution was added and the tubes were incubated for 15 minutes at 69 °C with shaking every 5 minutes. After incubation, 2.0 ml of ammonium molybdate solution 0.25% (m/v) and perchloric acid 2.5% (v/v) (kept in a refrigerator) were added to inactivate the reaction and precipitate the unhydrolyzed RNA. The tubes were then incubated in an ice bath for 10 minutes and then centrifuged for 10 minutes at 956 x G (Heraeus Megafuge 16R, Thermo Scientific, Osterode am Harz, Germany). The supernatant was collected and diluted 50-fold with deionized water and the absorbance was measured at 260 nm in a spectrophotometer (UV-M51, Bel Photonics, Piracicaba, Brazil) using quartz cuvettes with 1 cm of optical path. The blank was prepared by substituting 100 μl of enzyme solution with deionized water in the reaction mixture, followed by the same procedures. The activity of the enzyme was calculated according to Equation 1. ( )* * . * . * 260nm 260nmA Test A Blank 2 50U5 PDE mL 10 6 0 1 15 ′ −  =    (1) Where: 2 = volume (ml) of stopped reaction; 50 = dilution factor; 10.6 = millimolar extinction coefficient ξ (µmol/ml) of hydrolyzed ribonucleic acid at 260 nm; 0.1 = volume (ml) of enzyme used and 15 = time (in minutes) of assay as per the Unit Definition. One unit of 5'-PDE activity was defined as the amount of enzyme required to hydrolyze 1 μmol of substrate per minute. 2.3 5′-PDE characterization To determine the optimum reaction temperature, the enzyme activity was performed under the same conditions previously mentioned, changing only the temperature values to 30, 40, 45, 50, 55, 60, 65, 70, 80 and 90 °C. 5'-Ribonucleotides production using 5'-phosphodiesterase from spent malt roots Alves, E. M. et al. Braz. J. Food Technol., Campinas, v. 24, e2020246, 2021 | https://doi.org/10.1590/1981-6723.24620 4/13 To determine the optimal pH conditions of the reaction, the following 0.125 mol/L buffers were used: glycine-HCl (for pH values in the range of 2.0 to 3), sodium acetate (for pH values in the range of 4.0 to 5.5) and sodium phosphate (for pH values in the range of 6.0 to 8). The enzyme activity was determined as previously described altering only the buffer solution and the pH during the reaction. The enzyme Michaelis’ constant (Km) was determined by varying the initial concentration of RNA between 0.1 and 30 mg/ml during the reaction. The kinetic model proposed by Michaelis-Menten with substrate inhibition (Equation 2) was adjusted to the experimental results of enzyme activities during the reaction as a function of substrate concentration by means of nonlinear regression using MATLAB software R2017b (Mathworks®), so that the inhibition constant Ki was also determined. *max 2 m i V SV SK S K = + + (2) The effect of heating on the thermostability of 5'-PDE was investigated at 60, 65 and 70 °C for different periods up to 24 hours. 2.4 Enzymatic RNA hydrolysis Enzymatic RNA hydrolysis for 5'-ribonucleotides production was performed by incubating RNA from spent brewer's yeast with the crude enzyme extract from malt rootlet. Approximately 10 g of centrifuged spent brewer’s yeast, having a moisture content of 75%, were weighed and then added water (25.8 ml) so that the autolysis was performed at a ratio of yeast/water (w/v) of 7.5%, on yeast dry basis. After homogenization, the cell suspension was subjected to autolysis for 60 minutes at temperature 60 °C and then centrifugated for 10 minutes at 956 x G (Heraeus Megafuge 16R, Thermo Scientific, Osterode am Harz, Germany). The RNA hydrolysis assay was performed by mixing the supernatant (7.2 ml) with the crude enzyme (0.8 ml) for up to 24 hours. Samples were collected at intervals for 5’-ribonucleotides and ribonucleosides content determination. 2.5 5’-Ribonucleotides and ribonucleosides determination High Performance Liquid Chromatography (HPLC) was used to verify and quantify the 5’-ribonucleotides and ribonucleosides formed during RNA hydrolysis. Standard solutions containing 10μg/ml of 5'-GMP (G8377-5G, Sigma-Aldrich Brazil Ltda), 5'-CMP (C1006-5G, Sigma-Aldrich Brazil Ltda), 5'-UMP (U6375-5G, Sigma- Aldrich Brazil Ltda.), 5'-IMP (I4625-5G, Sigma-Aldrich Brazil Ltda), 5'-AMP (01930-5G, Sigma-Aldrich Brazil Ltda), Guanosine (G6752-25G, Sigma-Aldrich Brazil Ltda), Cytidine (C4654-5G, Sigma-Aldrich Brazil Ltda), Uridine (U3750-1G, Sigma-Aldrich Brazil Ltda), Inosine (I4125-5G, Sigma-Aldrich Brazil Ltda) and Adenosine (A9251-5G, Sigma-Aldrich Brazil Ltda) were prepared and used for standard curve preparation. The separation of molecules was achieved on reverse phase column Acclaim C30 (2.1 x 150 mm, 3 μm) at 15 °C using an HPLC system (2475 - Multi λ Fluorecence Detector, Waters®) with UV detector at 260 nm and elution gradient. The mobile phase consisted of 3 components: A) deionized water; B) 100 mM ammonium acetate buffer, pH 5 and C) Methanol. The entire run lasted 28 minutes and the elution gradient events are detailed in Table 1. From this methodology, chromatograms of each compound were obtained to determine the retention time of the molecules. Table 1. Elution gradient. Time (min) Solvent A (%) Solvent B (%) Solvent C (%) Flux (mL/min) 0.0 80 20 0 0.25 3.0 80 20 0 0.40 5.0 70 20 15 0.40 8.5 40 20 40 0.25 15.0 40 20 40 0.25 15.1 80 20 0 0.25 23.0 80 20 0 0.25 23.1 80 20 0 0.40 Solvent A: deionized water; Solvent B: 100 mM ammonium acetate buffer, pH 5; Solvent C: Methanol. 5'-Ribonucleotides production using 5'-phosphodiesterase from spent malt roots Alves, E. M. et al. Braz. J. Food Technol., Campinas, v. 24, e2020246, 2021 | https://doi.org/10.1590/1981-6723.24620 5/13 3 Results and discussion 3.1 Enzyme extraction Figure 1 shows the visual appearance of the fractions obtained after malt rootlets sieving. The influence of granulometry, water proportion and time for the enzyme extraction are shown in Figure 2. Figure 1. Fractions obtained after granulometric separation of malt rootlets: (a) mesh>16, (b) 16