Editorial for Special Issue "Yeasts Biochemistry and Biotechnology".

Yeasts, both Saccharomyces and non-conventional strains, are currently the focus of active research due to their impressive applications in biotechnological bioprocesses, such as being hosts to produce recombinant proteins or main actors of the fermentation industries [...].

Effects of Sand Surface Plyometric and Sprint Training on Physical and Technical Skill Performance in Beach Handball Players.

This study compared the effects of a 6-week combined plyometric and sprint-training program on the sand to regular preseason training, on the athletic performance and technical actions of beach handball (BH) players. Athletes were randomly assigned either to the control (CG, n = 12; BH training only) or the experimental group (EG, n = 12; plyometric + sprint + BH training). Assessments conducted before and after the training period included a squat jump, a countermovement jump, the Abalakov jump, a 15-m sprint, a modified Course-Navette endurance test, and four sport-specific BH throwing speed tests: a standing penalty throw, a 3-step running throw, a jump throw, and a 360º jump throw. The training intervention enhanced all athletic performance measures (all, p < 0.05). In contrast, the only improvement in the CG included endurance performance (p< 0.05). Significant time-group differences were noted in favor of the EG compared to the CG (p< 0.05) in the squat jump, the countermovement jump, the Abalakov jump, the jump throw velocity and 360º jump throw velocity. In conclusion, compared to BH regular training, 6 weeks of sand surface preseason plyometric and sprint training combined with regular BH training induced greater improvements in athletic performance and specific skills in BH players.

AMWEst, a new thermostable and detergent-tolerant esterase retrieved from the Albian aquifer.

A fosmid library was constructed with the metagenomic DNA from the high-temperature sediment-rich water of the Albian aquifer (Algeria). Functional screening of this library was subsequently done looking for genes encoding lipolytic enzymes. We identified a novel gene named AMWEst (1209 base pairs) encoding a protein of 402 amino acids with a predicted molecular weight of 43.44 kDa and conferring esterase activity. AMWEst was successfully overexpressed in the yeast mesophilic host Saccharomyces cerevisiae, and the expression system used proved to be efficient and produced sufficient activity for its biochemical characterization. Multiple sequence alignment indicated that AMWEst contained a conserved pentapeptide motif (Gly120-His121-Ser122-Gln123-Gly124). The optimum pH and temperature of the recombinant esterase AMWEst were 8 and 80 °C, respectively. Additionally, AMWEst showed higher activity towards short carbon substrates and showed maximum activity for p-nitrophenyl hexanoate (C6). Notably, AMWEst has a remarkable thermostability, and the enzyme retains almost maximum activity at 70 °C after incubation for 1 h. Moreover, enzyme activity was enhanced by high concentrations of SDS and Triton X-100 detergents. KEY POINTS: • A novel thermostable esterase has been retrieved through functional metagenomics • The esterase is detergent-tolerant, which is attractive for some applications • The esterase can be expressed in a yeast mesophilic host to enhance its yield.

Effect of a High-Intensity Handball-Specific Fatigue Protocol Focused on the Leg Contralateral to the Throwing Arm on Interlimb Asymmetries.

ABSTRACT: Janicijevic, D, Pérez-Castilla, A, Miras-Moreno, S, Ortega-Becerra, M, Morenas-Aguilar, MD, Smajla, D, Sarabon, N, and García-Ramos, A. Effect of a high-intensity handball-specific fatigue protocol focused on the leg contralateral to the throwing arm on interlimb asymmetries. J Strength Cond Res 37(7): 1382-1389, 2023-This study aimed to elucidate which countermovement jump (CMJ) variant (unilateral or bilateral) is more sensitive to detect the decrement in kinetic CMJ-derived variables of the leg more actively involved in a handball-specific fatigue protocol. Seventeen female handball players (age: 20.6 ± 2.5 years) performed a fatigue protocol consisting of 8 repetitions of the following circuit separated by 10 seconds: 10 m sprint, 180° change of direction with the leg contralateral to the throwing arm, 10 m sprint with ball reception at 7 m, and handball throw preceded by a jump over a 40 cm hurdle with the leg contralateral to the throwing arm. Before and after the fatigue protocol, 6 unilateral CMJs (3 with each leg) and 3 bilateral CMJs were performed on a dual Kistler force platform (model 9260AA6). Bilateral CMJ height was reduced by 5.4% after the fatigue protocol (p < 0.001). However, the fatigue protocol did not promote any significant change in peak force, mean force, and propulsive impulse or in their asymmetry values (17 of 18 comparisons). The decrement of 6.1% during the unilateral CMJ in the propulsive impulse developed by the leg ipsilateral to the throwing arm (less involved in the fatigue protocol) was the only variable that reached statistical significance (p = 0.038). The pre-post fatigue changes in asymmetry values presented negligible correlations between both CMJ variants (rs = 0.01 to -0.19). These results suggest that neither unilateral nor bilateral CMJs are able to detect changes in interlimb asymmetries after a high-intensity handball-specific fatigue protocol.

Novel Microbial Enzymes with Industrial Applications.

Eberhardt et al [...].

Insights on Microbial Communities Inhabiting Non-Volcanic Hot Springs.

The northwest of Spain has an abundance of non-volcanic hot springs that, until recently, had only been used for thermalism activities. One of such hot springs, Muiño da Veiga, has now been explored using metagenomics to study the microbial community that inhabits these high-temperature circumneutral continental waters. Sequencing of the metagenome allowed the characterization of its composition, diversity, metabolic connections and potential as a source for thermozymes, as well as its ability to assemble MAGs. A diverse microbial community dominated by Bacteria domain members was revealed, particularly from the early-branching Aquificales group. The most abundant genus was Sulfurihydrogenibium, known for its implication in sulfur cycling and for forming mats that enable novel niches. The variety of primary producers with autotrophic pathways (and specifically the sulfur oxidizing pathway) expands the range of available nutrients, and the increase in biomass forms thicker mats, resulting in more available niches and broader microbial diversity. Nonetheless, certain metabolic pathways were attributed to less abundant members of the microbial community, reinforcing the idea that the rare biosphere plays important roles in the network of interactions present in an ecosystem and acts as genetic reservoirs. In addition, three of the assembled MAGs represent novel microbial diversity found in this hot spring. Moreover, the presence of enzymes and microorganisms with possible biotechnological applications was confirmed, including proteases, lipases and cell-wall degrading enzymes, pointing to the potential for the hot spring as a source for thermozymes.

Characterization of a novel thermophilic metagenomic GH5 endoglucanase heterologously expressed in Escherichia coli and Saccharomyces cerevisiae.

BACKGROUND: Endoglucanases from thermophilic microorganisms are a valuable resource as they can be used in a wide variety of biotechnological applications including the valorisation of biomass and the production of biofuels. In the present work we analysed the metagenome from the hot spring Muiño da Veiga, located in the northwest of Spain (in the Galicia region), in search for novel thermostable endoglucanases. RESULTS: Sequence analysis of the metagenome revealed a promising enzyme (Cel776). Predictions on protein structure and conserved amino acid sequences were conducted, as well as expression in heterologous systems with Escherichia coli and Saccharomyces cerevisiae as the host. Cel776Ec was correctly expressed and purified by taking advantage of the His-Tag system, with a yield of 0.346 U/mL in the eluted fraction. Cel776Sc was expressed extracellulary and was easily recovered from the supernatant without the need of further purification, requiring only a concentration step by ultrafiltration, with a significantly higher yield of 531.95 U/mL, revealing a much more suitable system for production of large amounts of the enzyme. Their biochemical characterization revealed biotechnologically interesting enzymes. Both Cel776Ec and Cel776Sc had an optimal temperature of 80 °C and optimal pH of 5. Cel776Ec exhibited high thermostability maintaining its activity for 24 h at 60 °C and maintained its activity longer than Cel776Sc at increasing incubation temperatures. Moreover, its substrate specificity allowed the degradation of both cellulose and xylan. Whereas Cel776Ec was more active in the presence of calcium and magnesium, manganese was found to increase Cel776Sc activity. A stronger inhibitory effect was found for Cel776Ec than Cel776Sc adding detergent SDS to the reaction mix, whereas EDTA only significantly affected Cel776Sc activity. CONCLUSIONS: Our study reports the discovery of a new promising biocatalyst for its application in processes, such as the production of biofuel and the saccharification of plant biomass, due to its bifunctional enzymatic activity as an endoglucanase and as a xylanase, as well as highlights the advantages of a yeast expression system over bacteria.

Bioprospecting for Thermozymes and Characterization of a Novel Lipolytic Thermozyme Belonging to the SGNH/GDSL Family of Hydrolases.

Functional screenings were conducted on two metagenomic libraries from hot springs in order to find novel thermozymes with potential biotechnological applications. These included enzymes acting on plant cell walls such as endoglucanases and exoglucanases, ß-glucosidases, xylanases, and ß-xylosidases, and broad application enzymes such as proteases and lipolytic hydrolases. Of all the enzymes found by this bioprospection, we selected a novel lipolytic enzyme for further characterization. The protein was found to belong to the SGNH/GDSL family of hydrolases. It was purified and its biochemical parameters determined. We found that the enzyme was most active at 60 °C and pH 9 using pNP-laurate as substrate and was highly thermostable. It also showed preference for short-chained substrates and activation with temperature and with certain detergents such as Tween 80. Proteins of this family of hydrolases are relevant for their broad substrate specificity, that coupled with this protein's high temperature optima, broad pH range, and thermostability further highlights its biotechnological potential.

Comparative Metagenomic Analysis of Two Hot Springs From Ourense (Northwestern Spain) and Others Worldwide.

With their circumneutral pH and their moderate temperature (66 and 68°C, respectively), As Burgas and Muiño da Veiga are two important human-use hot springs, previously studied with traditional culture methods, but never explored with a metagenomic approach. In the present study, we have performed metagenomic sequence-based analyses to compare the taxonomic composition and functional potential of these hot springs. Proteobacteria, Deinococcus-Thermus, Firmicutes, Nitrospirae, and Aquificae are the dominant phyla in both geothermal springs, but there is a significant difference in the abundance of these phyla between As Burgas and Muiño da Veiga. Phylum Proteobacteria dominates As Burgas ecosystem while Aquificae is the most abundant phylum in Muiño da Veiga. Taxonomic and functional analyses reveal that the variability in water geochemistry might be shaping the differences in the microbial communities inhabiting these geothermal springs. The content in organic compounds of As Burgas water promotes the presence of heterotrophic populations of the genera Acidovorax and Thermus, whereas the sulfate-rich water of Muiño da Veiga favors the co-dominance of genera Sulfurihydrogenibium and Thermodesulfovibrio. Differences in ammonia concentration exert a selective pressure toward the growth of nitrogen-fixing bacteria such as Thermodesulfovibrio in Muiño da Veiga. Temperature and pH are two important factors shaping hot springs microbial communities as was determined by comparative analysis with other thermal springs.

Creencias maleables y esperanza en apoyo a concesiones del acuerdo con las FARC-EP en un contexto polarizado en Colombia / Malleable Beliefs and Hope in Supporting Concessions under the Agreement with FARC-EP amidst a Polarized Context in Colombia

Resumen Introducción: Este estudio analizó el papel de creencias maleables y esperanza en la disposición para apoyar concesiones del acuerdo con las FARC-EP en un contexto de polarización política. Método: Se desarrolló un estudio no experimental con 562 ciudadanos. Resultados: Los resultados confirman que la esperanza de paz surge de una percepción general del mundo como cambiante; y por lo tanto, las personas que creen que el conflicto colombiano con las FARC-EP puede cambiar, desarrollan una actitud esperanzadora, presentando más apoyo a concesiones sobre la Jurisdicción Especial para la Paz (JEP) y la participación en política de exguerrilleros. Asimismo, se encontró que las personas con orientación política de izquierda y de centro apoyan más las concesiones del acuerdo en comparación con personas con orientación política de derecha. Conclusiones: El estudio refleja que la polarización política constituye un obstáculo para el desarrollo de creencias maleables y actitudes esperanzadoras que favorecen la desescalada del conflicto. Se presentan las implicaciones prácticas sobre la formación de actitudes que favorecen salidas constructivas del conflicto, y sobre la responsabilidad de actores políticos en la formación de creencias rígidas que constituyen barreras para la paz.

Abstract Introduction: This study analyzed the role of malleable beliefs and hope in the willingness to support controversial concessions of the peace agreement with FARC-EP, in a context of political polarization. Method: Non-experimental study with 562 citizens. Results: The results confirm that hope for peace arises from a general perception of the world as changing, and, therefore, people who believe that the Colombian conflict with FARC-EP can change develop a hopeful attitude, presenting more support for concessions on the Special Justice for Peace (JEP), and the participation of ex-guerrillas in politics. Likewise, results showed that people with a left-leaning political orientation, and those that identified as centrist, are more supportive of the concessions of the agreement, compared to people with right-wing political leanings. Conclusions: The study reflects that political polarization constitutes an obstacle to the development of malleable beliefs and hopeful attitudes that favor the de-escalation of the conflict. The practical implications are presented on the formation of attitudes that favor constructive solutions to the conflict, and on the responsibility of political actors in the formation of rigid beliefs that constitute barriers to peace.

The HMGB Protein KlIxr1, a DNA Binding Regulator of Kluyveromyces lactis Gene Expression Involved in Oxidative Metabolism, Growth, and dNTP Synthesis.

In the traditional fermentative model yeast Saccharomyces cerevisiae, ScIxr1 is an HMGB (High Mobility Group box B) protein that has been considered as an important regulator of gene transcription in response to external changes like oxygen, carbon source, or nutrient availability. Kluyveromyces lactis is also a useful eukaryotic model, more similar to many human cells due to its respiratory metabolism. We cloned and functionally characterized by different methodologies KlIXR1, which encodes a protein with only 34.4% amino acid sequence similarity to ScIxr1. Our data indicate that both proteins share common functions, including their involvement in the response to hypoxia or oxidative stress induced by hydrogen peroxide or metal treatments, as well as in the control of key regulators for maintenance of the dNTP (deoxyribonucleotide triphosphate) pool and ribosome synthesis. KlIxr1 is able to bind specific regulatory DNA sequences in the promoter of its target genes, which are well conserved between S. cerevisiae and K. lactis. Oppositely, we found important differences between ScIrx1 and KlIxr1 affecting cellular responses to cisplatin or cycloheximide in these yeasts, which could be dependent on specific and non-conserved domains present in these two proteins.

The force-velocity profile as determinant of spike and serve ball speed in top-level male volleyball players.

Understanding the relationship between mechanical variables derived from actions such as jumping, sprinting, or ballistic bench press throwing and sport-specific performance moves is of scientific and practical interest for strength and conditioning coaches for improving training programs. We examined the association between mechanical variables derived from the force-velocity (FV) profiles of the aforementioned actions and spike and serve ball speeds in elite volleyball players. Twenty-two male elite volleyball players (age: 24.3 ± 4.5 years; height: 1.89 ± 0.06 m; body mass: 86.3 ± 8.6 kg) were tested in two sessions. Squatting, sprinting, and bench press throwing FV profiles were determined in the first session, while spike and serve ball speeds were assessed in the second session. The theoretical maximal force (F0) of vertical jumping, the theoretical maximal velocity of sprinting, and the F0 of bench press throwing in ascending order, were strongly associated (rs range 0.53-0.84; p<0.05) with spike and serve ball speeds. These mechanical variables explained 20%-36% of the variability in spike and serve ball speeds, with a greater influence on the serve speed. These results suggest that assessing jumping, sprinting, and bench press throwing force-velocity profiles might help provide player-specific training programs and optimize performance in these technical-tactical actions in male elite volleyball players.

Biochemical and Structural Characterization of a novel thermophilic esterase EstD11 provide catalytic insights for the HSL family.

A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60°C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester. The tridimensional structures of EstD11 and the inactive mutant have been determined at atomic resolutions. Structural and bioinformatic analysis, confirm that EstD11 belongs to the family IV, the hormone-sensitive lipase (HSL) family, from the α/ß-hydrolase superfamily. The canonical α/ß-hydrolase domain is completed by a cap domain, composed by two subdomains that can unmask of the active site to allow the substrate to enter. Eight crystallographic complexes were solved with different substrates and reaction products that allowed identification of the hot-spots in the active site underlying the specificity of the protein. Crystallization and/or incubation of EstD11 at high temperature provided unique information on cap dynamics and a first glimpse of enzymatic activity in vivo. Very interestingly, we have discovered a unique Met zipper lining the active site and the cap domains that could be essential in pivotal aspects as thermo-stability and substrate promiscuity in EstD11.

Effects of Cluster Set Configuration on Mechanical Performance and Neuromuscular Activity.

ABSTRACT: Ortega-Becerra, M, Sánchez-Moreno, M, and Pareja-Blanco, F. Effects of cluster set configuration on mechanical performance and neuromuscular activity. J Strength Cond Res 35(2): 310-317, 2021-The aim of this study was to compare the effects of different cluster set (CS) configurations on mechanical performance and electromyography (EMG) activity during the bench press (BP) exercise. Fourteen strength-trained men (age 23.0 ± 2.4 years; height 1.76 ± 0.08 m; body mass 78.3 ± 12.2 kg) performed 3 different protocols in the BP exercise consisting of 3 sets of 12 repetitions at 60% of 1 repetition maximum with interset rests of 2 minutes, differing in the set configuration: (a) traditional sets (TRDs), (b) cluster sets of 4 repetitions (CS4), and (c) cluster sets of 2 repetitions (CS2). Intraset rests of 30 seconds were interposed for CS protocols. The mean propulsive values of force, velocity, and power output were measured for every repetition by synchronizing a linear velocity transducer with a force platform. The root mean square (RMS) and median frequency (MDF) for pectoralis major (PM) and triceps brachii (TB) muscles were also recorded for every repetition. Force, velocity, and power values progressively increased as the number of intraset rests increased (TRD < CS4 < CS2). The CS2 protocol exhibited lower RMS-PM than CS4 and TRD for almost all sets. In addition, TRDs showed significantly lower MDF-TB than CS2 for all sets and lower MDF-TB than CS4 during the third set. In conclusion, more frequent intraset rests were beneficial for maintaining mechanical performance, which may be mediated, from a neuromuscular perspective, by lesser increases in EMG amplitude and attenuated reductions in EMG frequency.

Analysis of the Load-Velocity Relationship in Deadlift Exercise.

The aim of this study was to analyze the relationship between movement velocity and relative load (%1RM) in the deadlift exercise. Fifty men (age = 23.8 ± 3.6 years, body mass = 78.2 ± 8.3 kg, height = 1.78 ± 0.06 m) performed a first evaluation (T1) consisting of a one-repetition maximum (1RM) test. Forty-two subjects performed a second evaluation (T2) after 6 weeks. Mean (MV), mean propulsive (MPV) and peak (PV) velocity measures of the concentric phase were analyzed. Load-velocity relationships were studied by fitting first order equations to the data using loads from 30-100% of 1RM. A comprehensive set of statistics for assessing bias and level of agreement to estimate the 1RM value from the different models was used. Stability of these relationships was assessed using the coefficient of variation (CV) and the intraclass correlation coefficient (ICC). General load-velocity equations provided good adjustments (R2 ~; 0.91-0.93), however individual load-velocity regressions provided better adjustments (R2 ~; 0.97). Individual estimations also showed higher agreement and more regular variation than general equations. Moreover, MPV showed smaller bias than the other velocity parameters (MV and PV). The stability analysis of the load-velocity relationships resulted in ICC values higher than 0.82 and CV lower than 3.0%. Monitoring repetition velocity allows estimation of the %1RM in the deadlift exercise. More accurate predictions of relative load can be obtained when using individualized regression equations instead of general equations.

Effects of velocity loss in the bench press exercise on strength gains, neuromuscular adaptations, and muscle hypertrophy.

OBJECTIVE: This study aimed to compare the effects of four velocity-based training (VBT) programs in bench press (BP) between a wide range of velocity loss (VL) thresholds-0% (VL0), 15% (VL15), 25% (VL25), and 50% (VL50)-on strength gains, neuromuscular adaptations, and muscle hypertrophy. METHODS: Sixty-four resistance-trained young men were randomly assigned into four groups (VL0, VL15, VL25, and VL50) that differed in the VL allowed in each set. Subjects followed a VBT program for 8-weeks using the BP exercise. Before and after the VBT program the following tests were performed: (a) cross-sectional area (CSA) measurements of pectoralis major (PM) muscle; (b) maximal isometric test; (c) progressive loading test; and (d) fatigue test. RESULTS: Significant group x time interactions were observed for CSA (P < .01) and peak root mean square in PM (peak RMS-PM, P < .05). VL50 showed significantly greater gains in CSA than VL0 (P < .05). Only the VL15 group showed significant increases in peak RMS-PM (P < .01). Moreover, only VL0 showed significant gains in the early rate of force development (RFD, P = .05), while VL25 and VL50 improved in the late RFD (P ≤ .01-.05). No significant group × time interactions were found for any of the dynamic strength variables analyzed, although all groups showed significant improvements in all these parameters. CONCLUSION: Higher VL thresholds allowed for a greater volume load which maximized muscle hypertrophy, whereas lower VL thresholds evoked positive neuromuscular-related adaptations. No significant differences were found between groups for strength gains, despite the wide differences in the total volume accumulated by each group.

Physical and Physiological Demands During Handball Matches in Male Adolescent Players.

This study aimed to describe the physical and physiological demands of adolescent handball players and compare movement analysis and exercise intensities between the first and second halves and between the different periods of the match. Fourteen adolescent handball players (age 15.7 ± 0.8 years, body mass: 65.6 ± 3.4 kg, body height: 169.5 ± 3.9 cm), played two friendly matches, in which no substitutions were made. The analysis was carried out with a Global Positioning System technology. The following physical variables were analyzed: Total distance covered (TD); distance covered at faster velocities than 18 km·h-1 (TDC>18km·h-1); number of accelerations (Accel) and decelerations (Decel); number of accelerations and decelerations higher than 2.78 m·s-2 (Accel>2.78 m·s-2 and Decel>2.78 m·s-2); number of sprints (Sprints); accelerations interspersed with a maximum of 30 s between them (RAS≤30s) and as a physiological variable the heart rate (HR) was examined. Significant differences (p < 0.01 -p < 0.001) between the first and the second half in all variables mentioned were observed, except in Accel>2.78 m·s-2 and Decel>2.78 m·s-2. This trend was also observed when comparing performance between the different 10-min periods. The 5th period (period 40-50 min) was the one that showed differences with respect to the previous ones. Adolescent handball players showed lower levels of exercise intensity, assessed by both time-motion and HR data, in the second half of matches, especially in the middle of this period.

Velocity Loss as a Critical Variable Determining the Adaptations to Strength Training.

PURPOSE: This study aimed to compare the effects of four resistance training (RT) programs with different velocity loss (VL) thresholds: 0% (VL0), 10% (VL10), 20% (VL20), and 40% (VL40) on sprint and jump performance, muscle strength, neuromuscular, muscle hypertrophy, and architectural adaptations. METHODS: Sixty-four young resistance-trained men were randomly assigned into four groups (VL0, VL10, VL20, and VL40) that differed in the VL allowed in each set. Subjects followed an RT program for 8 wk (two sessions per week) using the full-squat (SQ) exercise, with similar relative intensity (70%-85% 1-repetition maximum), number of sets (3), and interset recovery period (4 min). Before and after the RT program, the following tests were performed: 1) muscle hypertrophy and architecture of the vastus lateralis (VLA), 2) tensiomyography, 3) 20-m running sprint, 4) vertical jump, 5) maximal voluntary isometric contraction in SQ, 6) progressive loading test in SQ, and 7) fatigue test. RESULTS: No between-group differences existed for RT-induced gains in sprint, jump, and strength performance despite the differences in the total volume performed by each group. VL20 and VL40 showed significant increases (P < 0.001) in muscle hypertrophy (group-time interaction, P = 0.06). However, only VL40 exhibited a significant slowing (P < 0.001) of the delay time in the VLA muscle (group-time interaction, P = 0.05). Moreover, VL40 showed a significant decrease in the early rate of force development (P = 0.04). CONCLUSIONS: Higher VL thresholds (i.e., VL20 and VL40) maximized hypertrophic adaptations, although an excessive VL during the set (i.e., VL40) may also induce negative neuromuscular adaptations. Therefore, moderate VL thresholds should be chosen to maximize strength adaptations and to prevent negative neuromuscular adaptations.

Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides.

BACKGROUND: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. RESULTS: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-D-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with ß-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. CONCLUSIONS: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.