ß-glucosidases from Saccharomyces cerevisiae: production, protein precipitation, characterization, and application in the enzymatic hydrolysis of delignified sugarcane bagasse.

ß-glucosidase is an essential enzyme for the enzymatic hydrolysis of lignocellulosic biomass, as it catalyzes the final stage of cellulose breakdown, releasing glucose. This paper aims to produce ß-glucosidase from Saccharomyces cerevisiae and evaluate the enzymatic degradation of delignified sugarcane bagasse. S. cerevisiae was grown in yeast peptone dextrose medium. Partial purification of the enzyme was achieved through precipitating proteins with ethanol, and the optimal activity was measured by optimizing pH and temperature. The effects of ions, glucose tolerance, and heat treatment were evaluated. Delignified sugarcane bagasse was hydrolyzed by the enzyme. ß-glucosidase showed a specific activity of 14.0712 ± 0.0207 U mg-1. Partial purification showed 1.22-fold purification. The optimum pH and temperature were 6.24 and 54 °C, respectively. ß-glucosidase showed tolerance to glucose, with a relative activity of 71.27 ± 0.16%. Thermostability showed a relative activity of 58.84 ± 0.91% at 90 °C. The hydrolysis of delignified sugarcane bagasse showed a conversion rate of 87.97 ± 0.10% in the presence of Zn2+, an ion that promoted the highest increase in enzymatic activity. S. cerevisiae produced an extracellular ß-glucosidase with good stability at pH and temperatures conventionally applied in the hydrolysis of lignocellulosic biomass, showing viability for industrial application.

Antinociceptive and anti-inflammatory properties of α-D-mannan from the yeast Kluyveromyces marxianus: evidence for a role in interleukin-6 inhibition.

The management of inflammatory states typically involves non-steroidal anti-inflammatory drugs (NSAIDs) and opiates. Understanding the mechanisms underlying the processing of nociceptive information from potential alternatives such as some polysaccharides may enable new and meaningful therapeutic approaches. In this study, α-D-mannan isolated from the Kluyveromyces marxianus cell wall produced antinociceptive effects in models of inflammatory pain (formalin and complete Freund's adjuvant tests). Furthermore, α-D-mannan reduced paw edema and interleukin-6 (IL-6) production after carrageenan-induced inflammation. The polysaccharide α-D-mannan was characterized by gas chromatography-mass spectrometry, methylation analysis, and spectroscopic techniques. Moreover, the Doehlert experimental design was applied to find the optimal conditions for biomass production, with the best conditions being 10.8 g/L and 117 h for the glucose concentration and the fermentation time, respectively. These results indicate that α-D-mannan from K. marxianus exerts anti-inflammatory and antinociceptive effects in mice, possibly via a mechanism dependent on the inhibition of IL-6 production.

The improvement of guava (Psidium guajava) juice quality using crude multi-enzymatic extracts obtained from yeasts.

Guava juice is cloudy and viscous, which hinders filtration, decreases yield, and causes the loss of quality after its processing and during storage. This study aimed to evaluate enzymatic treatment effects using crude multi-enzymatic extracts (CME) obtained from Rhodotorula mucilaginosa, Rhodotorula orizycola, and Pseudozyma sp. produced by submerse fermentation in the extraction of juice guava. Mixtures of 100 ml of guava pulp and multi-enzymatic extracts proposed by Doehlert planning were incubated under constant agitation at 150 rpm and 50°C, and a Doehlert design was applied as a multivariate optimization strategy. The optimal conditions using the multi-enzymatic extract were: 0.4% (v/v) of CME for 131 min for the multi-enzymatic treatment using Pseudozyma sp.; 3.0% (v/v) of CME for 154 min using the R. mucilaginosa CME; and 5.0% (v/v) of CME for 90 min using R. oryzicola. The maximum viscosity reduction values for the juices treated with the CME of yeasts were 10.33%, 86.38%, and 13.33% for the juices treated with the CME of Pseudozyma sp., R. mucilaginosa, and R. orizycola, respectively. The physical-chemical properties were improved after treatment with CMEs, yielding a reduction of clarity, increase of total soluble solids and reducing sugars, and decreasing the acidity (pH) for all treatments with enzymatic extracts of all strains. The yeasts studied showed a potential for CME production to be applied to juice, improving the quality of the juice, and R. mucilaginosa was the most prominent yeast due to most significant reduction of viscosity in guava juice.

α-D-mannan from Aureobasidium pullulans (CCMB 324): optimization extraction.

Mannans has been attracted the interest in various sectors due to its promising applications. The low toxicity of mannans allows for their use in cosmetics, pharmaceutical, and biomedical industries. In this study, the α-D-mannan extraction conditions from Aureobasidium pullulans by alkaline extraction were optimized using a Box-Behnken design (BBD). The effect of temperature (°C), pH and extraction time (hours) on the yield of α-mannan was investigated. The conditions that produced the highest yield (26%) were a temperature of 92 °C, extraction time of 3 h and pH 13. In addition, the α-D-mannan structure was confirmed by methylation analysis, 1D and 2D NMR spectroscopic analysis, and GC-MS.

Immobilization of Fungal Cellulases Highlighting ß-Glucosidase: Techniques, Supports, Chemical, and Physical Changes.

ß-Glucosidase is widely used in several industrial segments, among which we can highlight the pharmaceutical industry, beverages, biofuels, animal feed production, and the textile industry. The great applicability of this enzyme, associated with the high cost of its production, justifies the need to find ways to make its use economically viable on an industrial scale. Through enzyme immobilization, the biocatalyst can be reused more than once, without great impact on its catalytic activity, and higher operational and storage stabilities can be achieved as compared to the free form. Accordingly, this review brings information about different techniques and supports that have been studied in the immobilization of cellulases with a focus on ß-glucosidase, as well as the application of these immobilized systems to supplement commercial mixtures.

Inulinase from Rhodotorula mucilaginosa: immobilization and application in the production of fructooligosaccharides.

The crude extract containing inulinase from Rhodotorula mucilaginosa was obtained by submerged fermentation. Inulinase was immobilized on chicken eggshell by physical adsorption and covalent crosslinking, using glutaraldehyde as a crosslinking reagent, and Celite by adsorption. Fructooligosaccharides production was performed using immobilized inulinase (5%, w/v) and inulin substrate solution under experimental conditions evaluated through Doehlert experimental design. The production of inulinase was optimized for concentrations of D-glucose and yeast extract at 12.5 and 0.5 g/L, respectively, resulting in an optimal activity of 0.62 U. The optimal pH and temperature for enzyme activity were 8.0 and 75 °C, respectively, leading to an optimal activity of 3.54 U. The highest immobilization efficiency (46.27%) was obtained upon immobilization on Celite. Immobilization by adsorption to eggshell allowed for specific activity of 4.15 U/g, and adsorption to Celite resulted in specific activity of 3.70 U/g. The highest titer in fructooligosaccharides was obtained with an initial inulin concentration of 250 g/L (25%, w/v), and a reaction time of 16 h. Hence, immobilized inulinase proved to be a promising catalyst for fructooligosaccharides production since the formulation is performed through a simple, low-cost, and large-scale applicable methodology.

Antinociceptive and anti-inflammatory activity of α-d-mannan from Pseudozyma sp.

Pseudozyma sp. are yeasts that are commercially important due to their production of glycolipid biosurfactants, squalene, itaconic acid, and exopolysaccharide. The search for other analgesia inducing drugs, such as opiates and non-steroidal anti-inflammatory drugs (NSAIDs), as alternatives is beneficial. In this study, the antinociceptive and anti-inflammatory actions of α-d-mannan were studied using acetic acid-induced writhing, open field test, formalin test, and carrageenan-induced paw oedema tests in mice. The α-d-mannan obtained from Pseudozyma sp. was confirmed by methylation analysis, 1D and 2D NMR spectroscopic analysis, and GC-MS. The results show that α-d-mannan from Pseudozyma sp. has analgesic and anti-inflammatory activities. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02635-1.

Clarification of tangerine juice using cellulases from Pseudoyma sp.

Tangerine juice was treated with crude extract containing cellulase from Pseudozyma sp. obtained by liquid fermentation. The thermal stability of cellulase was investigated by incubating crude extract at different temperatures and times. The pulp, obtained from tangerine, was pasteurized at 85 °C for 5 min and then used in a clarification process with a Doehlert experimental design. The results showed that the cellulase obtained from Pseudozyma sp. is thermostable at temperatures of 60, 70 and 90 °C and retained 98%, 88% and 80% of activity, respectively, after a 1-h incubation time. The optimum conditions for clarification were verified by varying the enzyme extract concentration (%, v v-1) and the time (minutes) in a shaker at 150 rpm, at 50 °C. The optimum condition for clarification was obtained in the 80th min with a 1.25% enzymatic extract concentration (v v-1), resulting in a reduction of tangerine juice viscosity by 65%. The analysis of physical and chemical parameters of tangerine juice after clarification showed that the enzyme extract improved the process responsible for the clarification of tangerine juice. The results are promising since this is a methodology that can be used in the citrus juice industry.

Production, characterization, and immobilization of protease from the yeast Rhodotorula oryzicola.

The protease was produced extracellularly in submerged fermentation by the yeast Rhodotorula oryzicola using different sources of nitrogen and maximum activity (6.54 × 10-3 U/mg) was obtained in medium containing 2% casein (w/v). Purification of the protease by gel filtration chromatography resulted in a 3.07-fold increase of specific protease activity. The optimal pH and temperature for enzyme activity were 6.51 and 63.04 °C, respectively. Incubation in the presence of some salts enhanced enzyme activity, which peaked under 0.01 M BaCl2 . The enzyme retained about 90% of enzymatic activity at temperatures 50-60 °C. The commercially available enzyme carriers evaluated, silica gel, Celite 545, and chitosan effectively immobilized the protease. The enzyme immobilized in Celite 545 retained 73.53% of the initial activity after 15 reuse cycles. These results are quite promising for large-scale production and immobilization of protease from R. oryzicola, as the high operational stability of the immobilized enzyme lowers production costs in biotechnological applications that require high enzymatic activity and stability under high temperatures.

Characterization and immobilization of protease secreted by the fungus Moorella speciosa.

Protease was extracellularly produced in submerged fermentation by the fungus Moorella speciosa with maximum activity of 8.6 × 103 U/mL. The optimal pH and temperature for enzyme activity were 6.78 and 60.88 °C, respectively. The enzyme was incubated in the presence of several ions at concentrations of 0.1 M and 0.01 M to address the effect on enzyme activity. Enzyme activity was increased by 56% and 130% in the presence of 0.1 M BaCl2 and of 0.01 M Na2SO4, respectively. The V max and K m values were 0.01474 U/min/mg protein and 0.04190 mg/mL, respectively. The enzyme retained about 90% of enzymatic activity at 90 °C. Among the methods tested for enzyme immobilization, adsorption onto MAT540 carrier led to the most promising results, since after 15 reuse cycles up to 60% of the initial catalytic activity was retained. Entrapment in calcium alginate matrix allowed to retain up to 51% of the initial catalytic activity after 8 reuse cycles. This protease from M. speciosa, in either free or immobilized form, can be foreseen as a useful biocatalytic tool in process design by reducing operating costs, decreasing the use of chemical processing and, consequently, meeting the global demand for clean technologies.

Production and partial characterization of ß-1,3-glucanase obtained from Rhodotorula oryzicola.

The current study aims to assess the kinetics of population growth of Rhodotorula oryzicola and the production of ß-1,3-glucanase (EC 3.2.1.39) enzyme by this yeast. It also aims to obtain the optimum conditions of ß-1,3-glucanase enzymatic activity by varying the pH as well as to study the enzyme thermostability. R. oryzicola population doubled within 12 hr. During this period, 9.26 generations were obtained, with 1 hr and 29 min of interval from one generation to the other, with specific growth rate (µ) of 0.15 (hr-1). The entire microorganism growth process was monitored during ß-1,3-glucanases production, and the maximum value was obtained in the stationary phase in the 48-hr fermentation period. pH and temperature optimum values were 4.7 and 96°C, respectively. The enzyme maintained 88% of its activity when submitted to the temperature of 90°C for an incubation period of 1 hr. The results show that the enzyme can be used in industrial processes that require high temperatures and acidic pH.

Kinetic and thermodynamic parameters, and partial characterization of the crude extract of tannase produced by Saccharomyces cerevisiae CCMB 520.

Tannase can be used in different industrial sectors such as in food (juices and wine) and pharmaceutical production (trimethoprim) because it catalyses the hydrolysis of hydrolysable tannins. The aim of the current study is to assess the tannase found in the crude extract of Saccharomyces cerevisiae CCMB 520, and to set its catalytic and thermodynamic properties. The enzyme was optimally active at pH 6.0 and temperature 30 °C. Tannase was activated by Na+, Ca2+, K+ at 5 × 10-3 mol/L. The half-life at 30 °C was 3465.7 min. The activation energy was 40.32 kJ/mol. The Gibbs free energy, enthalpy and entropy at 30 °C were 85.40, 48.10 and -0.12 kJ/mol K, respectively. Our results suggest that the tannase found in the crude extract of S. cerevisiae is an attractive enzyme for industrial applications, such as for beverage manufacturing and gallic acid production, due its catalytic and thermodynamic properties (heat-stable and resistant to metal ions).

Comparison between the univariate and multivariate analysis on the partial characterization of the endoglucanase produced in the solid state fermentation by Aspergillus oryzae ATCC 10124.

Endoglucanase production by Aspergillus oryzae ATCC 10124 cultivated in rice husks or peanut shells was optimized by experimental design as a function of humidity, time, and temperature. The optimum temperature for the endoglucanase activity was estimated by a univariate analysis (one factor at the time) as 50°C (rice husks) and 60°C (peanut shells), however, by a multivariate analysis (synergism of factors), it was determined a different temperature (56°C) for endoglucanase from peanut shells. For the optimum pH, values determined by univariate and multivariate analysis were 5 and 5.2 (rice husk) and 5 and 7.6 (peanut shells). In addition, the best half-lives were observed at 50°C as 22.8 hr (rice husks) and 7.3 hr (peanut shells), also, 80% of residual activities was obtained between 30 and 50°C for both substrates, and the pH stability was improved at 5-7 (rice hulls) and 6-9 (peanut shells). Both endoglucanases obtained presented different characteristics as a result of the versatility of fungi in different substrates.

Application of aqueous biphasic systems as strategy to purify tannase from Aspergillus tamarii URM 7115.

The aims of the current study are to assess the influence of polyethylene glycol (PEG) concentration, molar mass, pH, and citrate concentrations on aqueous biphasic systems based on 24 factorial designs, as well as to check their capacity to purify tannase secreted by Aspergillus tamarii URM 7115. Tannase was produced through submerged fermentation at 26°C for 67 h in Czapeck-Dox modified broth and added with yeast extract and tannic acid. The factorial design was followed to assess the influence of PEG molar mass (MPEG 600; 4,000 and 8,000 g/ mol), and PEG (CPEG 20.0; 22.0 and 24.0% w/w) and citrate concentrations (CCIT 15.0, 17.5, and 20.0%, w/w), as well as of pH (6.0, 7.0, and 8.0) on the response variables; moreover, partition coefficient (K), yield (Y), and purification factor (PF) were analyzed. The most suitable parameters to purify tannase secreted by A. tamarii URM 7115 through a biphasic system were 600 (g/mol) MPEG, 24% (w/w) CPEG, 15% (w/w) CCIT at pH 6.0 and they resulted in 6.33 enzyme partition, 131.25% yield, 19.80 purification factor and 195.08 selectivity. Tannase secreted by A. tamarii URM 7115 purified through aqueous biphasic systems composed of PEG/citrate can be used for industrial purposes, since it presents suitable purification factor and yield.

The biochemical characterization, stabilization studies and the antiproliferative effect of bromelain against B16F10 murine melanoma cells.

The current study aims to extract bromelain from different parts (stem, crown, peels, pulp and leaves) of Ananas comosus var. comosus AGB 772; to determine of optimum pH and temperature; to test bromelain stability in disodium EDTA and sodium benzoate, and to investigate its pharmacological activity on B16F10 murine melanoma cells in vitro. The highest enzymatic activity was found in bromelain extracted from the pulp and peel. The optimum bromelain pH among all studied pineapple parts was 6.0. The optimum temperature was above 50 °C in all bromelain extracts. The fluorescence analysis confirmed the stability of bromelain in the presence of EDTA and sodium benzoate. Bromelain was pharmacologically active against B16F10 melanoma cells and it was possible verifying approximately 100% inhibition of tumor cell proliferation in vitro. Since bromelain activity was found in different parts of pineapple plants, pineapple residues from the food industry may be used for bromelain extraction.

Structure-based drug design studies of UDP-N-acetylglucosamine pyrophosphosrylase, a key enzyme for the control of witches' broom disease.

BACKGROUND: The witches' broom disease is a plague caused by Moniliophthora perniciosa in the Theobroma cacao, which has been reducing the cocoa production since 1989. This issue motivated a genome project that has showing several new molecular targets, which can be developed inhibitors in order to control the plague. Among the molecular targets obtained, the UDP-N-acetylglucosamine pyrophosphorylase (UNAcP) is a key enzyme to construct the fungal cell wall. The inhibition of this enzyme results in the fungal cell death. RESULTS: The results show that the molecular recognition of the enzyme with the substrates occurs mainly by hydrogen bonds between ligands and Arg116, Arg383, Gly381, and Lys408 amino acids; and few hydrophobic interactions with Tyr382 and Lys123 residues. CONCLUSIONS: Among the compounds analyzed, the NAG5 showed the best binding energy (-95.2 kcal/mol). The next steps for the control of witches' broom plague involve the synthesis and biological evaluation of these compounds, which are in progress.

Antioxidant activity, ascorbic acid and total phenol of exotic fruits occurring in Brazil.

The antioxidant activity, ascorbic acid and phenolic content were studied in 10 exotic fruits from Brazil: abiu, acerola, wax jambu, cashew, mamey sapote, carambola or star fruit, Surinam cherry, longan, sapodilla and jaboticaba. The ascorbic acid was determined by 2,6-dichloroindophenol titrimetic methods and total phenols were measured colorimetrically using the Folin-Ciocalteu reagent. The antioxidant activity was investigated with three different methods: hypochlorous acid scavenging activity, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization assay, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging method. The highest content of vitamin C (1,525.00 mg/100 g pulp) occurred in acerola. The total phenol content was higher in abiu, acerola, Surinam cherry and sapodilla. In relation to antioxidant activity, acerola has showed the great values in all three different methods tested. It was found that the fruits have a significant antioxidant effect when tested by each method, respectively, and these antioxidant capacities are promising. The sample concentration also influenced its antioxidant power.

Immobilization of pectinmethylesterase from acerola (Malpighia glabra L.) in porous silica.

The total and partially purified enzyme pectinmethylesterase from acerola fruit was covalently immobilized on porous silica particles. These efficiency values were 114% for the total PME and 351% for the partially purified PME. In both forms the immobilization resulted in compounds with high thermal stability.

Partial purification and characterization of pectin methylesterase from acerola (Malpighia glabra L.).

The enzyme pectin methylesterase (PME) is present in acerola fruit and was partially purified by gel filtration on Sephadex G-100. The results of gel filtration showed different PME isoforms. The total PME (precipitated by 70% salt saturation) and one of these isoforms (fraction from Sephadex G-100 elution) that showed a molecular mass of 15.5 +/- 1.0 kDa were studied. The optimum pH values of both forms were 9.0. The total and the partially purified PME showed that PME specific activity increases with temperature. The total acerola PME retained 13.5% of its specific activity after 90 min of incubation at 98 degrees C. The partially purified acerola (PME isoform) showed 125.5% of its specific activity after 90 min of incubation at 98 degrees C. The K(m) values of the total PME and the partially purified PME isoform were 0.081 and 0.12 mg/mL, respectively. The V(max) values of the total PME and the partially purified PME were 2.92 and 6.21 micromol/min/mL/mg of protein, respectively.