Amanda Reges de Sena

Antimicrobial activity of Marcetia DC species (Melastomataceae) and analysis of its flavonoids by reverse phase-high performance liquid chromatography coupled-diode array detector.

Background: Marcetia genera currently comprises 29 species, with approximately 90% inhabiting Bahia (Brazil), and most are endemic to the highlands of the Chapada Diamantina (Bahia). Among the species, only M. taxifolia (A.St.-Hil.) DC. populates Brazil (state of Roraima to Paraná) and also Venezuela, Colombia, and Guyana. Objective: This work evaluated the antimicrobial activity of hexane, ethyl acetate, and methanol extracts of three species of Marcetia (Marcetia canescens Naud., M. macrophylla Wurdack, and M. taxifolia A.StHil) against several microorganism. In addition, the flavonoids were analyzed in extracts by HPLC-DAD. Materials and methods: The tests were made using Gram-positive (three strains of Staphylococcus aureus) and Gram-negative (two strains of Escherichia coli, a strain of Pseudomonas aeruginosa and another of Salmonella choleraesius) bacteria resistant and nonresistant to antibiotics and yeasts (two strains of Candida albicans and one of C. parapsilosis) by the disk diffusion method. Solid-phase extraction (SPE) was performed on the above extracts to isolate flavonoids, which were subsequently analyzed by high performance liquid chromatography coupled diode array detector (HPLC-DAD). Results: Results showed that extracts inhibited the Gram-positive bacteria and yeast. The hexane extracts possessed the lowest activity, while the ethyl acetate and methanolic extracts were more active. Conclusion: Marcetia taxifolia was more effective (active against 10 microorganisms studied), and only its methanol extract inhibited Gram-negative bacteria (P. aeruginosa and S. choleraesius). SPE and HPLC-DAD analysis showed that M. canescens and M. macrophylla contain glycosylated flavonoids, while the majority of extracts from M. taxifolia were aglycone flavonoids.

Production, purification and characterization of a thermostable β-1,3-glucanase (laminarinase) produced by Moniliophthora perniciosa

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70%) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L(-1) and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20% of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

Application of Doehlert experimental design in the optimization of experimental variables for the Pseudozyma sp. (CCMB 306) and Pseudozyma sp. (CCMB 300) cell lysis

This study aimed to verify the influence of pH and temperature on the lysis of yeast using experimental design. In this study, the enzymatic extract containing β-1,3-glucanase and chitinase, obtained from the micro-organism Moniliophthora perniciosa, was used. The experiment showed that the best conditions for lysis of Pseudozyma sp. (CCMB 306) and Pseudozyma sp. (CCMB 300) by lytic enzyme were pH 4.9 at 37 oC and pH 3.9 at 26.7 oC, respectively. The lytic enzyme may be used for obtaining various biotechnology products from yeast.

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

Abstract 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).

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

ABSTRACT The current study aims to assess the kinetics of population growth of Rhodotorula oryzicola and the production of β-1,3-glucanase (EC 3.2.1.39) enzyme by this yeast. It also aims to obtain the optimum conditions of β-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 β-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, thermodynamic parameters and in vitro digestion of tannase from Aspergillus tamarii URM 7115

ABSTRACT Tannase is an enzyme used in various industries and produced by a large number of microorganisms. The aim of this study was to evaluate tannase production to determine the biochemical, kinetic, and thermodynamic properties and to simulate tannase in vitro digestion. The tannase-producing fungal strain was isolated from “jamun” leaves and identified as Aspergillus tamarii. Temperature at 26°C for 67 h was the best combination for maximum tannase activity (6.35-fold; initial activity in Plackett–Burman design—15.53 U/mL and average final activity in Doehlert design—98.68 U/mL). The crude extract of tannase was optimally active at 40°C, pH 5.5 and 6.5. Moreover, tannase was stimulated by Na+, Ca2+, Mg2+, and Mn2+. The half-life at 40°C lasted 247.55 min. The free energy of Gibbs, enthalpy, and entropy, at 40°C, was 81.47, 16.85, and −0.21 kJ/mol · K, respectively. After total digestion, 123.95% of the original activity was retained. Results suggested that tannase from A. tamarii URM 7115 is an enzyme of interest for industrial applications, such as gallic acid production, additive for feed industry, and for beverage manufacturing, due to its catalytic and thermodynamic properties.

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

ABSTRACT 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.