Guillermo R. Castro

Antimicrobial activity of flavonoids from leaves of Tagetes minuta

The total extract and fractions with different solvents, obtained from leaves of Tagetes minuta, showed several degrees of antimicrobial activity against Gram positive and Gram negative microorganisms. The same fractions were inactive against Lactobacillus, Zymomonas and Saccharomices species. The major component of the extract: quercetagetin-7-arabinosyl-galactoside, showed significant antimicrobial activity on pathogen microorganisms tested. Correlation results were carried out using chloramphenicol as standard antibiotic.

Thermostable alkaline proteases of Bacillus licheniformis MIR 29: isolation, production and characterization

Abstract Bacillus licheniformis MIR 29 has been isolated and produces extracellular proteases. It is able to grow at temperatures up to 60 °C and at pH values up to 9.0. Casein was the best carbon source for production of a thermostable protease activity which, in some conditions, is 90% extracellular. The synthesis of alkaline protease is not constitutive; different levels of production were found with different carbon and nitrogen sources. Casein was thought to be an inducer of enzyme synthesis. The optimal pH and temperature of the enzyme activity were 12 °C and 60 °C, respectively. The enzyme was stable up to 60 °C in the absence of stabilizers. The protease activity was inhibited with phenylmethylsulphonyl fluoride, indicating a serine-protease activity. The proteolytic activity was lowered by molecules present in the culture supernatant, which include amino acids and peptides, indicating end-product inhibition. Electrophoresis assay on denaturating gels showed two bands with alkaline protease activity, in the 25 to 40-kDa molecular mass range.

Recent trends in biocatalysis engineering

During the last 30 years the scope of biocatalysis has been expanding due to the advances in several technological fields. Diverse techniques as structural enzyme improvement (e.g. protein engineering, direct evolution), engineering approaches (e.g. ionic liquids, supercritical fluids) and physical stabilization (e.g. immobilization, CLEAS) have been developed, which in combination are powerful tools to improve biotransformation and to synthesize new products. In the present work, recent advances in biocatalysis are reviewed.

Homogeneous Biocatalysis in Organic Solvents and Water-Organic Mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.

Purification and characterization of a thermostable xylanase from Bacillus amyloliquefaciens

Abstract A Bacillus amyloliquefaciens strain isolated from soil produced xylanolytic enzymes in the extracellular medium when grown on xylan and nitrate. No cellulase activity was detected. Xylanase was purified from the culture supernatant in three steps which comprised anion-exchange adsorption, ammonium sulfate precipitation, and hydrophobic interaction chromatography. The pure enzyme appeared as two close protein bands on SDS-PAGE with molecular weights of 18.4 and 19.6 kD, respectively. The molecular weight obtained by sedimentation equilibrium under native conditions was about 18.5 kD. The isoelectric point was 10.1. The enzyme contains a relatively high content of aspartate, glycine, and threonine. Tryptophan seems to be essential for xylanase activity. The presence of carbohydrate was noted in the pure enzyme. Circular dichroism studies indicated that the protein contains almost equal proportions of α-helix, β-sheet, and β-turns. The optimum pH of activity was 6.8–7.0. The enzyme exhibited good stability even at pH 9.0. Excellent stability was observed at 50°C even though optimal activity was at 80°C. The activity was completely inhibited by Hg 2+ ions and was reduced drastically in the presence of Cu 2+ and Fe 3+ ions. Mn 2+ ions, EDTA, β-mercaptoethanol, and dithiothreitol up to 5 m m stimulated the enzyme activity.

Organic solvent adaptation of Gram positive bacteria: Applications and biotechnological potentials

Organic-solvent-tolerant bacteria are considered extremophiles with different tolerance levels that change among species and strains, but also depend on the inherent toxicity of the solvent. Extensive studies to understand the mechanisms of organic solvent tolerance have been done in Gram-negative bacteria. On the contrary, the information on the solvent tolerance mechanisms in Gram-positive bacteria remains scarce. Possible shared mechanisms among Gram-(-) and Gram-(+) microorganisms include: energy-dependent active efflux pumps that export toxic organic solvents to the external medium; cis-to-trans isomerization of unsaturated membrane fatty acids and modifications in the membrane phospholipid headgroups; formation of vesicles loaded with toxic compounds; and changes in the biosynthesis rate of phospholipids to accelerate repair processes. However, additional physiological responses of Gram-(+) bacteria to organic solvents seem to be specific. The aim of the present work is to review the state of the art of responsible mechanisms for organic solvent tolerance in Gram-positive bacteria, and their industrial and environmental biotechnology potential.

Isolation of four aquatic streptomycetes strains capable of growth on organochlorine pesticides.

Ninety-three wild-type isolates identified as actinomycetes were tested against 11 organochlorine pesticides (OPs): aldrin, chlordane, DDD, DDE, DDT, dieldrin, heptachlor, and heptachlor epoxides, lindane, and methoxychlor. Qualitative screening agar assays displayed 62-78% tolerance of strains to OPs. Four strains designed M4, M7, M9 and M15 were selected based on multi-OP-tolerance, and identified as members of the streptomycetes group. Different growth profiles were observed in cultures of the four selected streptomycetes cultured in synthetic medium containing 5-50 microg x l(-1) aldrin or chlordane or lindane. Increase of aldrin removal by the selected microorganisms was concomitant with the 4.8-36.0 microg x l(-1) pesticide concentration range. After 72 h of streptomycete M7 growth in synthetic medium containing 48.0 microg x l(-1) aldrin, the remaining OP concentration in the supernatant was approximately 10% of the initial concentration. Also, in stationary growth phase less than 2.5 microg x l(-1) aldrin residual concentration was detected in the medium.

Chromium accumulation by two Streptomyces spp. isolated from riverine sediments

Strains designated R22 and R25, isolated from Salí River sediments, Argentina, were highly resistant to chromium. These strains were shown by 16S rRNA sequencing studies to be Streptomyces spp.; this affiliation was consistent with morphological and chemical characteristics. Growth of strains R22 and R25 in medium containing 100 mg l−1 chromate was reduced by only 23% and 34%, respectively, compared with growth in medium without added chromium. Streptomyces sp. strains R22 and R25 both accumulated chromium with yields of 10.0 and 5.6 mg Cr g−1 of dry weight, respectively, and a chromate concentration of 50 mg ml−1. Cell fractionation studies with strain R22 showed that the great majority of the chromium were associated with the cell wall fraction. Streptomyces strains R22 and R25 may have applications in bioremediation of chromium contamination. Journal of Industrial Microbiology & Biotechnology (2001) 26, 210–215.

Novel Biopolymer Matrices for Microencapsulation of Phages: Enhanced Protection Against Acidity and Protease Activity

Phage therapy by oral administration requires enhanced resistance of phages to the harsh gastric conditions. The aim of this work is the microencapsulation of phages in natural biopolymeric matrices as a protective barrier against the gastric environment. Alginate and pectin are used as base polymers. Further emulsification with oleic acid or coating with a different biopolymer is also studied. Emulsified pectin shows the maximum encapsulation efficiency and the highest protection against acidity, leaving more than 10(3) active phages after 30 min exposure at pH = 1.6, and protects phage from pepsin activity (4.2 mg mL(-1)). Non-encapsulated phages are fully inactivated at pH = 1.6 or with pepsin (0.5 mg mL(-1)) after 10 min.

Milk kefir: composition, microbial cultures, biological activities, and related products

In recent years, there has been a strong focus on beneficial foods with probiotic microorganisms and functional organic substances. In this context, there is an increasing interest in the commercial use of kefir, since it can be marketed as a natural beverage that has health promoting bacteria. There are numerous commercially available kefir based-products. Kefir may act as a matrix in the effective delivery of probiotic microorganisms in different types of products. Also, the presence of kefir’s exopolysaccharides, known as kefiran, which has biological activity, certainly adds value to products. Kefiran can also be used separately in other food products and as a coating film for various food and pharmaceutical products. This article aims to update the information about kefir and its microbiological composition, biological activity of the kefir’s microflora and the importance of kefiran as a beneficial health substance.