A comparative profile of bactericidal action of a partially purified bacteriocin from lactic acid bacteria with antibiotics

Aims@#The menace of antibiotic resistance has led to the search for alternatives, which in turn has diverted the attention to bacteriocins, antimicrobial peptides (AMPs) produced by bacteria for their bactericidal properties. The aim of our study was to isolate and partially purify bacteriocin from lactic acid bacteria (LAB) and comparing its antimicrobial activity with antibiotics.@*Methodology and results@#Among 38 LAB screened using agar spot assay, LAB 28D1 showed the highest antimicrobial activity against the test bacterial strains. The proteinaceous nature of the antimicrobial compound extracted from LAB 28D1 was confirmed by its inactivation after treatment with proteolytic enzymes. The crude bacteriocin was found to be stable over a wide range of temperatures (60-100 °C) and pH (4-9). The bacteriocin was partially purified by ammonium sulfate precipitation (ASP) and the activity units were 204,800 AU/mL. The total protein and the specific activity of partially purified bacteriocin were found to be 24.585 mg and 124,954.24 AU/mg respectively. The molecular weight of partially purified bacteriocin was determined to be 8.5 kDa approximately. The efficacy of the partially purified bacteriocin against indicator bacterial strains was compared with antibiotics by the disc diffusion method and minimum inhibitory concentration (MIC). According to our study, the hospital waste isolate Enterococcus spp. was found to be multidrug-resistant (MDR) but sensitive to bacteriocin from LAB (MIC 0.06 ± 0 µg/mL).@*Conclusion, significance and impact of the study@#Bacteriocin from LAB has potential in combating MDR enterococcal infections.

In vitro α-glucosidase inhibition and antioxidant activities of partially purified Antidesma bunius fruit and Gynura nepalensis leaf extracts.

Diabetes mellitus is a metabolic disorder characterized by increased blood glucose levels. Current treatments involve the use of sulfonylureas, α-glucosidase inhibitors, and other synthetic drugs. The study demonstrated the α-glucosidase inhibition and antioxidant properties of partially purified ethanolic extracts of Antidesma bunius fruits and Gynura nepalensis leaves, as possible herbal drug candidates. After ethanol extraction, the extracts were fractionated using normal phase liquid column chromatography, with elution solvents ethyl acetate, methanol and water. Fractionation resulted in five fractions for A. bunius (A1, A2, A3, A4 and A5) and seven fractions for G. nepalensis (G1, G2, G3, G4, G5, G6 and G7). Fraction G1 showed the highest α-glucosidase inhibition activity (90.61±8.05%) and possibly acted via a mixed mode of inhibition. For the antioxidant activities, Fraction A1 exhibited highest radical scavenging activity via DPPH assay (97.39±2.48%), Fraction G7 exhibited highest iron (II)-chelating activity (95.85±1.46%) and Fraction G6 exhibited highest ferric-ion reducing activity via FRAP assay (272.60 μg/mL FeSO4 equivalents). Phytochemical screening revealed that flavonoids and tannins were common among all fractions. The results demonstrated the potential of these plants as an antidiabetic herbal treatment. However, further studies needs to be done, specifically focusing on isolating the active component(s), structure and mechanism elucidation and toxicity assays.

Production and partial purification of antifungal chitinase from Bacillus cereus VITSD3

The current work was designed to isolate and characterize chitin degrading bacteria. Among the 55 bacterial colonies isolated from 7 different soil samples, 4 isolates were capable of degrading chitinase, among which one strain VITSD3 was found to be potent. Based on the morphological, biochemical and molecular characterization of VITSD3 the isolate was confirmed as Bacillus cereus (Genbank accession number: KC961638), designated as Bacillus cereus VITSD3. The crude enzyme had a total activity of 220 U, precipitated with 44.8 U and 22.5 U for dialysed sample. The hydrolysed product NAG (N-Acetyl Glucosamine) from chitin was analysed by high-pressure liquid chromatography (HPLC).The molecular weight of the chitinase was determined using SDS PAGE and found to be 55 kDa. The partially purified chitinase produced from Bacillus cereus VITSD3 showed antifungal activity against Aspergillus fumigatus (18 mm), Aspergillus niger (6 mm) and Aspergillus flavus (15 mm). Hence the investigation suggests a potential benefit of partially purified chitinase extracted from Bacillus cereus VITSD3 which will serve as an excellent antifungal potential with therapeutic use.

O presente trabalho atual foi delineado para isolar e caracterizar bactérias degradadoras de quitina. Entre as 55 colónias bacterianas isoladas a partir de 7 amostras de solo diferentes, quatro isolados foram capazes de degradar quitinase, entre os quais uma estirpe, VITSD3, mostrou-se potente. Com base na caracterização morfológica, bioquímica e molecular de VIT D3 a soluto foi confirmada como Bacillus cereus (número de acesso Genbank: KC961638), designada como Bacillus cereus VITSD3. A enzima bruta tinha uma actividade total de 220 L, precipitou-se com 44,8 L e 22,5 L de amostra dialisada. O produto hidrolisado NAG (N-acetil-glucosamina) a partir de quitina foi analisado por cromatografia líquida de alta pressão (HPLC) .O peso molecular da quitinase foi determinado, utilizando-se SDS-PAGE e verificou-se ser 55 kDa. A quitinase parcialmente purificada produzida a partir de Bacillus cereus VITSD3 mostrou actividade antifúngica contra Aspergillus fumigatus (18 mm), Aspergillus niger (6 mm) e Aspergillus flavus (15 mm). Por isso, a investigação sugere um potencial benefício de quitinase parcialmente purificada extraída de Bacillus cereus VITSD3 o que poderá servir como um excelente potencial antifúngico para uso terapêutico.

Optimization of Borassus flabellifer Amylase Extraction Procedure Using Box-Behnken Design and Development of Simple Affinity Chromatographic Technique for Purification of Amylases.

This work is aimed to study the optimal conditions of extraction and development of novel purification process for α-Amylase from pericarp of Borassus flabellifer fruit. Optimization was done by Response Surface Methodology (RSM) using Box-Behnken design. The variables chosen for this purpose are pH (X1), temperature (X2, °C) and CaCl2 concentration (X3, ppm). A total of 9 runs were conducted, varying each factor while others were kept constant. The contribution of each factor was established giving raise to significant t-values and p-values. This revealed that the three variables are essential for activity of the enzyme. Based on the obtained optimal conditions from RSM studies, enzyme formation was successfully achieved with an activity of 28.8 U and concentration of 1.4 mg/ml for the 90% ammonium sulfate precipitated dialyzed enzyme sample. A novel affinity chromatographic process was developed for purification process and by this process the enzyme concentration was found to be 0.032mg/ml and activity was determined as 4.76 U.

Obtaining partial purified xylose reductase from Candida guilliermondii / Obtenção de xilose redutase de Candida guilliermondii parcialmente purificada

The enzymatic bioconversion of xylose into xylitol by xylose reductase (XR) is an alternative for chemical and microbiological processes. The partial purified XR was obtained by using the following three procedures: an agarose column, a membrane reactor or an Amicon Ultra-15 50K Centrifugal Filter device at yields of 40 percent, 7 percent and 67 percent, respectively.

A bioconversão enzimática da xilose em xilitol pela xilose redutase (XR) é uma alternativa para as vias química e microbiológica. Avaliouse a purificação parcial da XR, utilizando os três seguintes procedimentos: uma coluna de agarose, um reator com membrana ou tubos de ultracentrifugação Amicon Ultra-15 50K, com rendimento de 40 por cento, 7 por cento ou 67 por cento, respectivamente.

Partial purification of protein farnesyl cysteine carboxyl methyltransferase from bovine brain

C-terminal farnesyl cysteine carboxyl methylation has been known to be the last step in the post-translational modification processes of several important signal transduction proteins in eukaryotes including ras related GTP binding proteins and the gamma-subunit of heterotrimeric G proteins. Protein farnesyl cysteine carboxyl methyltransferase (PFCCMT; EC, 2.1.1.100) catalyzing the reaction is well characterized as being stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and suppressed by N-acetyl-S-farnesyl-L-cysteine (AFC). As an initial step to understand the physiological significance of the process, we attempted to purify the enzyme, which was partially purified 130-fold (specific activity, 143 pmol of methyl group transferred/min/mg of protein) with yield of 1.8% after purification by fast protein liquid chromatography (FPLC) on a Superdex 75 column. The enzyme was further purified with non denaturing polyacrylamide gel electrophoresis (ND-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of PFCCMT was determined to be about 30 kDa based on Superdex 75 FPLC as well as photoaffinity labelling with S-adenosyl-L-[methyl-3H] methionine ([methyl-3H]SAM). The partially purified enzyme (Superdex 75 eluate) was found to be characteristically affected by GTP gamma S, being activated about 40-fold in 2 mM, in contrast to ATP which did not show any effect on enzyme activity. Meanwhile, the enzyme was found to be markedly inhibited by AFC, reaching 0 activity in 2 mM. These observations strongly suggested that the partially purified enzyme was PFCCMT.