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Despite the importance of chitinases as potential biocontrol agents, their applications have not attracted significant attention due to the lack of stable enzyme formulations, high production costs and low yields from both wild and recombinant sources. However, through protein engineering techniques, these limitations can be overcome. Engineered mini-proteins for instance, could possess; enhanced stability, low production costs and simplified structural and functional mechanisms. Thus, this study was focused on developing miniaturized variants of Streptomyces griseus HUT6037 chitinase C that could serve as simpler yet potentially stable antifungal agents. Through computational techniques involving sequence analysis, docking and molecular dynamics simulation important residues and/or motifs within the catalytic cleft of SgChiC were identified as essential for recognition and binding to complex or crystalline chitin. Residues outside the catalytic clefts were thus considered targets for miniaturization. Five (5) SgChiC variants namely: M159, M140, M139, M109 and M101 were designed in silico, their genes were subsequently synthesised and cloned into pET-22b(+). The variants were then expressed in Escherichia coli BL21(DE3) and purified through on-column refolding. Biochemical assays revealed that all variants although had lower activities towards colloidal chitin when compared with the wild-type, retained the optimum temperature at 40 C. The optimum pH for activities of variants however varied with M101 and M139 drifting towards acidic pH of 5.0 and 6.0 respectively, while M159 and M109 had optimum activities at pH 8. Interestingly, all variants retained 40-50% of the specific activity of the WT towards colloidal chitin, with M159 displaying the highest specific activity at 31.6 Umg-1 compared to the WT with 52.3 Umg-1. Contrastingly, with chitosan, the smallest variant M101, displayed high chitosanase activity comparable with the WT with 59.6 and 61.4 Umg-1 respectiv

Despite the importance of chitinases as potential biocontrol agents, their applications have not attracted significant attention due to the lack of stable enzyme formulations, high production costs and low yields from both wild and recombinant sources. However, through protein engineering techniques, these limitations can be overcome. Engineered mini-proteins for instance, could possess; enhanced stability, low production costs and simplified structural and functional mechanisms. Thus, this study was focused on developing miniaturized variants of Streptomyces griseus HUT6037 chitinase C that could serve as simpler yet potentially stable antifungal agents. Through computational techniques involving sequence analysis, docking and molecular dynamics simulation important residues and/or motifs within the catalytic cleft of SgChiC were identified as essential for recognition and binding to complex or crystalline chitin. Residues outside the catalytic clefts were thus considered targets for miniaturization. Five (5) SgChiC variants namely: M159, M140, M139, M109 and M101 were designed in silico, their genes were subsequently synthesised and cloned into pET-22b(+). The variants were then expressed in Escherichia coli BL21(DE3) and purified through on-column refolding. Biochemical assays revealed that all variants although had lower activities towards colloidal chitin when compared with the wild-type, retained the optimum temperature at 40 C. The optimum pH for activities of variants however varied with M101 and M139 drifting towards acidic pH of 5.0 and 6.0 respectively, while M159 and M109 had optimum activities at pH 8. Interestingly, all variants retained 40-50% of the specific activity of the WT towards colloidal chitin, with M159 displaying the highest specific activity at 31.6 Umg-1 compared to the WT with 52.3 Umg-1. Contrastingly, with chitosan, the smallest variant M101, displayed high chitosanase activity comparable with the WT with 59.6 and 61.4 Umg-1 respectiv

Zincophorin is a polyketide antibiotic that possesses potent activity against Gram-positive bacteria, including human pathogens. While a number of total syntheses of this highly functionalized natural product were reported since its initial discovery, the genetic basis for the biosynthesis of zincophorin has remained unclear. In this study, the co-linearity inherent to polyketide pathways was used to identify the zincophorin biosynthesis gene cluster in the genome of the natural producer Streptomyces griseus HKI 0741. Interestingly, the same locus is fully conserved in the streptomycin-producing actinomycete S. griseus IFO 13350, suggesting that the latter bacterium is also capable of zincophorin biosynthesis. Biological profiling of zincophorin revealed a dose-dependent inhibition of the Gram-positive bacterium Streptococcus pneumoniae . The antibacterial effect, however, is accompanied by cytotoxicity. Antibiotic and cytotoxic activities were completely abolished upon esterification of the carboxylic acid group in zincophorin.

Proteolytic activity of enzymatic extracts generated from rumen microorganisms cultivated in vitro was evaluated. The incubation of rumen fluid used different substrates to generate a higher enzyme concentration and promote a broad spectrum of hydrolytic activity. The composition of the substrates used in the cultivation of the fluid was enriched in protein, starch or cell wall. Enzyme preparations were evaluated by incubating in 30 mL of buffer 50 mM Tris-HCl (pH 6.5) at 39 °C during 48 hours, 100 mg of crude protein from feeds soybean meal, canola meal, sunflower meal, gluten feed, dehydrated alfalfa meal, berseem clover, oat forage and perennial ryegrass. Enzyme extracts from cultivated rumen fluid showed an average protein breakdown of 75.5%, in eight feed samples tested. This value was very close to that measured with the technique of proteases from Streptomyces griseus (74.6% CP), but significantly lower (P≤0.05) than the one obtained by the in situ methodology (84.8% CP). The technique with extracted rumen enzymes showed higher level of proteolysis in the early hours of incubation (6 H) compared to the other techniques. These results suggest that the enzyme preparations of ruminal origin have the ability to predict degradability of feed proteins in the rumen, particularly in the first phase when most of proteins are hydrolyzed and become available for microbial utilization., Se evaluó la actividad proteolítica de extractos enzimáticos generados a partir de microorganismos ruminales cultivados in vitro. Esta incubación de fluido ruminal se realizó con diferentes sustratos por separado con el objeto de generar una mayor concentración enzimática y promover un amplio espectro de actividad hidrolítica. La composición de los sustratos empleados en el cultivo del fluido fueron enriquecidos en proteínas, almidones o paredes celulares. Los preparados enzimáticos fueron evaluados incubando en 30 mL de buffer Tris-HCl 50 mM (pH 6,5) a 39 °C, durante 48h, 100 mg de proteína cruda de los alimentos afrecho de soya, afrecho de canola, afrecho de maravilla, harina de gluten de maíz, harina de alfalfa, trébol alejandrino, ballica perenne y avena forrajera. Las enzimas ruminales mostraron una degradación promedio de 75,5% de proteína cruda, considerando los ocho alimentos. Este valor fue muy similar al medido con la técnica proteasas de Streptomyces griseus (74,6% PC), pero significativamente menor (P≤0,05) al exhibido por la metodología in situ (84,8% PC). La técnica con extractos de enzimas ruminales mostró un nivel de proteolisis superior en las primeras horas de incubación (6 H) respecto al resto de las técnicas comparadas. Estos resultados permiten sugerir que los preparados enzimáticos de origen ruminal tienen la capacidad de predecir in vitro la degradabilidad de las proteínas de los alimentos en el rumen.

Proteolytic activity of enzymatic extracts generated from rumen microorganisms cultivated in vitro was evaluated. The incubation of rumen fluid used different substrates to generate a higher enzyme concentration and promote a broad spectrum of hydrolytic activity. The composition of the substrates used in the cultivation of the fluid was enriched in protein, starch or cell wall. Enzyme preparations were evaluated by incubating in 30 mL of buffer 50 mM Tris-HCl (pH 6.5) at 39 °C during 48 hours, 100 mg of crude protein from feeds soybean meal, canola meal, sunflower meal, gluten feed, dehydrated alfalfa meal, berseem clover, oat forage and perennial ryegrass. Enzyme extracts from cultivated rumen fluid showed an average protein breakdown of 75.5%, in eight feed samples tested. This value was very close to that measured with the technique of proteases from Streptomyces griseus (74.6% CP), but significantly lower (P≤0.05) than the one obtained by the in situ methodology (84.8% CP). The technique with extracted rumen enzymes showed higher level of proteolysis in the early hours of incubation (6 H) compared to the other techniques. These results suggest that the enzyme preparations of ruminal origin have the ability to predict degradability of feed proteins in the rumen, particularly in the first phase when most of proteins are hydrolyzed and become available for microbial utilization., Se evaluó la actividad proteolítica de extractos enzimáticos generados a partir de microorganismos ruminales cultivados in vitro. Esta incubación de fluido ruminal se realizó con diferentes sustratos por separado con el objeto de generar una mayor concentración enzimática y promover un amplio espectro de actividad hidrolítica. La composición de los sustratos empleados en el cultivo del fluido fueron enriquecidos en proteínas, almidones o paredes celulares. Los preparados enzimáticos fueron evaluados incubando en 30 mL de buffer Tris-HCl 50 mM (pH 6,5) a 39 °C, durante 48h, 100 mg de proteína cruda de los alimentos afrecho de soya, afrecho de canola, afrecho de maravilla, harina de gluten de maíz, harina de alfalfa, trébol alejandrino, ballica perenne y avena forrajera. Las enzimas ruminales mostraron una degradación promedio de 75,5% de proteína cruda, considerando los ocho alimentos. Este valor fue muy similar al medido con la técnica proteasas de Streptomyces griseus (74,6% PC), pero significativamente menor (P≤0,05) al exhibido por la metodología in situ (84,8% PC). La técnica con extractos de enzimas ruminales mostró un nivel de proteolisis superior en las primeras horas de incubación (6 H) respecto al resto de las técnicas comparadas. Estos resultados permiten sugerir que los preparados enzimáticos de origen ruminal tienen la capacidad de predecir in vitro la degradabilidad de las proteínas de los alimentos en el rumen.

Laboratory data on the behaviour of the pathogenic form of the prion protein (PrP(Sc)) in environmental matrices such as sewage sludge is scarce. Direct experiments with this misfolded protein require strict safety measures, pathogen class-3 facilities and costly reagents. However, preliminary data can be generated by non-pathogenic model systems that involve lower costs and simpler manipulation. We chose amyloid-like fibrils formed from the well-studied protein lysozyme as a model because, in the case of an accidental contamination of sewage sludge, PrP(Sc) would most likely be in the form of amyloid fibrils. All amyloid fibrils have similar structural features and tend to bind to thioflavin-T, thereby enhancing the fluorescence yield of the dye. We used this fluorescence enhancement to monitor amyloid fibrils introduced into activated sludge. We observed that, in the presence of sludge flocs, the concentration of amyloid fibrils that are detectable through the enhancement of thioflavin-T fluorescence decreased as a function of time, most likely due to hydrolysis of the fibrils by sludge proteases. Some of the fluorescence loss seems also due to the binding of sludge exopolymers to amyloid fibrils.

Cr6+ can be reduced to the less toxic and mobile Cr3+ by microorganisms. During studies on Cr toxicity in actinomycetes, a strain of the fungus-like bacterium Streptomyces griseus was found to reduce Cr6+ to Cr3+ from media containing 5-60 ppm Cr6+. The concentration of Cr3+ increased and that of Cr6+ simultaneously decreased during growth with complete conversion in 3-4 days. Reduction was faster when Cr was added after 24 h growth rather than at the time of inoculation, particularly when high concentrations of Cr were involved. Conversion of Cr6+ to Cr3+ was complete within 24-48 h of Cr addition with significant uptake in the biomass. Reduction of Cr by resting cells and the effects of various factors on Cr reduction are discussed., Cr6+ can be reduced to the less toxic and mobile Cr3+ by microorganisms. During studies on Cr toxicity in actinomycetes, a strain of the fungus-like bacterium Streptomyces griseus was found to reduce Cr6+ to Cr3+ from media containing 5-60 ppm Cr6+. The concentration of Cr3+ increased and that of Cr6+ simultaneously decreased during growth with complete conversion in 3-4 days. Reduction was faster when Cr was added after 24 h growth rather than at the time of inoculation, particularly when high concentrations of Cr were involved. Conversion of Cr6+ to Cr3+ was complete within 24-48 h of Cr addition with significant uptake in the biomass. Reduction of Cr by resting cells and the effects of various factors on Cr reduction are discussed.