Queratinases são proteases de especial interesse devido a sua ação sobre a queratina insolúvel, que por sua vez, é o principal componente das penas. As penas de aves são fonte de aminoácidos, e, portanto, são transformadas em farinha de pena e utilizadas em ração animal. Entretanto, este produto possui baixo valor nutricional devido a sua baixa digestibilidade, já que a queratina não é degradada por enzimas proteolíticas comuns. Nesse contexto, as queratinases microbianas são uma abordagem promissora para conversão de queratina em hidrolisados proteicos. Os objetivos deste trabalho foram selecionar e identificar bactérias termófilas provenientes do bioma Caatinga (Brasil) capazes de produzir queratinases termoativas, e caracterizar a queratinase produzida pelo isolado selecionado. Oito bactérias proteolíticas foram isoladas de uma amostra de solo. Destas, o Anoxybacillus sp. PC2 produziu queratinase com maior atividade em meio composto por penas (2%), KH2PO4 (0,04%), K2HPO4 (0,2%) e NaCl (0.05%) em pH 6,5 e 60 ºC. A protease queratinolítica foi parcialmente purificada por concentração em dispositivo de filtração (10.000 NMWL) e cromatografia de troca aniônica. A protease parcialmente purificada foi ativa em uma faixa de pH de 5 a 10, com alta atividade entre as temperaturas de 50 e 80 ºC. A atividade ótima foi observada em pH 7 e entre 50 e 60 ºC, tornando-a adequada para utilização em processos biotecnológicos para bioconversão de queratina. Um estudo molecular conduzido para identificar a provável protease queratinolítica produzida pelo Anoxybacillus sp. PC2 identificou uma metaloprotease neutra, cujo gene apresentou 98% de similaridade com o gene nprS de Geobacillus stearothermophilus. / Keratinases are proteases of special interest due to their action on insoluble keratin, which in turn, is the major component of feathers. The poultry feathers are a source of amino acids, and therefore, are converted to feather meal to be used as animal feedstuff. However, this product has low nutritional value due to its poor digestibility, since keratin is not degraded by common proteolytic enzymes. In this context, microbial keratinases are a promising approach to convert keratin into protein hydrolysates. The aims of this study were to select and identify thermophilic bacteria from Caatinga biome (Brazil) capable to produce thermoactive keratinases, and characterize the keratinase produced by the selected isolate. Eight proteolytic bacteria were isolated from a soil sample. Of these, Anoxybacillus sp. PC2 produced keratinase with higher activity in a medium composed of 2% feather waste, 0.04% KH2PO4, 0.2% K2HPO4 and 0.05% NaCl at pH 6.5 incubated at 60 ºC. The keratinolytic protease was partially purified by concentration in a centrifugal filter device (10,000 NMWL) and anion-exchange chromatography. The partially purified protease was active in a pH range of 5.0-10.0 with high activity in temperature range of 50-80 ºC. The optimum activity was observed at pH 7.0 and 50-60 ºC, making it suitable for biotechnological processes for keratin bioconversion. A molecular study conducted to identify the presumable keratinolytic protease produced by Anoxybacillus sp. PC2 identified a neutral metalloprotease whose gene showed 98% similarity with Geobacillus stearothermophilus nprS gene.

Queratinases são proteases de especial interesse devido a sua ação sobre a queratina insolúvel, que por sua vez, é o principal componente das penas. As penas de aves são fonte de aminoácidos, e, portanto, são transformadas em farinha de pena e utilizadas em ração animal. Entretanto, este produto possui baixo valor nutricional devido a sua baixa digestibilidade, já que a queratina não é degradada por enzimas proteolíticas comuns. Nesse contexto, as queratinases microbianas são uma abordagem promissora para conversão de queratina em hidrolisados proteicos. Os objetivos deste trabalho foram selecionar e identificar bactérias termófilas provenientes do bioma Caatinga (Brasil) capazes de produzir queratinases termoativas, e caracterizar a queratinase produzida pelo isolado selecionado. Oito bactérias proteolíticas foram isoladas de uma amostra de solo. Destas, o Anoxybacillus sp. PC2 produziu queratinase com maior atividade em meio composto por penas (2%), KH2PO4 (0,04%), K2HPO4 (0,2%) e NaCl (0.05%) em pH 6,5 e 60 ºC. A protease queratinolítica foi parcialmente purificada por concentração em dispositivo de filtração (10.000 NMWL) e cromatografia de troca aniônica. A protease parcialmente purificada foi ativa em uma faixa de pH de 5 a 10, com alta atividade entre as temperaturas de 50 e 80 ºC. A atividade ótima foi observada em pH 7 e entre 50 e 60 ºC, tornando-a adequada para utilização em processos biotecnológicos para bioconversão de queratina. Um estudo molecular conduzido para identificar a provável protease queratinolítica produzida pelo Anoxybacillus sp. PC2 identificou uma metaloprotease neutra, cujo gene apresentou 98% de similaridade com o gene nprS de Geobacillus stearothermophilus. / Keratinases are proteases of special interest due to their action on insoluble keratin, which in turn, is the major component of feathers. The poultry feathers are a source of amino acids, and therefore, are converted to feather meal to be used as animal feedstuff. However, this product has low nutritional value due to its poor digestibility, since keratin is not degraded by common proteolytic enzymes. In this context, microbial keratinases are a promising approach to convert keratin into protein hydrolysates. The aims of this study were to select and identify thermophilic bacteria from Caatinga biome (Brazil) capable to produce thermoactive keratinases, and characterize the keratinase produced by the selected isolate. Eight proteolytic bacteria were isolated from a soil sample. Of these, Anoxybacillus sp. PC2 produced keratinase with higher activity in a medium composed of 2% feather waste, 0.04% KH2PO4, 0.2% K2HPO4 and 0.05% NaCl at pH 6.5 incubated at 60 ºC. The keratinolytic protease was partially purified by concentration in a centrifugal filter device (10,000 NMWL) and anion-exchange chromatography. The partially purified protease was active in a pH range of 5.0-10.0 with high activity in temperature range of 50-80 ºC. The optimum activity was observed at pH 7.0 and 50-60 ºC, making it suitable for biotechnological processes for keratin bioconversion. A molecular study conducted to identify the presumable keratinolytic protease produced by Anoxybacillus sp. PC2 identified a neutral metalloprotease whose gene showed 98% similarity with Geobacillus stearothermophilus nprS gene.

Queratinases são proteases de especial interesse devido a sua ação sobre a queratina insolúvel, que por sua vez, é o principal componente das penas. As penas de aves são fonte de aminoácidos, e, portanto, são transformadas em farinha de pena e utilizadas em ração animal. Entretanto, este produto possui baixo valor nutricional devido a sua baixa digestibilidade, já que a queratina não é degradada por enzimas proteolíticas comuns. Nesse contexto, as queratinases microbianas são uma abordagem promissora para conversão de queratina em hidrolisados proteicos. Os objetivos deste trabalho foram selecionar e identificar bactérias termófilas provenientes do bioma Caatinga (Brasil) capazes de produzir queratinases termoativas, e caracterizar a queratinase produzida pelo isolado selecionado. Oito bactérias proteolíticas foram isoladas de uma amostra de solo. Destas, o Anoxybacillus sp. PC2 produziu queratinase com maior atividade em meio composto por penas (2%), KH2PO4 (0,04%), K2HPO4 (0,2%) e NaCl (0.05%) em pH 6,5 e 60 ºC. A protease queratinolítica foi parcialmente purificada por concentração em dispositivo de filtração (10.000 NMWL) e cromatografia de troca aniônica. A protease parcialmente purificada foi ativa em uma faixa de pH de 5 a 10, com alta atividade entre as temperaturas de 50 e 80 ºC. A atividade ótima foi observada em pH 7 e entre 50 e 60 ºC, tornando-a adequada para utilização em processos biotecnológicos para bioconversão de queratina. Um estudo molecular conduzido para identificar a provável protease queratinolítica produzida pelo Anoxybacillus sp. PC2 identificou uma metaloprotease neutra, cujo gene apresentou 98% de similaridade com o gene nprS de Geobacillus stearothermophilus. / Keratinases are proteases of special interest due to their action on insoluble keratin, which in turn, is the major component of feathers. The poultry feathers are a source of amino acids, and therefore, are converted to feather meal to be used as animal feedstuff. However, this product has low nutritional value due to its poor digestibility, since keratin is not degraded by common proteolytic enzymes. In this context, microbial keratinases are a promising approach to convert keratin into protein hydrolysates. The aims of this study were to select and identify thermophilic bacteria from Caatinga biome (Brazil) capable to produce thermoactive keratinases, and characterize the keratinase produced by the selected isolate. Eight proteolytic bacteria were isolated from a soil sample. Of these, Anoxybacillus sp. PC2 produced keratinase with higher activity in a medium composed of 2% feather waste, 0.04% KH2PO4, 0.2% K2HPO4 and 0.05% NaCl at pH 6.5 incubated at 60 ºC. The keratinolytic protease was partially purified by concentration in a centrifugal filter device (10,000 NMWL) and anion-exchange chromatography. The partially purified protease was active in a pH range of 5.0-10.0 with high activity in temperature range of 50-80 ºC. The optimum activity was observed at pH 7.0 and 50-60 ºC, making it suitable for biotechnological processes for keratin bioconversion. A molecular study conducted to identify the presumable keratinolytic protease produced by Anoxybacillus sp. PC2 identified a neutral metalloprotease whose gene showed 98% similarity with Geobacillus stearothermophilus nprS gene.

This diploma thesis is focused on isolation and identification of thermophilic microorganisms capable of production of polyhydroxyalkanoates (PHA) in the sample of activated sludge from wastewater treatment. 6 culture samples were isolated from activated sludge by means of cultivation technics and methods of molecular biology. They were closer specified by comparing nucleotide sequences of 16S-rRNA gene and assigned to bacterial genus Anoxybacillus. The production of PHA by this genus was not reported in literature so far. Samples were confirmed to contain phaC gene that codes the enzyme PHA-synthase and they also gave a positive response to staining colonies with Nile red, what refers to presence of intracellular lipidic structures. However, the PHA production by isolates was not successful. The reason should be an inappropriate production medium or conditions. The positive phenotype result of Nile red dyeing was probably achieved by production of huge amount of lipids by bacterial cells that provides similar fluorescence than PHA granules.

Thesis (Ph. D. (Microbiology)) -- University of Limpopo, 2020 / The use of lignocellulosic biomass for bioethanol production is largely dependent on cost effective production of cellulase enzymes and most importantly, the availability of cellulases with sufficient β-glucosidase activity for complete hydrolysis of cellulose to glucose. Commercial cellulase preparations are often inefficient in the complete hydrolysis of cellulose to glucose. The addition of β-glucosidases to commercial cellulase preparations may enhance cellulolytic activity in the saccharification of cellulose to fermentable sugars. A β-glucosidase producing thermophilic bacterium, Anoxybacillus sp. KTC2 was isolated from a hot geyser in the Zambezi Valley, Zimbabwe. The bacterium identified through biochemical tests and 16S rDNA sequencing, had an optimal growth temperature and pH of 60˚C and pH 8, respectively. The β-glucosidase enzyme had an optimal temperature of 60˚C and a broad pH range for activity, between 4.5 and 7.5 with an optimum at pH 7. The β-glucosidase enzyme retained almost 100% activity after 24 hours’ incubation at 50˚C. The Anoxybacillus sp. KTC2 β-glucosidase was partially purified and a partial amino acid sequence obtained through MALDI-TOF analysis. The whole genome of Anoxybacillus sp KTC2 β-glucosidase was sequenced and a β-glucosidase gene identified. The deduced amino acid sequence corresponded to the peptide sequences obtained through MALDI-TOF, confirming the presence of the a β glucosidase on the genome of Anoxybacillus sp KTC2. Analysis of the deduced amino acid sequence revealed that the β-glucosidase enzyme belongs to the GH family 1. The β-glucosidase gene was isolated by PCR and successfully cloned into an E. coli expression system. The saccharification efficiency of the β-glucosidase enzyme was evaluated through the creation of enzyme cocktails with the commercial cellulase preparation, CelluclastTM. CelluclastTM with the Anoxybacillus sp KTC2 β-glucosidase were used to hydrolyse pure Avicel cellulose, at 50˚C over a 96 hour reaction time. The Anoxybacillus sp KTC2 β-glucosidase enabled a 25% decrease in the total cellulose loading without a decrease in the amount of glucose released. / University of Limpopo staff development programme and VLIR

The growth of thermophiles during the manufacture of milk powder leads to a progressive increase in the number of thermophilic bacteria contaminating the final product. The limited residence time of the milk in the plant during milk powder manufacture and the concentration effect of converting milk into milk powder cannot explain the number of thermophiles found in the final product. This suggests that thermophiles are attaching to the large surface area of stainless steel found within a milk powder plant and then growing and developing into biofilms, with individual cells and/or biofilm fragments sloughing off into the product line and thus contaminating the final product. The aim of the present study was to investigate the attachment mechanisms that enable the thermophile Anoxybacillus flavithermus (B 1 2) to attach to stainless steel surfaces. Passing a B 1 2 culture through a column of stainless steel chips, collecting the first cells to pass through, re-culturing and repeating the process six times, resulted in the isolation of a mutant, labelled X7, with lO-fold reduced ability to attach to stainless steel as well as a reduced ability to attach to plastic and glass. A comparison of bacterial cell surface properties indicated that X7 was less hydrophobic than its parental strain B 1 2 . Cell surface charge measurements also suggest that X7 has less net negative surface charge. Disruption of extracellular polysaccharides and DNA appeared to have no effect on the attachment process. Removal of surface proteins caused a reduction in attachment of B 1 2 and X7 as well as a reduction in surface hydrophobicity suggesting surface protein involvement in both. Analysis by two-dimensional gel electrophoresis of lysozyme/mutanolysin extracted surface proteins revealed two proteins expressed at reduced levels in X 7 compared with B 1 2 . One protein was identified by mass spectrometry as the cytoplasmic enzyme Formate acetyltransferase. The role of Formate acetyltransferase and the second unidentified protein on the attachment process of Anoxybacillus flavithermus remains unclear.