Purification, biochemical characterization, and biotechnological applications of a multifunctional enzyme from the Thermoascus aurantiacus PI3S3 strain.

The filamentous Thermoascus aurantiacus fungus characterized by its thermophilic nature, is recognized as an exceptional producer of various enzymes with biotechnological applications. This study aimed to explore biotechnological applications using polygalacturonase (PG) derived from the Thermoascus aurantiacus PI3S3 strain. PG production was achieved through submerged fermentation and subsequent purification via ion-exchange chromatography and gel filtration methods. The crude extract exhibited a diverse spectrum of enzymatic activities including amylase, cellulase, invertase, pectinase, and xylanase. Notably, it demonstrated the ability to hydrolyze sugarcane bagasse biomass, corn residue, and animal feed. The purified PG had a molecular mass of 36 kDa, with optimal activity observed at pH 4.5 and 70 °C. The activation energy (Ea) was calculated as 0.513 kJ mol-1, highlighting activation in the presence of Ca2+. Additionally, it displayed apparent Km, Vmax, and Kcat values of at 0.19 mg mL-1, 273.10 U mL-1, and 168.52 s-1, respectively, for hydrolyzing polygalacturonic acid. This multifunctional PG exhibited activities such as denim biopolishing, apple juice clarification, and demonstrated both endo- and exo-polygalacturonase activities. Furthermore, it displayed versatility by hydrolyzing polygalacturonic acid, carboxymethylcellulose, and xylan. The T. aurantiacus PI3S3 multifunctional polygalacturonase showed heightened activity under acidic pH, elevated temperatures, and in the presence of calcium. Its multifunctional nature distinguished it from other PGs, significantly expanding its potential for diverse biotechnological applications.

Biopolishing of denim by the recombinant xylanase II of Caulobacter crescentus.

Denim, also known as jeans, is a fabric made up of braided cotton threads dyed indigo blue, whose fibers contain approximately 10% of non-cellulosic impurities that reduce its commercial value. Microbial enzymes can act in the cleaning and desizing processes of jeans, improving their color, softness, and covering capacity. The recombinant Xylanase II (XynA2) from the aquatic bacterial Caulobacter crescentus (C. crescentus), previously characterized in terms of its biochemical features, was applied to the biotreatment of jeans to clean and degum it. The biotreatment performance was evaluated in terms of tissue weight loss, amount of reducing sugars released and analysis of the images obtained by scanning electron microscopy (SEM). Biotreated tissues, at 12 and 24 h, showed a dry weight loss of 4.9 and 6.6%, respectively. The reducing sugars amount released after XynA2 action over the jean's fibers showed statistically significant values when compared with each other and with their respective controls. SEM images clearly shown that the fabric treated for 12 h presented a smooth and polished surface, while the fabric treated for 24 h showed the cotton fibers broken, displaying severe damage to the textile. The best treatment for the jeans was in the presence of 1 U mg-1 XynA2 at pH 8 and 60 °C during 12 h. In conclusion, XynA2 of C. crescentus was satisfactorily applied for the biopolishing of denim jeans being a more sustainable alternative to the use of chemical and abrasive processes to obtain the same effects.

Prebiotic effect of sorghum biomass xylooligosaccharides employing immobilized endoxylanase from Thermomyces lanuginosus PC7S1T.

Purified endoxylanase from Thermomyces lanuginosus PC7S1T was immobilized in calcium alginate, resulting in a yield of 78.5% and a reusability for 11 cycles. The stability of the immobilized enzyme was given for a pH range of 4 to 9 for 96 h. Endoxylanase immobilized in calcium alginate at 65 °C exhibited thermal stability equal to the soluble enzyme for 5 h, and at high temperatures of 75 °C and 85 °C showed half-lives of 4 and 3 h, respectively. Both soluble endoxylanase and immobilized forms were able to hydrolyze hemicellulose, obtained from low-lignin sorghum biomass pretreated with 5% H2O2 and 2% NaOH, after 1 h of incubation at 65 °C, releasing a mixture of short-chain xylooligosaccharides (X2-X6). The highest amounts of XOS generated were those for X5 (24 to 40%), X4 (33 to 39%), and X3 (11 to 22%). These XOS acted as prebiotics, promoting the growth of the probiotic L. acidophilus, similar to glucose in the MRS broth. These results show the potential of low-lignin sorghum to generate XOS with prebiotic activity, suggesting the application of these compounds in the food industry.

Spike protein of SARS-CoV-2 variants: a brief review and practical implications.

The scientific community has been alarmed by the possible immunological evasion, higher infectivity, and severity of disease caused by the newest variants of SARS-CoV-2. The spike protein has an important role in the cellular invasion of viruses and is the target of several vaccines and therapeutic resources, such as monoclonal antibodies. In addition, some of the most relevant mutations in the different variants are on the spike (S) protein gene sequence that leads to structural alterations in the predicted protein, thus causing concern about the protection mediated by vaccines against these new strains. The present review highlights the most recent knowledge about COVID-19 and vaccines, emphasizing the different spike protein structures of SARS-CoV-2 and updating the reader about the emerging viral variants and their classifications, the more common viral mutations described and their distribution in Brazil. It also compiles a table with the most recent knowledge about all of the Omicron spike mutations.

Production, immobilization and application of invertase from new wild strain Cunninghamella echinulata PA3S12MM.

AIMS: The objective of this study was to determine the best conditions to produce invertase by Cunninghamella echinulata PA3S12MM and to immobilize and apply the enzyme. METHODS AND RESULTS: The maximum production was verified in 8 days of cultivation at 28°C supplemented with 10 g L-1 apple peel, reaching 1054.85 U ml-1 . The invertase was purified from the DEAE-Sephadex column. The derivative immobilized in alginate-gelatin-calcium phosphate showed reusability >50% for 19 cycles. The derivative immobilized in glutaraldehyde-chitosan showed greater thermostability and at a different pH. The hydrolysis of 15 ml of sucrose 500 g L-1 in a fixed bed reactor (total volume of 31 ml) produced 24.44 µmol min-1 of glucose and fructose at a residence time of 30 min and a conversion factor of 0.5. CONCLUSIONS: The new wild strain C. echinulata PA3S12MM presents high invertase production in medium supplemented with an agro-industrial residue and the immobilized enzyme showed high thermal stability and resistance at a different pH. SIGNIFICANCE AND IMPACT OF THE STUDY: The fungus C. echinulata PA3S12MM is an excellent producer of invertases in Vogel medium supplemented with apple peel. The enzyme is promising for industrial application since it has good performance in reusability and inverted sugar production.

Cunninghamella echinulata PA3S12MM invertase: Biochemical characterization of a promiscuous enzyme.

The Cunninghamella echinulata PA3S12MM fungus is a great producer of invertases in a growth medium supplemented by apple peels. The enzyme was purified 4.5 times after two chromatographic processes, and it presented a relative molecular mass of 89.2 kDa. The invertase reached maximum activity at pH of 6 and at 60°C, in addition to presenting stability in alkaline pH and thermal activation at 50°C. The enzymatic activity increased in the presence of Mn2+ and dithiothreitol (DTT), while Cu2+ and Z2+ ions inhibited it. Also, DTT showed to protect enzymatic activity. The apparent values for Km , Vmáx , and Kcat for the sucrose hydrolysis were, respectively, 173.8 mmol/L, 908.7 mmol/L min-1 , and 1,388.79 s-1 . The carbohydrate content was of 83.13%. The invertase presented hydrolytic activity over different types of glycosidic bonds, such as α1 â†” 2ß (sucrose), α1 â†’ 4 (polygalacturonic acid), α1 â†’ 4 and α1 â†’ 2 (pectin), and α1 â†” 1 (trehalose), indicating that the enzyme is multifunctional. Thus, the biochemical properties showed by the C. echinulata PA3S12MM suggest a broad industrial application, such as in the biomass hydrolysis or in the food industry. PRACTICAL APPLICATIONS: Invertases are hydrolytic enzymes employed in several industrial sectors. Given their great importance for the economy and several industrial sectors, there is a growing interest in microorganisms producing this enzyme. The analysis of the biochemical properties of invertase in C. echinulata PA3S12MM suggest applications in the food industry. Due to its increased hydrolytic activity, the hydrolysis process of the sucrose may employ invertase for the production of invert sugar. The stability at alkaline pH suggests an application in the development of enzymatic electrodes for the quantification of sucrose in food and beverage. The multifunctional activity may work in the biomass hydrolysis or saccharification of by-products for the extraction of fermentable sugars. The high level of invertase N-linked glycosylation of invertase grants this enzyme thermal stability at high temperatures, in addition to resistance against the action of proteases, which are desirable characteristics for the application of this enzyme in industrial processes.

Cloning, expression and characterization of C. crescentus xynA2 gene and application of Xylanase II in the deconstruction of plant biomass.

Biotechnology offers innovative alternatives for industrial bioprocesses mainly because it uses enzymes that biodegrade the hemicellulose releasing fermentable sugars. Caulobacter crescentus (C. crescentus) has seven genes responsible for xylanolytic cleavage, 5 to ß-xylosidases (EC 3.2.1.37) and 2 for endoxylanases, like xynA2 (CCNA_03137) that encodes Xylanase II (EC 3.2.1.8) of the glycohydrolases-GH10 group. The xynA2 gene was amplified by PCR, cloned into the pTrcHisA vector e efficiently overexpressed in E. coli providing a His-tag fusion protein. Recombinant xylanase (XynA2) was purified by affinity chromatography using a nickel sepharose column and exhibited a single 43 kDa band on SDS-PAGE gel. XynA2 showed an optimum alkaline pH (8) and stability at alkaline pH for 24 h. Although C. crescentus is mesophilic, XynA2 has optimum temperature of 60 °C and is thermo-resistance at 65 °C. XynA maintains 66% of the enzymatic activity at high temperatures (90 °C) without being denatured.The enzyme displayed a xylanolitic activity free of cellulase to xylan from beechwood and it was not inhibited in the presence of 50 µmol mL-1 of xylose. In addition, dithiothreitol (DTT) induced XynA2 activity, as it improved its kinetic parameters by lowering the KM (5.78 µmol mL-1) and increasing the KCat/KM ratio (1.63 U s-1). Finally, C. crescentus XynA2 efficiently hydrolyzed corn straw with high release of reducing sugars that can be applied in different branches of the industry.

Upregulation of the clpB gene in response to heat shock and beta-lactam antibiotics in Acinetobacter baumannii.

The role of the clpB gene encoding HSP/chaperone ClpB was evaluated in the multiresistant antibiotic cells of Acinetobacter baumannii (RS4 strain) under stress-induced heat shock and different beta-lactams. The expression of the clpB gene was assessed by qPCR during heat shock at 45 °C and subinhibitory concentrations of ampicillin (30 µg mL-1), amoxicillin + sulbactam (8/12 µg mL-1), cefepime (30 µg mL-1), sulfamethoxazole + trimethoprim (120/8 µg mL-1) and meropenem (18 µg mL-1). The results indicated a transient increase in clpB transcription in all treatments except cefepime. Both in the presence of ampicillin and amoxicillin/sulbactam for 20 min, the mRNA-clpB synthesis was 1.4 times higher than that of the control at time zero. Surprisingly, the mRNA-clpB levels were more than 30-fold higher after 10 min of incubation with meropenem and more than eightfold higher in the presence of trimethoprim/sulfamethoxazole. In addition, western blot assays showed that the RS4 strain treated with meropenem showed a marked increase in ClpB protein expression. Our data indicate that during exposure to beta-lactams, A. baumannii adjusts the transcription levels of the clpB mRNA and protein to respond to stress, suggesting that the chaperone may act as a key cellular component in the presence of antibiotics in this bacterium.

Enhance of Cellulase Production and Biomass Degradation by Transformation of the Trichoderma reesei RUT-C30∆ zface1 Strain

Abstract The second-generation bioethanol employs lignocellulosic materials degraded by microbial cellulases in their production. The fungus Trichoderma reesei is one of the main microorganisms producing cellulases, and its genetic modification can lead to the optimization in obtaining hydrolytic enzymes. This work carried out the deletion of the sequence that encodes the zinc finger motif of the transcription factor ACE1 (cellulase expression repressor I) of the fungus T. reesei RUT-C30. The transformation of the RUT-C30 lineage was confirmed by amplification of the 989 bp fragment relative to the selection marker, and by the absence of the zinc finger region amplification in mutants, named T. reesei RUT-C30Δzface1. The production of cellulases by mutants was compared to RUT-C30 and measured with substrates carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel®) and Whatman filter paper (PF). The results demonstrated that RUT-C30Δzface1 has cellulolytic activity increased 3.2-fold in Avicel and 2.1-fold in CMC and PF. The mutants presented 1.4-fold higher sugar released in the hydrolysis of the biomass assays. These results suggest that the partial deletion of ace1 gene is an important strategy in achieving bioethanol production on an industrial scale at a competitive price in the fuel market.

Immobilization and stabilization of different ß-glucosidases using the glutaraldehyde chemistry: Optimal protocol depends on the enzyme.

Three ß-glucosidases (Pectinex Ultra SP-L, Pectinex Ultra Clear and homemade preparation from Aspergillus niger) were immobilized using different strategies: ionic adsorption on aminated (MANAE)-agarose beads at pH 5, 7, and 9, followed by biocatalysts modification with glutaraldehyde, or on glutaraldehyde pre-activated supports. The pH of the immobilization was altered to allow different enzyme molecule orientations on the support surface. The biocatalysts from Pectinex Ultra SP-L showed the highest thermal and operational stabilities when immobilized on MANAE-agarose-glutaraldehyde at pH 7. The ß-glucosidase from Pectinex Ultra Clear and from A. niger produced best results when immobilized on MANAE-agarose beads at pH 5 and 7, respectively, which was later treated with glutaraldehyde. The best immobilization results using pre-activated supports were observed for the enzyme present in Pectinex Ultra SP-L, to which the highest thermal stabilities were obtained. Remarkably, the enzyme from A. niger, immobilized on MANAE-agarose at pH 9 and subsequently treated with glutaraldehyde, produced the highest stabilization (approximately 560 times more stable than soluble enzyme at 60 °C). Results showed that optimal protocol for ß-glucosidases immobilizations using the glutaraldehyde chemistry must be individually tested and tailored to each type of enzyme.

High levels of ß-xylosidase in Thermomyces lanuginosus: potential use for saccharification

ABSTRACT A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and its β-xylosidases optimization in response to agro-industrial residues was performed. Using statistical approach as a strategy for optimization, the induction of β-xylosidases activity was evaluated in residual corn straw, and improved so that the optimum condition achieved high β-xylosidases activities 1003 U/mL. According our known, this study is the first to show so high levels of β-xylosidases activities induction. In addition, the application of an experimental design with this microorganism to induce β-xylosidases has not been reported until the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60 °C showing better thermostability at 55 °C. The saccharification ability of β-xylosidase in the presence of hemicellulose obtained from corn straw raw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50 °C. Our data strongly indicated that the β-xylosidases activities was not subjected to the effects of potential enzyme inhibitors often produced during fermentation process. These data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production.

ß-(1→3)-Glucan of the Southern Bracket Mushroom, Ganoderma australe (Agaricomycetes), Stimulates Phagocytosis and Interleukin-6 Production in Mouse Peritoneal Macrophages.

Ganoderma australe was studied to determine the composition of the cell wall, and polysaccharide fraction SK5 was obtained after freeze-thawing an aqueous 5% potassium hydroxide extraction. The monosaccharide composition of the SK5 fraction revealed by gas chromatography-mass spectrometry showed 81.3% glucose, and analyses by 13C nuclear magnetic resonance spectroscopy confirmed a ß-glucan with glycosidic links of the (1→3)-ß type and most likely 4-O substituted. In addition, the biological effect of the ß-glucan from G. australe was evaluated via in vitro cell cultures of peritoneal macrophages isolated from Swiss mice. Biological assays were assessed for toxicity and cell activation, interleukin-6 cytokine concentrations, and the ability to stimulate phagocytic activity. There was an increase in interleukin-6 by approximately 111% with 1.0 µg/mL of polysaccharide, and phagocyte activity was increased in all concentrations examined, obtaining 52.3% with 0.25 µg/mL polysaccharide. The results indicate that a ß-(1→3)-glucan isolated from G. australe can be classified as a biological response modifier.

High levels of ß-xylosidase in Thermomyces lanuginosus: potential use for saccharification.

A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and its ß-xylosidases optimization in response to agro-industrial residues was performed. Using statistical approach as a strategy for optimization, the induction of ß-xylosidases activity was evaluated in residual corn straw, and improved so that the optimum condition achieved high ß-xylosidases activities 1003U/mL. According our known, this study is the first to show so high levels of ß-xylosidases activities induction. In addition, the application of an experimental design with this microorganism to induce ß-xylosidases has not been reported until the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60°C showing better thermostability at 55°C. The saccharification ability of ß-xylosidase in the presence of hemicellulose obtained from corn straw raw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50°C. Our data strongly indicated that the ß-xylosidases activities was not subjected to the effects of potential enzyme inhibitors often produced during fermentation process. These data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production.

Proteomic profile of hemolymph and detection of induced antimicrobial peptides in response to microbial challenge in Diatraea saccharalis (Lepidoptera: Crambidae).

Insects are organisms extremely well adapted to diverse habitats, primarily due to their innate immune system, which provides them with a range of cellular and humoral responses against microorganisms. Lepidoptera hemolymph proteins involved in humoral responses are well known; however, there is a lack of knowledge about the sugarcane borer Diatraea saccharalis. In this present work, the hemolymph proteins of this pest insect were studied by applying proteomic methodologies. Two-dimensional electrophoresis (2-DE) gels of proteins extracted from naive larvae and larvae challenged with Escherichia coli (ATCC 11224) and Bacillus subtilis (ATCC 6623) showed an average of 300 spots, and 92 of these spots corresponded in all three 2-DE gels. Forty-one spots were excised and digested with trypsin and analyzed using mass spectrometry. After analysis, 10 proteins were identified, including some proteins of the immune system: ß-defensin-like protein, Turandot A-like protein, attacin-like protein, peptidoglycan recognition protein and cyclophilin-like protein. Nine proteins were present in both experimental conditions; however, ß-defensin-like protein was present only in hemolymph challenged by B. subtilis. Notably, attacin-like protein was strongly induced by challenge with E. coli, suggesting an immune response against the infection. However, antimicrobial activity was observed in the test zone of microbial growth inhibition of B. subtilis solely with the hemolymph extract of the larvae challenged with B. subtilis. We made for the first time a proteomic profile of the hemolymph of D. saccharalis in which it was possible to identify the presence of important proteins involved in the immune response.

Analysis of the xynB5 gene encoding a multifunctional GH3-BglX ß-glucosidase-ß-xylosidase-α-arabinosidase member in Caulobacter crescentus.

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted ß-glucosidase-ß-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), ß-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for ß-glucosidase (pNPG: p-nitrophenyl-ß-D-glucoside) ß-xylosidase (pNPX: p-nitrophenyl-ß-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for ß-glucosidase and α-L-arabinosidase and 60 °C for ß-xylosidase. BglX-V-Ara predominantly presented ß-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (Km 0.24 ± 0.0005 mM, Vmax 0.041 ± 0.002 µmol min(-1) mg(-1) and Kcat/Km 0.27 mM(-1) s(-1)), followed by ß-xylosidase (Km 0.64 ± 0.032 mM, Vmax 0.055 ± 0.002 µmol min(-1) mg(-1) and Kcat/Km 0.14 mM(-1)s(-1)) and finally α-L-arabinosidase (Km 1.45 ± 0.05 mM, Vmax 0.091 ± 0.0004 µmol min(-1) mg(-1) and Kcat/Km 0.1 mM(-1) s(-1)). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.

Short-chain fructooligosaccharides do not alter glucose homeostasis but improve the lipid profile in obese rats / Frutooligossacarídeos de cadeia curta na dieta não altera homeostase glicêmica, porém melhora o perfil lipídico em ratos obesos

The present study investigated the effects of short-chain fructooligosaccharides (scFOS) feeding on body weight, fat accumulation, glucose homeostasis and lipid profile in cafeteria (CAF) obese rats. Male Wistar rats were divided randomly into two groups: control group (CTL, n = 10), which received a chow diet and water and CAF (n = 20), which received the cafeteria diet, standard chow and soda. After 30 weeks of diet, 10 animals of CAF group received scFOS in the diet (50 g kg-1 of diet) over a period of 50 days, forming the CAF FOS group. Were evaluated the body weight, fat pad as well as, quantity of feces, glucose tolerance, insulin resistance (IR) and serum lipids levels. Animals submitted to the CAF diet displayed obesity, hyperglycemia, glucose intolerance, hyperinsulinemia and IR. The scFOS feeding not altered obesity, glucose intolerance, hyperinsulinemia and IR. CAF rats also presented hypertriglyceridemia and lower levels of HDL-cholesterol. The CAF FOS animals had reduced serum triglycerides (TG) and increased HDL-cholesterol. Thus, the use of scFOS in the diet can be considered as a hypolipidemic agent in the obese state.

O presente estudo investigou os efeitos da adição de frutooligossacarídeos de cadeia curta (scFOS) sobre o peso corporal, acúmulo de gordura, homeostase glicêmica e perfil lipídico em ratos obesos pela dieta de cafeteria (CAF). Ratos Wistar foram divididos em dois grupos: controle (CTL, n = 10), que receberam dieta padrão e água e CAF, que receberam dieta de CAF, ração padrão e refrigerante (n = 20). Após 30 semanas, dez animais do grupo CAF receberam 50 g kg-1 de dieta de scFOS na ração padrão durante 50 dias, formando o grupo CAF FOS. Foram avaliados o peso corporal e o peso das gorduras, bem como, quantidade de fezes, homeostase glicêmica e concentração de lipídios séricos. Animais do grupo CAF apresentaram obesidade, hiperglicemia, intolerância à glicose, hiperinsulinemia e RI. A adição scFOS não alterou a obesidade, intolerância à glicose, hiperinsulinemia e RI no grupo CAF FOS comparado ao grupo CAF. Animais CAF também apresentaram hipertrigliceridemia e redução na concentração de HDL-colesterol. Os animais CAF FOS apresentaram redução na concentração sérica de triglicerídeos (TG) e aumento no HDL-colesterol. Desta forma, a utilização de scFOS na dieta pode ser considerado como um agente hipolipidêmico nos estados de obesidade.

Cloning and expression of the xynA1 gene encoding a xylanase of the GH10 group in Caulobacter crescentus.

Caulobacter crescentus (NA1000 strain) are aquatic bacteria that can live in environments of low nutritional quality and present numerous genes that encode enzymes involved in plant cell wall deconstruction, including five genes for ß-xylosidases (xynB1-xynB5) and three genes for xylanases (xynA1-xynA3). The overall activity of xylanases in the presence of different agro-industrial residues was evaluated, and it was found that the residues from the processing of corn were the most efficient in inducing bacterial xylanases. The xynA1 gene (CCNA_02894) encoding a predicted xylanase of group 10 of glyco-hydrolases (GH10) that was efficiently overexpressed in Escherichia coli LMG194 using 0.02 % arabinose, after cloning into the vector pJet1.2blunt and subcloning into the expression vector pBAD/gIII, provided a fusion protein that contained carboxy-terminal His-tags, named XynA1. The characterization of pure XynA1 showed an enzymatic activity of 18.26 U mL(-1) and a specific activity of 2.22 U mg-(1) in the presence of xylan from beechwood as a substrate. XynA1 activity was inhibited by EDTA and metal ions such as Cu(2+) and Mg(2+). By contrast, ß-mercaptoethanol, dithiothreitol (DTT), and Ca(2+) induced recombinant enzyme activity. Kinetic data for XynA1 revealed K M and V max values of 3.77 mg mL-(1) and 10.20 µM min-(1), respectively. Finally, the enzyme presented an optimum pH of 6 and an optimum temperature of 50 °C. In addition, 80 % of the activity of XynA1 was maintained at 50 °C for 4 h of incubation, suggesting a thermal stability for the biotechnological processes. This work is the first study concerning the cloning, overexpression, and enzymatic characterization of C. crescentus xylanase.

Expression and characterization of a GH39 ß-xylosidase II from Caulobacter crescentus.

In the present work, the gene xynB2, encoding a ß-xylosidase II of the Glycoside Hydrolase 39 (GH39) family, of Caulobacter crescentus was cloned and successfully overexpressed in Escherichia coli DH10B. The recombinant protein (CcXynB2) was purified using nickel-Sepharose affinity chromatography, with a recovery yield of 75.5 %. CcXynB2 appeared as a single band of 60 kDa on a sodium dodecyl sulfate polyacrylamide gel and was recognized by a specific polyclonal antiserum. The predicted CcXynB2 protein showed a high homology with GH39 ß-xylosidases of the genus Xanthomonas. CcXynB2 exhibited an optimal activity at 55 °C and a pH of 6. CcXynB2 displayed stability at pH values of 4.5-7.5 for 24 h and thermotolerance up to 50 °C. The K (M) and V (Max) values were 9.3 ± 0.45 mM and 402 ± 19 µmol min(-1) for ρ-nitrophenyl-ß-D-xylopyranoside, respectively. The purified recombinant enzyme efficiently produced reducing sugars from birchwood xylan and sugarcane bagasse fibers pre-treated with a purified xylanase. As few bacterial GH39 family ß-xylosidases have been characterized, this work provides a good contribution to this group of enzymes.

The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus ß-Xylosidase I.

The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved ß-Xylosidase and α-L-Arabinofuranosidase (ß-Xyl I-α-L-Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant ß-Xyl I-α-L-Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant ß-Xyl I-α-L-Ara exhibited a specific ß-Xylosidase I activity of 1.25 U mg(-1) to oNPX and a specific α-L-Arabinofuranosidase activity of 0.47 U mg(-1) to pNPA. The predominant activity of the recombinant enzyme was its ß-Xylosidase I activity, and the enzymatic characterization was focused on it. The ß-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45 °C, and a high degree of stability was verified over 240 min at this temperature. Moreover, ß-Xylosidase activity was inhibited in the presence of the metals Zn(2+) and Cu(2+), and the enzyme exhibited K(M) and V(Max) values of 2.89 ± 0.13 mM and 1.4 ± 0.04 µM min(-1) to oNPX, respectively. The modeled structure of ß-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form.

DnaK and GroEL are induced in response to antibiotic and heat shock in Acinetobacter baumannii.

We studied the expression of DnaK and GroEL in Acinetobacter baumannii cells (strains ATCC 19606 and RS4) under stress caused by heat shock or antibiotics. A Western blot assay showed that DnaK and GroEL levels increased transiently more than 2-fold after exposure of bacterial cells to heat shock for 20 min at 50 degrees C. Heat induction of DnaK and GroEL was blocked completely when an inhibitor of transcription, rifampicin, was added 1 min before a temperature upshift to 50 degrees C, suggesting that the induction of these chaperones depends on transcription. A. baumannii cells pretreated at 45 degrees C for 30 min were better able to survive at 50 degrees C for 60 min than cells pretreated at 37 degrees C, indicating that A. baumannii is able to acquire thermotolerance. DnaK and GroEL were successfully induced in cells pre-incubated with a subinhibitory concentration of streptomycin. Moreover, bacterial cells pretreated for 30 min at 45 degrees C were better able to survive streptomycin exposure than cells pretreated at physiological temperatures. DnaK expression was upregulated in a multidrug-resistant strain of A. baumannii (RS4) in the presence of different antimicrobials (ampicillin+sulbactam, cefepime, meropenem and sulphamethoxazole+trimethoprim). This study is to the best of our knowledge the first to show that A. baumannii DnaK and GroEL could play an important role in the stress response induced by antibiotics.