A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40 degrees C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0-5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0-9.5. The temperature optimum was 65 degrees C and retained 100% activity after 240 min at 60 degrees C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-D-glucan glucohydrolase). The K(m) was calculated as 0.32 mg ml(-1). Circular dichroism spectroscopy estimated a secondary structure content of 33% alpha-helix, 17% beta-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.

Agro-industrial wastes are mainly composed of complex polysaccharides that might serve as nutrients for microbial growth and production of enzymes. The aim of this work was to study polygalacturonase (PG) production by Aspergillus niveus cultured on liquid or solid media supplemented with agro-industrial wastes. Submerged fermentation (SbmF) was tested using Czapeck media supplemented with 28 different carbon sources. Among these, orange peel was the best PG inducer. On the other hand, for solid state fermentation (SSF), lemon peel was the best inducer. By comparing SbmF with SSF, both supplemented with lemon peel, it was observed that PG levels were 4.4-fold higher under SSF. Maximum PG activity was observed at 55°C and pH 4.0. The enzyme was stable at 60°C for 90 min and at pH 3.0–5.0. The properties of this enzyme, produced on inexpensive fermentation substrates, were interesting and suggested several biotechnological applications.

An extracellular a-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS-PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical alpha-glucosidase activity, hydrolyzing p-nitrophenyl alpha-D-glucopyranoside and presented an optimum temperature and pH of 65 degrees C and 6.0, respectively. In the absence of substrate the purified alpha-glucosidase was stable for 60 min at 60 degrees C, presenting t(50) of 90 min at 65 degrees C. Hydrolysis of polysaccharide substrates by alpha-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and beta-ciclodextrin were poor substrates, and sucrose and alpha-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an a-helical content of 31% and a beta-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.

An extracellular amylase secreted by Aspergillus niveus was purified using DEAE fractogel ion exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5 % polyacrylamide gel electrophoresis (PAGE) and 10 % sodium dodecyl sulfate (SDS-PAGE). The enzyme exhibited 4.5 % carbohydrate content, 6.6 isoelectric point, and 60 and 52 kDa molar mass estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The amylase efficiently hydrolyzed glycogen, amylose, and amylopectin. The end-products formed after 24 h of starch hydrolysis, analyzed by thin layer chromatography, were maltose, maltotriose, maltotetraose, and maltopentaose, which classified the studied amylase as an α-amylase. Thermal stability of the α-amylase was improved by covalent immobilization on glyoxyl agarose (half-life of 169 min, at 70 °C). On the other hand, the free α-amylase showed a half-life of 20 min at the same temperature. The optima of pH and temperature were 6.0 and 65 °C for both free and immobilized forms.

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-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), β-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 β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-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 β-glucosidase and α-L-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-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 β-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.

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.

ABSTRACT

Aims The aim of this work was to characterize and apply a polygalacturonase of Penicillium janthinellum new strain VI2R3M. Methods and Results The polygalacturonase obtained from P. janthinellum VI2R3M was incubated in cultures of passion fruit peel and was partially purified by ion‐exchange chromatography and gel filtration. The enzyme showed a relative molecular mass of 102·0 kDa, maximum activity at pH 5·0, temperature of 50°C, 100% stablity at 50°C and 80% stablity at pH 3·0–5·0. The apparent Km, Vmax and Kcat values for hydrolyzing polygalacturonic acid were 2·56 mg ml−1, 163·1 U mg−1 and 277 s−1 respectively. The polygalacturonase presented exo activity and was activated by Mg2+. The juices treated with polygalacturonase presented increases in transmittance with reduction in colour. Conclusions The results suggest that the new lineage P. janthinellum VI2R3M presents a high yield of an exo‐polygalacturonase induced by agro‐industrial residues, with excellent activity and stability in acidic pH and at 50°C. Significance and Impact of the Study The use of agro‐industrial residue to obtain the polygalacturonase can contribute to a decrease enzyme production cost. The results of the activity, stability to acidic pH and excellent performance in the clarification of juices show that the enzyme is promising for industrial application.