Pedro Ricardo Vieira Hamann

Biofilm formation by Escherichia coli can contribute to persistence and reduced susceptibility to antimicrobial treatment. Here, we evaluated treatment-associated changes following individual exposure to preparations of the glycosidases SdBgl1B and dispersin B (DspB) and the cysteine protease papain in assays assessing changes in preformed biofilm biomass and in biomass formed during 24 h of enzyme exposure across four E. coli strains (042, 1007, P180, and 23699) using static crystal violet microtiter-plate assays. The assays comprised three independent biological replicates with three technical wells per replicate for each condition. Papain-treated wells showed reductions in preformed crystal-violet-stained biomass of up to 92.4% for strain 042 at 1.00 mg/mL and 81.4–90.8% for strain 23699 across the tested concentration range. DspB-treated wells showed 67–72% less preformed biomass for strain 23699 and approximately 89% less biomass formed over 24 h by strain 042 at 0.03–0.06 mg/mL. Exposure to SdBgl1B was associated with variable responses, including approximately 62% less biomass formed by strain 042 at the enzyme concentration of 0.50 mg/mL. These percentages describe changes relative to the corresponding controls, not statistically established rankings among strains or enzymes. Several enzyme–strain combinations displayed non-monotonic responses, and some treatments produced crystal violet signals above the control. The control scheme does not resolve vehicle- or preparation-dependent staining contributions, precluding enzyme-specific causal attribution. Because crystal violet staining quantifies total attached stained material rather than specific matrix cleavage or bacterial viability, these findings document changes in the attached biomass endpoint but do not establish biofilm eradication, a molecular degradation mechanism, or bactericidal activity. Further matrix characterization, viability, dispersal, and confocal microscopy assays are required to determine mechanism and assess translational potential.

Engineering broad‐spectrum disease resistance remains a major challenge in crop improvement because most resistance genes are effective against a limited range of pathogens. Pathogenesis‐related (PR) proteins, particularly endochitinases, are promising targets for enhancing plant immunity due to their antimicrobial activity and ability to activate defense responses. Here, we generated transgenic tomato ( Solanum lycopersicum L.) plants overexpressing an endochitinase gene to assess their potential for enhanced resistance against bacterial and fungal pathogens. Three independent overexpression lines and the wild type were evaluated for transgene expression, chitinase activity, defense‐related gene expression, and resistance after inoculation with Xanthomonas euvesicatoria pv. perforans (Xep) and Sclerotinia sclerotiorum . The transgene was strongly expressed in all transgenic lines, whereas chitinase activity increased by up to fivefold compared with the wild type. Endochitinase overexpression activated defense responses, with marked induction of PR1 expression, reaching a maximum increase of over 480‐fold, and enhanced expression of defense‐associated genes, including SlACS and SlACO . Compared with the wild type, the most responsive transgenic lines showed reductions of up to 79.7% in bacterial spot severity and 85.6% in white mold severity. These findings demonstrate that endochitinase overexpression activates defense signaling and improves broad‐spectrum resistance, representing a promising strategy for developing disease‐resistant tomato cultivars.

Starch-degrading enzymes are key biocatalysts in industrial applications, particularly when derived from thermophilic microorganisms with potential to operate under elevated temperatures. In this study, three recombinant starch-degrading enzymes were heterologously produced, purified, and biochemically characterized: an α-amylase from Geobacillus kaustophilus, and an α-glucosidase and a type I pullulanase from Geobacillus sp. G4, a thermophilic strain isolated from a geothermal field in southern Peru. The three enzymes were successfully expressed in soluble form in Escherichia coli and purified by one-step affinity chromatography. Biochemical characterization showed that α-glucosidase and α-amylase displayed optimum activity at pH 6–7, whereas pullulanase exhibited a broader pH profile, retaining high activity up to pH 9. All three enzymes reached maximum activity at 60 °C, although their thermal stability profiles differed markedly, with pullulanase showing the highest thermostability. Metal ion assays revealed enzyme-dependent effects, with pullulanase being stimulated by Ca²⁺ and Mg²⁺, while α-amylase and α-glucosidase showed limited responses to divalent ions. Kinetic analysis using soluble potato starch indicated that α-amylase had the most favorable catalytic profile, with the lowest K_m and the highest catalytic efficiency among the three enzymes. Functional hydrolysis assays demonstrated that all enzymes were active on soluble starch and pretreated potato peel, while the enzymatic mixture consistently released the highest concentration of reducing sugars. These results expand the biochemical knowledge of thermophilic amylolytic enzymes from Geobacillus and support their potential use in future enzymatic systems for the conversion of starch-rich residues.

Forests and natural environments are valuable sources of organisms capable of degrading plant cell wall carbohydrates. In the present study, two isolates, Penicillium polonicum and Penicillium chrysogenum , obtained from Brazilian Cerrado soil, along with a previously characterized isolate of Trichoderma harzianum , were evaluated for their ability to produce carbohydrate-active enzymes when grown in the presence of wheat bran. P. chrysogenum exhibited higher production of endoglucanases, xylanases, and pectinases. Biochemical characterization showed that most enzymes were active at pH 5 and within a temperature range of 30–50 °C, with the exception of P. chrysogenum endoglucanases, which displayed optimal activity at 60 °C. Hydrolysis of cellulose and carboxymethyl cellulose by the three enzyme sources demonstrated that supplementation with recombinant β-glucosidase from Clostridium thermocellum (BglA) significantly enhanced reducing sugar release; specifically, when applied to P. chrysogenum , the reducing sugar yield from cellulose hydrolysis increased by 2.53-fold. In the hydrolysis of wheat bran, enzymes from P. chrysogenum and T. harzianum supplemented with BglA resulted in a 1.6- and 3.93-fold increase in D-glucose yield, respectively. The results presented in this study contribute to the development of more robust enzyme cocktails for wheat bran valorization.

Residual lignin generated by pulp, paper, and biorefining industries is commonly burned for energy, despite its potential as a renewable source of aromatic compounds. Studies focusing on microbial lignin degradation contribute to lignin valorization and represent a sustainable strategy to enhance biomass circularity. Here, we report the isolation of Klebsiella sp. IL2_9 from a ruminal consortium and demonstrate its ability to degrade and metabolize organosolv lignin. After 24 h of cultivation, the strain removed 22% of the initial lignin content. FTIR analysis revealed alterations in functional groups associated with guaiacyl and syringyl units, indicating structural modification of the polymer. GC–MS analyses further showed the consumption of lignin-derived aromatics, including vanillin, 2-aminobenzoic acid, and 4-hydroxybenzoic acid, along with the formation of vanillyl alcohol and phenyllactic acid derivatives. Overall, these findings highlight the potential of Klebsiella sp. IL2_9 as a promising biotechnological candidate for lignin valorization under anaerobic conditions.

Pectin-degrading enzymes play a crucial role in various biotechnological applications, including food processing, lignocellulosic biomass deconstruction for biofuel production, and biorefinery processes. In recent years, there has been increasing demand for novel enzymes capable of efficiently hydrolyzing plant biomass. In this context, the present study investigated the production of pectinases by the filamentous fungus Trichoderma harzianum TR274. The enzymes produced were biochemically characterized, exhibiting optimal activity within a pH range of 5.0–6.0 and at a temperature of 50 °C. Although the pectinase-enriched enzymatic cocktail showed reduced thermal stability at 50 °C, it retained 55% of its activity at 40 °C after 4 h. Remarkably, plant cell wall-associated phenolic compounds, typically reported as enzyme inhibitors, such as trans-ferulic acid, p -coumaric acid, and syringic acid, significantly enhanced the activity of T. harzianum TR274 enzymes against apple pectin, leading to improved pectin hydrolysis. This result was not observed with a commercial enzyme. When applied to the hydrolysis of orange peel, the pectinase cocktail demonstrated an additive effect when combined with a thermostable xylanase. The findings reported here contribute valuable insights for the development of new pectinase-rich enzyme cocktails tailored for the deconstruction of fruit residues, with promising potential for future applications in biorefineries. Graphical Abstract

The genome of Geobacillus sp. G4, a thermophilic bacterium isolated from a geothermal field in Peru, was sequenced and analyzed to evaluate its taxonomic and biotechnological potential. This strain exhibits optimal growth at temperatures between 50 and 70 °C and at a pH range of 6.0–7.5. Phenotypic assays demonstrated extracellular enzymatic activities, including amylases, cellulases, pectinases, and xylanases, highlighting its potential for efficient polysaccharide degradation. The assembled genome comprises approximately 3.4 Mb with a G+C content of 52.59%, containing 3,490 genes, including coding sequences, rRNAs, and tRNAs. Functional annotation revealed genes associated with key metabolic pathways such as glycogen and trehalose biosynthesis, indicating adaptation to carbohydrate-rich environments. Phylogenetic analyses based on ANI and dDDH values identified Geobacillus thermoleovorans KCTC3570 as its closest relative, suggesting a strong evolutionary relationship. Additionally, the genome harbors gene clusters for secondary metabolites such as betalactone and fengycin, suggesting potential industrial and pharmaceutical applications, including bioremediation. The identification of antibiotic resistance genes, specifically those conferring glycopeptide resistance, underscores their relevance for antimicrobial resistance studies. The presence of enzymes like amylases and pullulanase further emphasizes its biotechnological potential, particularly in starch hydrolysis and biofuel production. Overall, this research highlights the significant potential of Geobacillus species as valuable sources of thermostable enzymes and biosynthetic pathways for industrial applications.

Glucanohydrolases have shown promise in degrading exopolysaccharides in cariogenic biofilms, making them a potential strategy for biofilm control without disrupting the oral microbiota. However, their direct antimicrobial effects remain unclear. Aim: To determine the antimicrobial activity on S. mutans of two newly discovered glucanases characterized by our group, PmGH87 (mutanase) from Prevotella melaninogenica and CoGH66 (dextranase) from Capnocytophaga ochracea, using a commercial dextranase from Penicillum sp. as a control. Methods: Their effects on growth were assessed using a luciferase reporter system coupled with the promoter of the ldh gene in Streptococcus mutans. Results: Quantification of optical density and luminescence over a 10-hour growth period revealed that the commercial dextranase exhibited inhibitory effects on S. mutans growth. However, these effects were neutralized by heat treatment, suggesting the presence of a heat-sensitive contaminant or an additional antimicrobial property associated with the commercial dextranase from Penicillum sp. On the other hand, the purified mutanase and dextranase enzymes had no inhibitory effect on S. mutans growth. Conclusion: In conclusion, the absence of inhibitory effects on S. mutans growth by the newly discovered enzymes emphasizes their potential for biofilm control while preserving the delicate balance of the oral microbiota and preventing the emergence of resistance.

Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products.

Lytic polysaccharide monooxygenases (LPMOs) are critical players in enzymatic deconstruction of cellulose. A number of LPMOs have been identified at a genomics level; however, they still need to be characterized and validated for use in industrial processes aimed at cellulose deconstruction. In the present study, we biochemically characterized a new LPMO, a member of auxiliary activities family 9 (AA9) from the filamentous fungus Aspergillus fumigatus (AfLPMO9D). This LPMO demonstrated higher efficiency against amorphous cellulose as compared to more recalcitrant forms of cellulose such as bacterial cellulose and Avicel. AfLPMO9D has a capacity to oxidize the substrate at either the C₁ or C₄ positions, with pH-dependent regioselectivity. Photoactivation experiments demonstrated that light-stimulated chlorophyllin triggers AfLPMO9D activation without requirements of an external electron donor. AfLPMO9D is capable of boosting phosphoric acid-swollen cellulose depolymerization via GH7 endoglucanase and cellobiohydrolase. The results of the present study might help to elucidate the role of different LPMOs in cellulosic fiber deconstruction.