Protease Production Research Articles

Low frequency magnetic field assisted production of acidic protease by Aspergillus niger.

Acid protease is widely used in industries such as food processing and feed additives. In the study, low frequency magnetic field (LF-MF) as an aid enhances acid protease production by Aspergillus niger (A. niger). The study assessed mycelial biomass, the enzymic activity of the acidic protease and underlying mechanism. At low intensities, alternating magnetic field (AMF) is more effective than static magnetic fields (SMF). Under optimal magnetic field conditions, acid protease activity and biomass increased by 91.44% and 16.31%, as compared with the control, respectively. Maximum 19.87% increase in enzyme activity after magnetic field treatment of crude enzyme solution in control group. Transcriptomics analyses showed that low frequency alternating magnetic field (LF-AMF) treatment significantly upregulated genes related to hydrolases and cell growth. Our results showed that low-frequency magnetic fields can enhance the acid protease production ability of A. niger, and the effect of AMF is better at low intensities. The results revealed that the effect of magnetic field on the metabolic mechanism of A. niger and provided a reference for magnetic field-assisted fermentation of A. niger.

Immobilization of alkaline protease produced by Streptomyces rochei strain NAM-19 in solid state fermentation based on medium optimization using central composite design.

This study evaluated Streptomyces rochei strain NAM-19 solid-state fermentation of agricultural wastes to produce alkaline protease. Alkaline protease production increased with flaxseed, rice bran, and cheese whey fermentation reaching 147U/mL at 48h. Statistical optimization of alkaline protease production was performed using the central composite design (CDD). Results of CDD and the optimization plot showed that 4.59g/L flaxseed, 4.31g/L rice bran, 4.17mL cheese whey, and a vegetative inoculum size of 7.0% increased alkaline protease production by 27.2% reaching 186U/mL. Using the 20-70% ammonium sulfate fractionation method, the optimally produced enzyme was partially purified to fivefold. The partially purified alkaline protease was then covalently immobilized on a biopolymer carrier, glutaraldehyde-polyethylene-imine-κ-carrageenan (GA-PEI-Carr), with 90% immobilization efficiency. Characterizations revealed that immobilization improved thermostability, reusability, optimum temperature, and sensitivity towards metal ions of the free enzyme. The optimal temperature for free and immobilized enzymes was 40 and 50°C, respectively. Both enzymes had the same optimum pH of 10. Immobilization increased Km from 19.73 to 26.52mM and Vmax from 56.7 to 62.5mmolmin-1L-1. The immobilized enzyme retained 35% of its initial activity at 70°C, while the free enzyme retained only 5%. The immobilized enzyme kept 80% of its initial activity at the 20th cycle. After 7weeks of storage, the free enzyme lost all its initial activity, whereas the immobilized enzyme retained 50%. The free and immobilized enzymes were able to hydrolyze gelatin, and azo-casein demonstrating different relative activity, 85, 80, 90 and 95%, respectively, compared to casein (100%).

Synergistic anti-virulence efficacy of citral and carvacrol against mixed vaginitis causing Candida albicans and Gardnerella vaginalis: An in vitro and in vivo study.

Mixed vaginitis due to bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) is the most prevalent form and presents a significant therapeutic challenge globally. Since, the administration of monotherapy leads to subsequent recurrent infections, synergistic therapy that completely eradicates both pathogens is of dire need to manage mixed vaginities scenario and to prevent its recurrence. The current investigation was focused on exploring the synergistic inhibitory efficacy of phytochemicals against the virulence traits of individual and mixed species of C. albicans and G. vaginalis in vitro and in vivo (Galleria mellonella). Out of five phytochemicals (carvacrol, thymol, cinnamaldehyde, eugenol, and borneol) screened for synergism with citral [(Ct) as the prime molecule owing to its myriad therapeutic potential], carvacrol (Ca) in combination with citral exhibited promising synergistic effect. Time-kill kinetics and one-minute contact-killing assays demonstrated the phenomenal microbicidal effect of Ct-Ca combination against both mono and dual-species within 30 min and one-minute time intervals, respectively. Furthermore, the sub-CMICs (synergistic combinatorial MIC) of Ct-Ca have significantly eradicated the mature biofilms and remarkably reduced the virulence attributes of both C. albicans and G. vaginalis (viz., yeast to hyphae transition, filamentation, protease production, and hydrophobicity index), in single and dual species states. The non-toxic nature of Ct-Ca combination was authenticated using in vitro (human erythrocyte cells) and in vivo (Galleria mellonella) models. In addition, the in vivo efficacy evaluation and subsequent histopathological investigation was done using the invertebrate model system G. mellonella, which further ascertained the effectiveness of Ct-Ca combination in fighting off the infection caused by individual and mixed species of C. albicans and G. vaginalis. Concomitantly, the current work is the first of its kind to delineate the in vitro interaction of C. albicans and G. vaginalis mixed species at their growth and biofilm states, together emphasizes the promising therapeutic potential of acclaimed phytochemicals as combinatorial synergistic therapy against mixed vaginitis.

Characterization of thermophilic bacteria from Ie Seum Hot Springs, Aceh Besar, Indonesia as producers of protease enzyme

Abstract. Sabaria E, Yasmin Y, Ismail YS, Bessania MA, Putri I, Fitri L. 2024. Characterization of thermophilic bacteria from Ie Seum Hot Springs, Aceh Besar, Indonesia as producers of protease enzyme. Biodiversitas 25: 1867-1874. Thermophilic bacteria are microorganisms that can survive in high-temperature environments exceeding 75°C. The Ie Seum Hot Springs in Aceh Besar, Indonesia are known to be a place where these thermophilic bacteria inhabit. In such extreme conditions, proteins and enzymes are often denatured, making these bacteria of interest due to their ability to adapt and produce enzymes such as proteases. Proteases are called proteolytic enzymes that can be applied both in the economic and medical fields. This study aimed to isolate and identify thermophilic bacteria species that have the highest potential for protease production in Ie Seum Hot Spring, Aceh Besar by analyzing the 16S rRNA gene and characterizing them using biochemical tests. Based on the result, seven thermophilic bacterial isolates, namely BT1, BT2, BT3, BT4, BT5, BT6, and BT7 were obtained, each showing different colony characteristics. The biochemical tests also revealed differences metabolic activity of each isolate. Of these isolates, BT4 exhibited the highest proteolytic index (4.65). Additionally, the 16S rRNA gene sequence analysis showed that the BT4 strain belongs to the genus Bacillus and has high similarities to Bacillus sp., B. licheniformis and B. sonorensis. These findings suggest that BT4 has significant potential as a source of thermophilic protease enzymes.

Lipase and Protease Production Ability of Multi-drug Resistant Bacteria Worsens the Outcomes of Wound Infections.

Surgical site infections are one of the major clinical problems in surgical departments that cost hundreds of millions of dollars to healthcare systems around the world. The study aimed to address the pressing issue of surgical site infections, which pose significant clinical and financial burdens on healthcare systems globally. Recognizing the substantial costs incurred due to these infections, the research has focused on understanding the role of lipase and protease production by multi-drug resistant bacteria isolated from surgical wounds in the development of post-surgical wound infections. For these purposes, 153 pus specimens were collected from patients with severe post-surgical wound infections having prolonged hospital stays. The specimens were inoculated on appropriate culture media. Gram staining and biochemical tests were used for the identification of bacterial growth on suitable culture media after 24 hours of incubation. The isolated pathogens were then applied for lipase and protease, key enzymes that could contribute to wound development, on tributyrin and skimmed milk agar, respectively. Following the CSLI guidelines, the Kirby-Bauer disc diffusion method was used to assess antibiotic susceptibility patterns. The results revealed that a significant proportion of the samples (127 out of 153) showed bacterial growth of Gram-negative (n = 66) and Gram-positive (n = 61) bacteria. In total, isolated 37 subjects were declared MDR due to their resistance to three or more than three antimicrobial agents. The most prevalent bacteria were Staphylococcus aureus (29.13%), followed by S. epidermidis (18.89%), Klebsiella pneumoniae (18.89%), Escherichia coli (14.96%), Pseudomonas aeruginosa (10.23%), and Proteus mirabilis (7.87%). Moreover, a considerable number of these bacteria exhibited lipase and protease activity with 70 bacterial strains as lipase positive on tributyrin agar, whereas 74 bacteria showed protease activity on skimmed milk agar with P. aeruginosa as the highest lipase (69.23%) and protease (76.92%) producer, followed by S. aureus (lipase 62.16% and protease 70.27%). The antimicrobial resistance was evaluated among enzyme producers and non-producers and it was found that the lipase and protease-producing bacteria revealed higher resistance to selected antibiotics than non-producers. Notably, fosfomycin and carbapenem were identified as effective antibiotics against the isolated bacterial strains. However, gram-positive bacteria displayed high resistance to lincomycin and clindamycin, while gram-negative bacteria were more resistant to cefuroxime and gentamicin. In conclusion, the findings suggest that lipases and proteases produced by bacteria could contribute to drug resistance and act as virulence factors in the development of surgical site infections. Understanding the role of these enzymes may inform strategies for preventing and managing post-surgical wound infections more effectively.

Exploring alkaline serine protease production and characterization in proteolytic bacteria Stenotrophomonas maltophilia: Insights from real-time PCR and fermentation techniques

This study focuses on identifying a potent microbe for efficient alkaline protease production, characterizing its properties, and investigating protease gene expression across diverse culture conditions. Stenotrophomonas maltophilia strain GEBN5 is methodically chosen and refined for peak alkaline protease production at 36 h (75.37 U/mL) in basal media employing precise adjustments in temperature, pH, incubation time, and carbon/nitrogen sources, achieving a noteworthy maximum protease activity of 97.03 U/mL in shake flask culture. Alkaline protease gene (StmPr1) up-regulation was explored via Real-time PCR under varied culture conditions (pH, temperature, carbon, and nitrogen sources) in a 2L fermenter at 150 rpm, resulting in peak protease production of 113 ± 0.53 U/mL in batch culture at pH 9.0, 35 °C, with sucrose and casein, respectively. The characterization data demonstrates exceptional stability of the current protease across a broad pH (9.0–11.0) and optimum temperature of 35 °C, highlighting its promising applications in diverse industrial and biotechnological settings. A higher instability complex of protein 28.59 was revealed by in silico analysis, indicating prolonged stability during hydrolysis and a wide range of potential uses. The purified protease demonstrates superior compatibility (45%–88% residual activity) with diverse detergent brands treated for 45 min at 40 °C, indicating its potential as a valuable additive for enhanced washing efficacy. Current research offers a novel strategy to boost protease production, presenting a promising alternative to chemical dehairing in leather industries and solid organic waste treatment. This approach holds the potential for mitigating environmental pollution through bioremediation.

Screening of Bacillus spp. bacterial endophytes for protease production, and application in feather degradation and bio-detergent additive

Research on proteases and secondary metabolites from endophytes is an area that requires attention from researchers. In this study, proteases from Bacillus sp. strain MHSD16 and Bacillus sp. strain MHSD17 endophytes were characterised, and their potential biotechnological applications were investigated. Optimum protease production was achieved when isolates were grown in media containing (g/L): glucose 10g, casein 5g, yeast extract 5g, KH2PO4 2g, Na2CO3 10g at pH 9. The crude protease extracts were active in alkaline environments, thus referred to as alkaline proteases with optimal pH of 10. Additionally, Bacillus sp. strain MHSD 16 and Bacillus sp. strain MHSD17 proteases were active at high temperatures, with optimum enzyme activity at 50 °C. Thermostability profiles of these proteases showed that the enzymes were highly stable between (40–60 °C), maintaining over 85 % stability after 120 min incubation at 60 °C. Furthermore, the enzymes were stable and compatible with various household and laundry detergents. In the presence of commercial laundry detergent, OMO® 68 % and 72 % activity was retained for Bacillus sp. strain MHSD16 and Bacillus sp. strain MHSD17, respectively, while 67 % and 68 % activity were retained in the presence of Sunlight®. The potential application for use in detergents was investigated through the removal of blood stains with the crude alkaline extracts displaying efficient stain removal abilities. Feather degradation was also investigated and Bacillus sp. MHSD17 exhibited feather keratin degrading properties more effectively than Bacillus sp. MHSD16.

Molecular Characterization and Screening of Protease Production from indigenous thermophilic fungi Malbranchea cinnamomea isolated from dairy farm compost soil

Compost environments represent a rich reservoir of microbial diversity, including thermophilic fungi with the potential to produce proteases. The dynamic and nutrient-rich nature of compost provides a conducive habitat for the growth and metabolism of diverse fungal species, including Malbranchea cinnamomea. In this study, Malbranchea cinnamomea was isolated from dairy farm compost soil and characterized based on its morphological characters and subsequently confirmed molecularly using internal transcribed spacer (ITS) sequencing. The Phylogenetic tree was inferred using Neighbour- joining method. The evolutionary distances were computed using the maximum composite likelihood method. The identified fungi was primarily screened for protease and further used for enzyme production in submerged fermentation for 4-6 days at 50OC. Culture filtrate obtained were used for the assessment of enzymatic activity. This study revealed thermophilic fungus Malbranchea cinnamomea holds significant potential for protease production. The enzyme activity was estimated at 50 °C, pH 7, six days incubation time and using 1.0 ml inoculum. Under submerged substrate cultivation, the best substrate lodge for maximal fungal biomass production was YPSs broth. Hence this isolate could be further exploited for numerous industrial applications that require thermophilic enzymes.

Isolation and Molecular Identification of Protease Producing Bacterium Associated with the Brown Algae Hydroclathrus sp. from Hoga Island of Wakatobi District

Isolation and Molecular Identification of Protease Producing Bacterium Associated with the Brown Algae Hydroclathrus sp. from Hoga Island of Wakatobi District

Bacillus velezensis strain NA16 shows high poultry feather-degrading efficiency, protease and amino acid production

Isolated Bacillus velezensis strain NA16, which produces proteases, amino acids and the transcription levels of different keratinolytic enzymes and disulfide reductase genes in whole gene sequencing, was evaluated during feather degradation. The result shows under optimum fermentation conditions, chicken feather fermentation showed total amino acid concentration of 7599 mg/L, degradation efficiency of 99.3% at 72 h, and protease activity of 1058 U/mL and keratinase activity of 288 U/mL at 48 h. Goose feather fermentation showed total amino acid concentration of 4918 mg/L (96 h), and degradation efficiency was 98.9% at 120 h. Chicken feather fermentation broth at 72 h showed high levels of 17 amino acids, particularly phenylalanine (1050 ± 1.90 mg/L), valine (960 ± 1.04 mg/L), and glutamic (950 ± 3.00 mg/L). Scanning electron microscopy and Fourier transform infrared analysis revealed the essential role of peptide bond cleavage in structural changes and degradation of feathers. Protein purification and zymographic analyses revealed a key role in feather degradation of the 39-kDa protein encoded by gene1031, identified as an S8 family serine peptidase. Whole genome sequencing of NA16 revealed 26 metalloproteinase genes and 22 serine protease genes. Among the proteins, S8 family serine peptidase (gene1031, gene1428) and S9 family peptidase (gene3132) were shown by transcription analysis to play major roles in chicken feather degradation. These findings revealed the transcription levels of different families of keratinolytic enzymes in the degradation of feather keratin by microorganisms, and suggested potential applications of NA16 in feather waste management and amino acid production.

INHIBIION ACTIVITY OF SOME PLANT SEEDS EXTRACTS AGAINST THE PROTEASE PRODUCED FROM LOCAL ISOLATE OF CANDIDA ALBICANS ISOLATE USING SOLID STATE FERMENTATION

This study was aimed at inhibition of purified protease produced by C. albicans using plant seeds extract. One hundred two local C. albicans isolates that were isolated and identified by microscopic and biochemical tests were submitted to primary and secondary screening techniques in order to select the qualified C. albicans isolate for protease synthesis. Among these isolates, nineteen isolates with the highest hydrolysis zone on skim milk media (primary screening) were chosen for secondary screening. It has been found that C. albicansV56 had the highest productivity of the enzyme (41.8U/mg protein). The optimum conditions of protease production by selected isolate using solid state fermentation by using wheat bran as best substrate , temperature 28 °C and pH 7, after 5 days of incubation . The enzyme was purified by concentration with sucrose, then used gel filtration chromatography using Sephadex G-75. The results show two peaks. The first peak has a purification folds 6.5 time with an enzyme yield of 13.08 %, while peak 2 has a purification folds 3.3 time with an enzyme yield of 20%. The purified enzyme exhibited maximal activity at pH 6.0 for peak 1 and 3.0 for peak 2, whereas the maximum stability was 7.0 for peak 1 and 8 for peak 2. The optimal temperature for purified enzyme activity was 40 for peak 1 and 30 for peak 2, and it was stable until 45°C. Protease activity was inhibited with local plant seed extracts. Lathyrus sativus extract inhibits approximately 50 % of protease activity.

Disposal and utilization of dead animals during breeding in livestock and poultry farming by means of synthetic microbiota

The disposal of animal remains resulting from breeding is a significant challenge that impacts the industry's growth. To address the issues with current treatment methods, such as the large space required for corpse storage, and the high energy consumption of pyrolysis. Three strains with high protease and lipase production and one strain with high keratinase production were screened. The virulence genes were evaluated, the synthesis gene clusters of degrading enzymes were mined, secondary metabolites of each strain were analyzed, and the bacterial community with both growth rate and enzyme production ability was developed. Therefore, a microbial degradation method with mild reaction conditions and rapid liquefaction of animal residues was developed. The liquid degradation of four common farm-raised animal residues (sheep, cattle, chickens, and pigs) was tested under laboratory conditions. The results showed that the liquid degradation of animal residues was achieved within 144 h, transforming the months-long anaerobic process of traditional compost fermentation process into a mere 6 days' anaerobic process. N, P, K plant nutrients accounted for 15% of the total matrix, pH value was 5.5–6.7, heavy metal content was less than 0.2 mg L−1. Designed and improved fermentation equipment, produced a 3 m³ fermentation equipment, used in chicken, pig two types of animal residues pilot test. The emissions of greenhouse gases such as CO2 in the entire degradation process were 1.6 × 104 ppm, which was 481 times less than that of composting by 7.7 × 106. This study provides a solution for the treatment of dead livestock and poultry, which has promotional and practical value.

Wheat bran submerged fermentation for protease production by a newly Streptomyces sp. isolate: optimization by factorial designs

Wheat bran submerged fermentation for protease production by a newly Streptomyces sp. isolate: optimization by factorial designs

Antifungal and Antivirulence Activity of Vanillin and Tannic Acid Against Aspergillus fumigatus and Fusarium solani.

Aspergillus fumigatus and Fusarium solani infections have become severe health threat; both pathogens are considered a priority due to the increasing emergence of antifungal-resistant strains and high mortality rates. Therefore, the discovery of new therapeutic strategies has become crucial. In this study, we evaluated the antifungal and antivirulence effects of vanillin and tannic acid against Aspergillus fumigatus and Fusarium solani. The minimum inhibitory concentrations of the compounds were determined by the microdilution method in RPMI broth in 96-well microplates according to CLSI. Conidial germination, protease production, biofilm formation, and in vivo therapeutic efficacy assays were performed. The results demonstrated that vanillin and tannic acid had antifungal activity against Aspergillus fumigatus, while tannic acid only exhibited antifungal activity against Fusarium solani. We found that vanillin and tannic acid inhibited conidial germination and secreted protease production and biofilm formation of the fungal pathogens using sub-inhibitory concentrations. Besides, vanillin and tannic acid altered the fungal membrane permeability, and both compounds showed therapeutic effect against aspergillosis and fusariosis in an infection model in Galleria mellonella larvae. Our results highlight the antivirulence effect of vanillin and tannic acid against priority pathogenic fungi as a possible therapeutic alternative for human fungal infections.

Purification, characterisation and visualisation of soybean protein hydrolysis by aspergillopepsin I from mangrove Aspergillus tubingensis

In order to obtain marine-derived enzymes suitable for protein hydrolysis in acid brewing system, seven species of fungi were obtained from mangrove soil. One of the strains, Aspergillus tubingensis, was determined to have a high acid protease production capacity. Its fermentation broth was purified and the obtained protease was identified as aspergillopepsin I (ATAP) with a molecular weight of 41205 Da. Homology modelling shows that ATAP contains three active sites, each containing to a Zn atom and amino acid residues. The enzyme had the highest relative activity at pH 3.5 and 35–40 °C, and exhibited pH and thermal stability at pH 3.0–3.5 and 20–50 °C. ATAP showed certain salt tolerance. K+ and Zn2+ promoted the enzyme activity, while Mg2+, Cu2+, Co2+, and Fe2+ slightly inhibited the enzyme activity. The results of inhibitors suggest that ATAP belongs to the class of aspartic proteases containing metal atoms. Furthermore, the results of enzymatic hydrolysis of defatted soybean powder showed that the contents of soluble protein, amino nitrogen, free amino acids and the proportion of small molecular peptides (<3 kDa) in the hydrolysate increased significantly. In addition, molecular docking of ATAP and aspergillopepsin I from Aspergillus oryzae 3.042 with soybean globulins (7S and 11S), respectively, showed better hydrolysis of soybean globulins by the former, especially for 7S globulin. Visual analysis indicated the interaction forces between enzymes and soybean globulins were dominated by hydrogen bond and hydrophobic interaction. In conclusion, ATAP may be a candidate as an enzyme preparation for the hydrolysis of soybean proteins.

The Interplay Between Endoplasmic Reticulum Stress and Oxidative Stress in Chondrocyte Catabolism.

Oxidative stress and endoplasmic reticulum (ER) stress play pivotal roles in disrupting the homeostasis of chondrocytes by producing catalytic proteases and enhancing chondrocyte senescence, consequently contributing to the progression of osteoarthritis (OA). Despite their close interaction, the underlying molecular mechanisms remain poorly understood. Here, we show that ER stress and oxidative stress reciprocally modulate each other to promote cartilage degradation. Primary chondrocytes were obtained from the articular cartilage of 5-day-old C57BL/6J mice by excising distal femur and proximal tibia. Tunicamycin was applied to induce ER stress in primary chondrocytes. Surgical OA was induced in 12-week-old male C57BL/6J mice by destabilizing the medial meniscus (DMM). Tunicamycin-induced ER stress led to an increase in the production of reactive oxygen species (ROS) and catalytic proteases, including MMP13 and Adamts5, in primary chondrocytes, and it was primarily dependent on the NADPH oxidase (NOX) system. ER stress directly increased the expression of NOX2, NOX3, NOX4, and p22phox. Specifically, the protein kinase RNA-like ER kinase (PERK) pathway is involved in the expression of NOX4 and p22phox, the inositol-requiring enzyme 1 alpha (IRE1α) pathway in NOX2 and NOX3 expression, and the activating transcription factor 6 (ATF6) pathway influences NOX3 expression in chondrocytes. Conversely, inhibiting NOX function significantly reduced both ER stress sensor-related signaling and chondrocyte catabolism, thereby decelerating the progression of surgically induced OA in vivo. Our findings highlight the positive feedback loop between ER stress and oxidative stress in OA pathogenesis, suggesting that targeting NOX isoforms is a promising therapeutic strategy for OA.

The Combination of Enzymes and Conidia of Entomopathogenic Fungi against Aphis gossypii Nymphs and Spodoptera frugiperda Larvae.

The slow action of fungi is one of the biggest challenges in using entomopathogenic fungi. A promising alternative to reduce the time of action is to combine conidia with extracellular enzymes. This study aimed to characterize the production of Pr1 subtilisin protease and lipases by Beauveria bassiana and Metarhizium anisopliae in different culture media and to evaluate the efficiency of the enzymatic treatment against Aphis gossypii and Spodoptera frugiperda. The isolates were cultivated in five different liquid cultures, and, after 7 days, the culture was filtered and centrifuged, and the activity of the Pr1 and lipases was measured. The fungi cultured in a Luria-Bertani broth medium had the highest activity of proteases and lipases. The mortality of A. gossypii nymphs treated with conidia 7 days after the treatment was 39% (JEF-410), 76.5% (JEF-492), 74.8% (ERL-836), and 70.9% (JEF-214). The B. bassiana JEF-410 supernatant combined with conidia increased the fungal virulence at day 5 and day 6 after treatment. When S. frugiperda larvae were treated with B. bassiana JEF-492 conidia combined with its supernatant, the time of infection was shorter compared to the larvae treated with conidia only. Once the supernatant was incubated at 37 °C, the relative activity decreased from 100% to 80% after 2 h and to 45% after 24 h. The results suggest that the supernatant of entomopathogenic fungi may be formulated and used as a biopesticide in an efficient strategy for the biological control of pests.

Unveiling the ocular battlefield: Insights into Pseudomonas aeruginosa virulence factors and their implications for multidrug resistance

The research investigates the virulence factors of Pseudomonas aeruginosa (P. aeruginosa), a pathogen known for its ability to cause human infections by releasing various exoenzymes and virulence factors. Particularly relevant in ocular infections, where tissue degeneration can occur, even after bacterial growth has ceased due to the potential role of secreted proteins/enzymes. Clinical isolates of P. aeruginosa, both ocular (146) and non-ocular (54), were examined to determine the frequency and mechanism of virulence factors. Phenotypic characterization revealed the production of alginate, biofilm, phospholipase C, and alkaline protease, while genotypic testing using internal uniplex PCR identified the presence of Exo U, S, T, Y, and LasB genes. Results showed a significant prevalence of Exo U and Y genes in ocular isolates, a finding unique to Indian studies. Additionally, the study noted that ocular isolates often contained all four secretomes, suggesting a potential link between these factors and ocular infections. These findings contribute to understanding the pathogenesis of P. aeruginosa infections, particularly in ocular contexts, and highlights the importance of comprehensive virulence factor analysis in clinical settings.

Co-regulation of Thermosensor Pathogenic Factors by C-di-GMP-Related Two-Component Systems and a cAMP Receptor-like Protein (Clp) in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a major threat to the food industry and human health owing to its strong protease production and biofilm formation abilities. However, information regarding regulatory factors or potential mechanisms is limited. Herein, we observed that temperature differentially regulates biofilm formation and protease production, and a cAMP receptor-like protein (Clp) negatively regulates thermosensor biofilm formation, in contrast to protease synthesis. Among four c-di-GMP-related two-component systems (TCSs), promoter fusion analysis revealed that clp transcription levels were predominantly controlled by LotS/LotR, partially controlled by both RpfC/RpfG and a novel TCS Sm0738/Sm0737, with no obvious effect caused by Sm1912/Sm1911. Biofilm formation in Δclp and ΔTCSs strains suggested that LotS/LotR controlled biofilm formation in a Clp-mediated manner, whereas both RpfC/RpfG and Sm0738/Sm0737 may occur in a distinct pathway. Furthermore, enzymatic activity analysis combined with c-di-GMP level indicated that the enzymatic activity of c-di-GMP-related metabolism proteins may not be a vital contributor to changes in c-di-GMP level, thus influencing physiological functions. Our findings elucidate that the regulatory pathway of c-di-GMP-related TCSs and Clp in controlling spoilage or the formation of potentially pathogenic factors in Stenotrophomonas expand the understanding of c-di-GMP metabolism and provide clues to control risk factors of S. maltophilia in food safety.

Investigation of the presence and enzymatic activity of fungi in stored corn (Zea mays L.) and soybean [Glycine max (L.) Merrill] grains

Soy [Glycine max (L.) Merrill] and corn (Zea mays L.) are important crops in Brazilian agribusiness. When stored improperly, these grains are susceptible to insects and microorganisms. Among the microorganisms capable of causing grain deterioration, fungi stand out. Despite the harm associated with fungi, they are biotechnologically appreciable due to their potential ability to produce hydrolytic enzymes, such as cellulases and proteases. Fungal hydrolytic enzymes have been applied in various processes in the food, textile, and detergent production industries, among others. The objective of this study was to verify the presence of fungi in samples of corn and soybean grains under storage conditions and characterize them regarding the production of cellulolytic and proteolytic enzymes. Among the results, there was a greater quantity and variety of filamentous fungi (35 morphotypes) in association with the grains evaluated, than of yeasts (three morphotypes). Representatives of the genera Aspergillus spp., Curvularia sp., and Penicillium spp. were detected, corroborating previous reports in the literature. Filamentous fungi that tested positive for cellulase production had an enzyme ratio index (ERI) varying between 1.06 and 2.57 (mean ± standard deviation = 1.37 ± 0.48), while for proteolytic activity, only the fungi isolated from soybean tested positive, with ERI (mean ± standard deviation) = 1.21 ± 0.01. Although the taxonomy of the isolates in this study has not been completely elucidated, it is possible to state that these fungi are promising for producing hydrolytic enzymes, with potential for use in the development of products and/or biotechnological processes.