Production Of Extracellular Enzymes Research Articles

Production of Extracellular Enzymes by Lentinula edodes Strains in Solid-State Fermentation on Lignocellulosic Biomass Sterilized by Physical and Chemical Methods.

Two methods of sterilization of lignocellulosic biomass were performed in this study. Eucalypt waste (EW) supplemented with rice bran (RB) was added in the proportions 80:20 and 90:10 in dry weight. The compositions were sterilized by physical method (autoclaving) and by chemical method (H2O2). The production of extracellular enzymes by Lentinula edodes strains was compared within the two methods. Inactivation of catalase present in RB was achieved with 250mM H2O2. The use of H2O2, when compared by physical method, favored high production of hydrolytic enzymes such as endoglucanase (1,600IU/kg), twofold higher, β-glucosidase (1,000IU/kg), fivefold higher, xylanase (55,000IU/kg), threefold higher and β-xylosidase (225IU/kg), similar results. Oxidative enzymes, MnP and laccase, were produced within a different profile between strains, with shorter times for laccase (2,200IU/kg) by SJC in 45days and MnP (2,000IU/kg) by CCB-514 in 30days. High production of extracellular enzymes is achieved by the use of the chemical method of sterilization of lignocellulosic biomass; in addition to no energy consumption, this process is carried out in a shorter execution time when compared to the physical process. The use of H2O2 in sterilization does not produce toxic compounds from the degradation of hemicellulose and cellulose such as furfural and hydroxy-methyl-furfural that cause inhibition of microorganisms and enzymes.

Design, synthesis, antibacterial and antiviral evaluation of chalcone derivatives containing benzoxazole

In this study, 24 chalcone derivatives containing benzoxazole were designed and synthesized, and the structures of all target compounds were confirmed by NMR and HRMS. The results of antibacterial activity showed that the median effective concentration (EC50) value of Z2 against Xanthomonas oryzae pv.oryzae (Xoo) was 8.10 μg/mL, which was better than thiodiazole copper (60.62 μg/mL), the antibacterial activity of Z2 was verified by scanning electron microscopy (SEM). Antibacterial mechanism of Z2 on Xoo was preliminarily explored, and the results showed concentrations were 100, 50 mg/L, which had a good cell membrane permeability to Xoo and can inhibit the EPS content and the production of extracellular enzymes, affecting the formation of biofilm, preliminary validation of the antibacterial effect of Z2 on Xoo. At 200 mg/L, Z2 had a better protective activity on harvested potatoes, which was comparable with thiodiazole copper. The activity against tobacco mosaic virus (TMV) was determined with half leaf dry spot method. The EC50 value of curative activity of Z15 was 101.97 μg/mL, which was superior to ningnanmycin (NNM) 294.27 μg/mL, and the EC50 value of protective activity of Z16 was 104.05 μg/mL, which was superior to NNM 185.73 μg/mL. Molecular docking results showed Z15, Z16 had a significantly higher affinity with tobacco mosaic virus coat protein (TMV-CP) than NNM. The above results indicated that chalcone derivatives containing benzoxazole have the potential to become antibacterial and antivirial agents.

Soil macroporosity modulates the extent of negative microbial associations during organic substance decomposition

Microbial species interactions are expected to influence the community-level properties, such as the production of extracellular enzymes and the degradation of organic substances. This work examined how microbial diversity, composition and the overall sign of microbial associations were altered with soil texture and structure following the amendment of organic substances. Two sets of microcosms (1:100 and 1:1) of a 4 × 3 factorial design were constructed, with four artificial textural classes (a sandy loam, two loams, and a (silty) clay loam) and three organics (TSB, tryptic soy broth; CA, a mixture of cellulose and humic/fulvic acids; BS, barley straw). As the ‘microbial inoculant’, an agricultural soil was added to the 1:100 and 1:1 microcosms at 1% and 50%, respectively. A few of microbial taxa were specifically enriched after soil addition of TSB, CA, or BS, but distributions across textural classes were inconsistent between microcosms or between organic amendments. Regardless, top abundant bacterial and fungal OTUs were overall negatively associated, suggesting that microbial competition for the shared resource dominated the decomposition of both simple and complex organics. Microbial associations were also modified by soil pore size distribution (PSD), being fewer negative (or more positive) in soils of greater macroporosity than in soils of lower macroporosity. The PSD-based differences in microbial associations were coordinated with PSD-based differences in the activities of exoglucanase and β-glucosidase in TSB-amended soils or soil respiration characteristics in CA-amended soils. Our results provide new insight into how soil structure regulates microbial interactions and, accordingly, the degradation of organic matter.

Insights into the plant growth promotion properties of bacterial endophytes isolated from Alternanthera philoxeroides and Eichhornia crassipes from Bellandur lake, India

The presence of macrophytes such as Alternanthera philoxeroides and Eichhornia crassipes in Bellandur Lake, India, has been observed despite the high pollution levels. Our research aims to explore the potential role of endophytes in promoting the growth of these macrophytes in such heavily contaminated environments. In current study, we isolated 20 endophytic bacteria from various parts of A. philoxeroides (12) and E. crassipes (8) plants, including shoots, roots, leaves, and flowers. We found that a significant proportion of endophytes from A. philoxeroides (42 %) and E. crassipes (25 %) produced more than 100 µg/mL of indole acetic acid (IAA). Similarly, the majority of the isolates possessed other plant growth promoting traits like ammonia production, nitrogen fixation, phosphate and potassium solubilization, siderophore production, and ACC deaminase production. Many of these isolates demonstrated extracellular enzyme production and halotolerance properties. We identified Acinetobacter soli ON529869 and Bacillus licheniformis ON506048 as the most effective plant growth promoters among the isolates, which also displayed antifungal properties against Fusarium solani and Cladosporium tenuissimum in vitro. Furthermore, greenhouse trials using these two endophytes revealed their significant plant growth promotion abilities in Amaranthus viridis. The pigment indices were evaluated using CI-710 Leaf Spectrometer. In conclusion, our findings provide compelling evidence for exploring the endophytic microbiomes of macrophytes in polluted areas for sustainable agriculture and bioprospecting of unique traits. This research could lead to the discovery of new and valuable resources for agricultural practices and other applications.

Constituents of Prokaryotic Organisms for Production of Extracellular Enzymes in the Development of Immunological Process

Constituents of Prokaryotic Organisms for Production of Extracellular Enzymes in the Development of Immunological Process

Assessment of biofilm, enzyme production and antibiotic susceptibility of bacteria from milk pre- and post-pasteurization pipelines in Algeria

Bacterial biofilm is a major concern of dairy industry due to its association with milk contamination and its derived products. Algerian pasteurized milk shelf-life does not exceed one day, which may reflect the high level of contamination of this product and presence of extracellular enzymes such as lipases and proteases. This work aimed to investigate the microbial biodiversity in milk-processing surfaces of a dairy plant in Algeria. Therefore, stainless steel cylinders were placed in piping system of the dairy system before and after pasteurization of the milk, being removed after 7 days, for biofilm maturation and microorganism isolation and identification by mass spectrometry.Fifty-nine Gram-positive isolates were identified, namely Bacillus altitudinis, Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Bacillus weithenstephanensis, Enterococcus casseliflavus, Enterococcus faecium, and Staphylococcus epidermidis. In addition, twenty-four Gram-negative isolates were identified, namely Acinetobacter schindleri Enterobacter cloacae, Enterobacter xiangfangensis, Leclercia adecarboxylata, and Raoultella ornithinolytica. Bacterial isolates showed ability for production of extracellular enzymes, being 49 % capable of both proteolytic and lipolytic activities. Milk isolates were tested for the ability to form biofilms on stainless steel. The cell numbers recovered on plate count agar plates from stainless steel biofilms ranged from 3.52 to 6.92 log10 CFU/cm2, being the maximum number detected for Enterococcus casseliflavus. Bacterial isolates showed intermediate and/or resistant profiles to multiple antibiotics. Resistance to amoxicillin, cefoxitin and/or erythromycin was commonly found among the bacterial isolates.

Optimization of L-glutaminase production by Monascus ruber URM 8542 isolated from ice cream industrial effluent.

L-glutaminase is a hydrolytic enzyme with wide biotechnological applications. Mostly, these enzymes are employed in the feed industry for flavor enhancement and acrylamide mitigation. Also, L-glutaminase may have antiviral and antineoplastic effects making it a good choice for pharmaceutical applications. In this study, the strain Monascus ruber URM 8542 was identified through classical and molecular taxonomy using partial sequencing of β-tubulin and calmodulin genes. Subsequently, the optimal culture conditions were evaluated by submerged fermentation (L-glutamine 10g.L- 1) for L-glutaminase excretion. The isolate was identified as M. ruber URM 8542 which showed significant extracellular enzyme production with a yield of 11.4 times in relation to the specific activity of intracellular L-glutaminase. Regarding the optimization experiments, several factors such as L-glutamine concentration, temperature, and pH were compared using a full factorial design (23). The concentrations greater than 1% proved to be significantly better for glutaminase production (R2 = 0.9077). Additionally, the L-glutaminase was optimally active at pH 7.0 and 30 ºC. The L-glutaminase was remarkably stable across an alkaline pH range (7.0-8.0) and had a thermal stability ranging from 30 ºC to 60 ºC for 1h. Taken together, these findings suggest that the L-glutaminase produced by M. ruber is a promising candidate for pharmacological application, although further studies need to be performed. To the best of our knowledge, this is the first report of L-glutaminase production by Monascus ruber.

Modeled energetics of bacterial communities in ancient subzero brines.

Cryopeg brines are isolated volumes of hypersaline water in subzero permafrost. The cryopeg system at Utqiaġvik, Alaska, is estimated to date back to 40 ka BP or earlier, a remnant of a late Pleistocene Ocean. Surprisingly, the cryopeg brines contain high concentrations of organic carbon, including extracellular polysaccharides, and high densities of bacteria. How can these physiologically extreme, old, and geologically isolated systems support such an ecosystem? This study addresses this question by examining the energetics of the Utqiaġvik cryopeg brine ecosystem. Using literature-derived assumptions and new measurements on archived borehole materials, we first estimated the quantity of organic carbon when the system formed. We then considered two bacterial growth trajectories to calculate the lower and upper bounds of the cell-specific metabolic rate of these communities. These bounds represent the first community estimates of metabolic rate in a subzero hypersaline environment. To assess the plausibility of the different growth trajectories, we developed a model of the organic carbon cycle and applied it to three borehole scenarios. We also used dissolved inorganic carbon and nitrogen measurements to independently estimate the metabolic rate. The model reconstructs the growth trajectory of the microbial community and predicts the present-day cell density and organic carbon content. Model input included measured rates of the in-situ enzymatic conversion of particulate to dissolved organic carbon under subzero brine conditions. A sensitivity analysis of model parameters was performed, revealing an interplay between growth rate, cell-specific metabolic rate, and extracellular enzyme activity. This approach allowed us to identify plausible growth trajectories consistent with the observed bacterial densities in the cryopeg brines. We found that the cell-specific metabolic rate in this system is relatively high compared to marine sediments. We attribute this finding to the need to invest energy in the production of extracellular enzymes, for generating bioavailable carbon from particulate organic carbon, and the production of extracellular polysaccharides for cryoprotection and osmoprotection. These results may be relevant to other isolated systems in the polar regions of Earth and to possible ice-bound brines on worlds such as Europa, Enceladus, and Mars.

Transcriptional Regulators Controlling Virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a pathogen capable of colonizing virtually every human tissue. The host colonization competence and versatility of this pathogen are powered by a wide array of virulence factors necessary in different steps of the infection process. This includes factors involved in bacterial motility and attachment, biofilm formation, the production and secretion of extracellular invasive enzymes and exotoxins, the production of toxic secondary metabolites, and the acquisition of iron. Expression of these virulence factors during infection is tightly regulated, which allows their production only when they are needed. This process optimizes host colonization and virulence. In this work, we review the intricate network of transcriptional regulators that control the expression of virulence factors in P. aeruginosa, including one- and two-component systems and σ factors. Because inhibition of virulence holds promise as a target for new antimicrobials, blocking the regulators that trigger the production of virulence determinants in P. aeruginosa is a promising strategy to fight this clinically relevant pathogen.

Candida palmioleophila: A New Emerging Threat in Brazil?

Human activity directly or indirectly causes climate change, promoting changes in the composition of the atmosphere. This change is beyond the variation of the natural climate. In this manner, climate change could create an environmental pressure which is enough to trigger new fungal diseases. In addition to climate alterations, the onset of the COVID-19 pandemic has also been associated with the emergence of fungal pathogens. Fungi showed that an inability to grow at high temperatures limits the capacity of fungi to infect mammals. However, fungi can develop thermotolerance, gradually adapting to rising temperatures due to climate change, and generating a greater number of disease-causing organisms. In the present study, we reported the detection and identification of Candida palmioleophila isolates recovered from raw sewage samples in Niteroi city, Rio de Janeiro State, Brazil, during a monitoring program for measuring SARS-CoV-2 presence and concentration. Using polyphasic taxonomy to identify the species and evaluating some virulence aspects of this species, such as biofilm formation and extracellular enzyme production, our data highlight this species as a possible emerging pathogen in Brazil, especially in the pandemic context.

Pre-harvest occurrence and physico-chemical characterization of Cladosporium cladosporioides on grape in India

Aim: The present study aimed to characterize Cladosporium cladosporioides C1 with respect to its pathogenicity, effect of different environmental factors and extracellular enzyme production. Methodology: A survey was conducted on C. cladosporioides in vineyards in Pune, Nashik and Sangli district of Maharashtra. The growth rate of C. cladosporioides was determined at different levels of temperature, pH and water activity. The pathogenicity of C. cladosporioides on both coloured and white table grape varieties with different total soluble solid (TSS) values was investigated along with its ability to produce different lytic enzymes required for pathogenicity. Results: The survey of the vineyards indicated that every vineyard was infected with C. cladosporioides irrespective of the infection level. The optimum growth of C. cladosporioides was noted at 25°C with pH 5 and 0.99 water activity (aw). Injured berries with high TSS were necessary to cause berry rot. Berries having 25% TSS developed significantly larger rot patches of 4.94±0.07 mm in white grapes as compared to berries having 15 % TSS. In the colored variety the berries having 25% TSS developed rot of 4.54±0.12 mm which was significantly higher as compared to berries having 15 % TSS. The fungi showed production of protease, lipase, amylase and pectinase enzymes. Quantitative analysis revealed that the yield of amylase was 71.67 %, which was significantly higher as compared to other enzymes. Interpretation: The information obtained on the growth of C. cladosporioides from the physico-chemical analysis would help to predict the development of the pathogen in the natural environment as well as formulate a strategy to control its pre-harvest infection. Key words: C. cladosporioides, Enzymes, Grapes, Pathogenicity

Bacillus strains exhibit various plant growth promoting traits and their biofilm-forming capability correlates to their salt stress alleviation effect on maize seedlings.

Soil salinity interferes with plant growth and development. Bacillus genus has been used to increase the growth and productivity of a wide variety of crops by alleviating the effects of salt stress. A total of thirty two Bacillus isolates were obtained from maize rhizosphere, and their plant growth-promoting (PGP) traits and biocontrol activities were tested. Bacillus isolates displayed varying degrees of PGP properties-the production of extracellular enzymes, indole acetic acid, hydrogen cyanide, phosphate solubilization, biofilm formation, and antifungal potential against several fungal pathogens. The phosphate-solubilizing isolates belong to B. safensis, B. thuringiensis, B. cereus, and B. megaterium species. Each Bacillus isolate demonstrated different levels of antifungal activity against the fungal pathogens tested. Biofilm production by some salt-tolerant isolates significantly increased at elevated levels of NaCl (p<0.05). The strains B. safensis B24, B. halotolerans B7/B18, B. subtilis B26, and B. thuringiensis B10 significantly increased the length of root (by 32.7-38.2%) and shoot (by 19.5-29.8%) of maize (p<0.05). Maize plants treated with some Bacillus strains displayed significantly greater chlorophyll content with an increase of 26.7-32.1% (p<0.05). Among PGP properties, enhanced biofilm formation played a more important role in maize growth under higher salinity. These salt-tolerant biofilm-forming strains could be efficiently used as bio-inoculant for maize under salinity stress.

Phosphate solubilization, siderophore production and extracellular enzyme production activities of endophytic fungi isolated from tea (Camellia sinensis) bushes of Assam, India

In the present study, a total of ten endophytic fungi were isolated from Camellia sinensis root, stem and leaf samples collected from twelve different tea gardens of Assam, India and evaluated for phosphate solubilization, siderophore production and extracellular enzyme producing activities. Out of ten, nine fungi were found positive for phosphate solubilization, siderophore production and cellulase activities. The highest fungal population was recorded in the root sample ORG-B (7.5 x 105) followed by ORG-A (7.4 x 105). Phosphate solubilization index was recorded highest in Penicillium sclerotiorum (2.87±0.01) followed by Penicillium sp F 7 (2.22±0.02) after 9 days of incubation. The highest siderophore percentage was observed in P. chrysogenum F 5 (61.09 ± 0.75%) followed by P. chrysogenum F1(56.29 ± 0.43) after 6 days of incubation. The highest cellulase (42.25±2.6 µg/ml/30 min), amylase (41.50±1.54 µg/ml/30 min) and xylanase (32.00±2.18 µg/ml/30 min) activites were recorded for P. chrysogenum F5, Aspergillus fumigates and A. niger respectively.

Synergistic Benefits: Exploring the Anti-Virulence Effects of Metformin/Vildagliptin Antidiabetic Combination against Pseudomonas aeruginosa via Controlling Quorum Sensing Systems.

The repurposing of drugs is one of the most competent strategies for discovering new antimicrobial agents. Vildagliptin is a dipeptidyl peptidase-4 inhibitor (DPI-4) that is used effectively in combination with metformin to control blood glucose levels in diabetic patients. This study was designed to evaluate the anti-virulence activities of this combination against one of the most clinically important pathogens, Pseudomonas aeruginosa. The current findings show a significant ability of the vildagliptin-metformin combination to diminish biofilm formation, bacterial motility, and the production of virulent extracellular enzymes and pyocyanin pigment. Furthermore, this drug combination significantly increased the susceptibility of P. aeruginosa to oxidative stress, indicating immunity enhancement in the eradication of bacterial cells. In compliance with the in vitro findings, the histopathological photomicrographs of mice showed a considerable protective effect of the metformin-vildagliptin combination against P. aeruginosa, revealing relief of inflammation due to P. aeruginosa-induced pathogenesis. P. aeruginosa mainly employs quorum sensing (QS) systems to control the production of its huge arsenal of virulence factors. The anti-virulence activities of the metformin-vildagliptin combination can be interrupted by the anti-QS activities of both metformin and vildagliptin, as both exhibited a considerable affinity to QS receptors. Additionally, the metformin-vildagliptin combination significantly downregulated the expression of the main three QS-encoding genes in P. aeruginosa. These findings show the significant anti-virulence activities of metformin-vildagliptin at very low concentrations (10, 1.25 mg/mL, respectively) compared to the concentrations (850, 50 mg/mL, respectively) used to control diabetes.

Wildfire severity alters soil microbial exoenzyme production and fungal abundances in the southern Appalachian Mountains

Climate change has increased drought frequency and duration that are exacerbated by increased temperatures globally. This effect has, and will continue, to increase fire occurrence across many regions of North America. In the southern Appalachian Mountains, wildfires with high burn severity occurred in 2016 due to increased drought and human activity. To investigate the effects of burn severity on soil physicochemical properties, microbial extracellular enzyme production, and microbial abundances in a temperate region, surface soils (0–15 cm) were collected from two sites (the Great Smoky Mountains National Park in Tennessee and the Nantahala National Forest in North Carolina, USA) spanning lightly, moderately, and severely burned areas, accompanied by adjacent unburned locations that act as controls. The soil samples were collected at three time points between 2017 and 2019 (i.e., 0.5, 1, and 2.5 years post-fire) among burn severity plots. Total hydrolytic enzyme production varied over time, with severe burn plots having significantly lower enzyme production at 2.5 years post-fire. Individual enzymes varied among burn severities and across time post-fire. Light burn plots showed greater carbon-specific (β-glucosidase and β-xylosidase) and phosphorus-specific (acid phosphatase) enzyme activities at 0.5 years post-fire, but this effect was transient. At 2.5 years post-fire, the β-xylosidase and acid phosphatase activities were lower in severe or moderate burn plots relative to the controls. In contrast, the activity of nitrogen-specific enzyme leucyl aminopeptidase was the lowest in severe burn plots at 0.5 years post-fire, but was the lowest in light burn plots at 2.5 years post-fire. The fungi:bacteria ratio declined with burn severity, indicating that fungi are sensitive or less resilient to high burn severity during recovery. These results suggest that wildfires alter trajectories for soil microbial structure and function within a 2.5-year timeframe, which potentially has long-term impacts on biogeochemical cycling.

Crinum latifolium mediated biosynthesis of gold nanoparticles and their anticandidal, antibiofilm and antivirulence activity

Candida spp. is one of the most common opportunistic human fungal pathogens, responsible for 90–100% of mucosal infections. Germ tube formation, hyphal morphogenesis, the production of tissue-damaging extracellular enzymes, and drug-resistant biofilm formation contribute to their pathogenicity, which can lead to systemic infections in the worst scenarios. Thus, there is an urgent need to discover new therapeutic agents to overcome the above virulence factors. Therefore, we aimed to prepare Crinum latifolium leaves-mediated biosynthesis of gold nanoparticles (AuNPs) that was characterized by various sophisticated techniques, and further their antifungal, antibiofilm, and anti-virulence activities was investigated. The AuNPs show a zone of inhibition between 19 and 22 mm for test strains of Candida spp. at 1000 µg/ml, whereas the MIC values were ranged from 250 to 500 µg/ml. AuNPs inhibit germ tube formation in C. albicans by 93.3% at 50 µg/ml. Furthermore, exposure to AuNPs significantly reduced the secretions of phospholipase, proteinase, hemolysin, esterase, and lipase. Confocal laser scanning microscopy (CLSM) investigation shows that 25 µg/ml of AuNPs significantly inhibit colonization and biofilm formation. Lastly, the interaction of Candidal cells with AuNPs revealed, ultrastructural changes in the cell wall and cell membranes as visualized by a transmission electron microscope (TEM).

Interpreting patterns of ecoenzymatic stoichiometry

The stoichiometry of extracellular enzyme activities has been both proposed and refuted to quantify resource use efficiency and resource limitations to soil microbial communities. The approach evaluates the acquisition of resources obtained through the enzyme-mediated catalysis of polymeric substrates. However, labile resources that do not require enzyme activity circumvent this catalytic pathway, can alter the balance of enzyme activities and skew their interpretation. More specifically, the microbial use of soluble resources can occur within minutes to hours of their addition whereas the use of polymeric resources depends on the production and turnover of extracellular enzymes (days to weeks). This temporal difference between potential, short-term microbial metabolic responses to soluble resources and longer-term community responses to enzyme-mediated acquisition of polymeric resources can produce seemingly conflicting interpretations of microbial resource limitations. However, stoichiometric tools provide insights to microbial resource use and limitations that differ in functional context from other measures of microbial community behavior.

The posttranscriptional regulator CsrA affects multidrug resistance and biocontrol activity in Lysobacter enzymogenes.

The posttranscriptional regulator CsrA regulates many cellular processes, including stress responses in diverse bacteria. However, the role of CsrA in multidrug resistance (MDR) and biocontrol activity in Lysobacter enzymogenes strain C3 (LeC3) remains unknown. In this study, we demonstrated that deletion of the csrA gene resulted in the initial slow growth of LeC3 and reduced its resistance to multiple antibiotics, including nalidixic acid (NAL), rifampicin (RIF), kanamycin (Km), and nitrofurantoin (NIT). Loss of the csrA gene also reduced its ability in inhibiting hypha growth of Sclerotium sclerotiorum and influenced its extracellular cellulase and protease activities. Two putative small noncoding regulatory RNAs (sRNAs), referred to as csrB and csrC, were also revealed in the genome of LeC3. Double deletion of csrB and csrC in LeC3 led to increased resistance to NAL, RIF, Km, and NIT. However, no difference was observed between LeC3 and the csrB/csrC double mutant in their suppression of S. sclerotiorum hypha growth and production of extracellular enzymes. These results suggest that CsrA in LeC3 not only conferred its intrinsic MDR, but also contributed to its biocontrol activity.

Isolation and characterization of halophilic and halotolerant fungi from man-made solar salterns in Pattani Province, Thailand.

The present study explored culturable halophilic and halotolerant fungi from man-made solar salterns in Pattani Province, Thailand. A total of 24 fungal isolates were discovered and characterized using morphological and molecular identification. Production of extracellular enzymes, secondary metabolites and mycoviruses was examined. Growth was observed in salinity and temperature ranges between 0%-20% and 28-40°C, respectively. Growth in different environmental conditions confirmed the halophilic or halotolerant nature of some strains. Fungal isolates were phylogenetically classified into seven different genera belonging to Aspergillus, Cladosporium, Curvularia, Diaporthe, Ectophoma, Fusarium and Penicillium. An enzymatic production test revealed that thirteen isolates could produce proteases and amylases at different levels. The presence of mycoviruses was detected in three isolates. Seventeen of the 24 isolates produced antimicrobial metabolites. The majority of these active isolates were identified as Aspergillus and Penicillium species. Crude extracts of the fungal mycelia and culture broths from these isolates had an inhibitory effect on both Gram-positive and Gram-negative bacteria and human pathogenic fungi. Research into fungi from saline environments could reveal fungal strains of biotechnological and industrial interest.

Thermoactive metallo-keratinase from Bacillus sp. NFH5: Characterization, structural elucidation, and potential application as detergent additive

In recent times, robust green technological developments have advanced the goal of a circular economy by minimizing waste generation. The study was undertaken to explore the keratinolytic activity of chicken feather-degrading bacteria from South African soil. Isolates coded as SSN-01 and HSN-01 were identified as Bacillus sp. NFH5 and Bacillus sp. FHNM and their sequences were deposited in GenBank, with accession numbers MW165830.1 and MW165831.1, respectively. Extracellular enzyme production and thiol group generation by Bacillus sp. NFH5 peaked at 120 h with 1879.09 ± 88.70 U/mL and 9.49 ± 0.78 mM, respectively. Glutamic acid (4.44%), aspartic acid (3.50%), arginine (3.23%), glycine (2.61%), serine (2.08%), and proline (2.08%) were relatively higher in concentration. Keratinase (KerBAN) activity was highest at pH 8.0 and 90 °C but was inhibited by both EDTA and 1,10-phenanthroline. In addition, the keratinase-encoding gene (kerBAN) accessioned OK033360 had 362 amino acid residues, with molecular weight and theoretical isoelectric point of 39 kDa and 8.81, respectively. Findings from this study highlight the significance of Bacillus sp. NFH5 in the bio-recycling of recalcitrant keratinous wastes to protein hydrolysates – potential dietary supplements for livestock feeds. The properties of KerBAN underscore its application potential in green biotechnological processes.