Protease Production Research Articles

A Study of the Characteristics and Properties of P. aeruginosa Isolated from Different Sources and their Resistance to Antibiotics

Pseudomonas aeruginosa, a common opportunistic bacterium, is a major source of human surface infections. Infections with these bacteria are difficult to cure and cause significant economic implications. Objectives The current study looked at the antibiotic resistance patterns of P. aeruginosa isolated from burns, wound swabs, and urinary tract infections that were referred to Iraq Hospitals' emergency health care units. From October 2022 to May 2023, 180 swabs were collected from burn and wound patients (110 burns, 67 wounds, and 3 urinary tract infections) of both sexes, (80) males and (100) females, aged 1-72 years, at Baghdad government hospitals: Ghazi Al-Hariri Hospital, Central Laboratories, Specialized Burns Hospital, and Yarmouk Hospital. Results:(180) diagnostic swabs from patients with burns and wound infections of both sexes, (80) males and (100) females, aged 1–72 years. The current study found 25(31.25) more P. aeruginosa positive cases in males than females, with 29(29) out of 54 positive cases. The study discovered that the age group 21-30 years had the highest rate of bacterial infection, followed by the age groups 11-20 and 1-10, with 11 (20.37%), 10 (18.51%), and 10 (18.51%), respectively. According to the current study's findings, the antibiotics Amikacin, Gentamicin, Ceftazidime, Cefepime, Netilmicin, and Tobramycin showed the highest resistance rates (81.4%, 81.4%, 79.6%, 72.2%, 74%, and 79.6%, respectively).The test findings showed that 52 isolates (96%) produced pyrocyanin and hemolysins. Bacterial colonies were white to gray with a sticky feel. All isolates exhibited β-hemolysis.Alkaline protease production All isolates exhibited a translucent halo surrounding the bacterial colonies.Lipase production All isolates exhibited a hazy halo surrounding the development zone of P.aeruginosa colonies. The current study discovered that the antibiotics Amikacin, Gentamicin, Ceftazidime, Cefepime, Netilmicin, and Tobramycin were the most resistant in cases of burn and wound patients, as well as urinary tract infections in Iraq.

Biological Control of Sclerotinia Rot Caused by Sclerotinia sclerotiorum in Lettuce Using Bacillus amyloliquefaciens HY

To test the antagonistic ability of <i>Bacillus amyloliquefaciens</i> HY against lettuce sclerotinia rot caused by <i>Sclerotinia sclerotiorum</i>, an <i>in vitro</i> test, greenhouse test and growth chamber test were conducted. As a result of <i>in vitro</i> dual culture test for sclerotia of <i>B. amyloliquefaciens</i> HY, the mycelial growth inhibition rate was 70.7%. Mycelial tip was swollen, black, and had an irregular and deformed shape. In I-plate test, the antagonistic volatile compounds produced by <i>B. amyloliquefaciens</i> HY, the mycelial growth inhibition rate was 57.7% and the sclerotia germination inhibition rate was 46.7%. In addition, <i>B. amyloliquefaciens</i> HY had sideropore production capacity, phosphate solubillibty, and protease production capability, but <i>B. amyloliquefaciens</i> HY did not produce chitinase. In growth chamber test, the lettuce survival rate was 0% in the control group treated with only the pathogen, and 80% in the treatment group treated with of <i>S. sclerotiorum</i> and <i>B. amyloliquefaciens</i> HY by diluting 10 times of the lettuce survival rate. In addition, the lettuce survival rate of 80% was shown in the treatment group treated with the culture medium of <i>B. amyloliquefaciens</i> HY 50 times 1 week later after inoculation with <i>S. sclerotiorum</i>.

Production of Tobacco Etch Virus Protease (TEV) Expressed in the Endotoxin-Free Bacillus subtilis and Its Application.

Tobacco Etch virus (TEV) protease is one of the most common tools for removing fusion tags, but no study has shown that TEV can be expressed at high levels in the GRAS host strain Bacillus subtilis and purified for further application. In this study, the fusion protein BsLysSN-TEV C/S-His-TEV consisting of a fusion tag, N-terminal domain of a lysyl-tRNA synthetase (BsLysSN) coded by B. subtilis lysS gene, placed at the N-terminus followed by an endoprotease TEV cleavage site and then the expression of this fusion protein in the cytoplasm of B. subtilis was investigated. The SDS-PAGE and Western-blot analysis demonstrated that His-TEV was overexpressed under the induction of IPTG. This result infers that His-TEV protease showed promising activity in the B. subtilis cytoplasm by the cleavage of the fusion protein. These cleavage products could be purified using the Ni-NTA column, which effectively cleaved the purified recombinant protein substrate, which can be applied in the protein purification process to remove the fusion tag. Significantly, since both His-TEV protease and the fusion recombinant protein substrate are expressed in the endotoxin-free host strain, the tag removal and purified product should be theoretically endotoxin-free, which could be a promising approach for producing therapeutic proteins and also for other relevant biomedical applications.

Enhanced production, artificial intelligence optimized three-phase partitioning extraction, and in silico characterization of extracellular neutral Bacillus cereus proteinase

The present study applied atmospheric and room temperature plasma (ARTP) mutagenesis to improve Bacillus cereus strain for neutral proteinase production. The selected improved stable strain, Phe−Tyr-ARTP-60-E demonstrated a 2.88-fold enhanced proteinase yield and 2.81-fold enhanced proteinase activity with shortened fermentation time. An artificial neural network-embedded genetic algorithm (ANN-GA) was employed to optimize the enzyme's three-phase partitioning (TPP) recovery using Python software. The optimized solution of the hybrid model recommended 38.05% wv−1 (NH4)2SO4, 1.0:2.0 crude extract/tert-butanol ratio, pH 5.84 at 23.4 °C, for the exclusive partitioning of proteinase in the intermediate phase, with 202% recovery, 13-fold purity with specific activity of 922 Umg−1. Sensitivity analysis of the model revealed that enzyme recovery was most sensitive to crude extract/tert-butanol ratio with total sensitivity (ST) of 0.253 while purification factor was most sensitive to pH with an ST of 0.444. In silico analysis using Expasy ProtParam tool revealed that mutant proteinase had 3 amino acid substitutions; Val-to-Cys; Val-to-His and Ile-to-Lys, at positions 152, 288, and 292, respectively, and 39.2 kDa molecular weight, 6.42 isoelectric point, and 33.35 instability index. The 3-Dimensional structure of the enzyme predicted by ModWeb v r273 Server revealed 39% sequence identity with Pseudoalteromonas lipolytica thermolysin. The macromolecule increased transmittance in lysozyme-coated contact lenses by 99.6% in 45 min. We conclude that ARTP mutagenesis and ANN-GA optimized TPP are effective and efficient recalcitrant strain improvement and product recovery methods, respectively. Further genetic tools and analysis of the neutral proteinase-producing gene may be required for increased improvement toward sustainable application.

Optimization of the production of a new milk clotting enzyme by Aspergillus flavus thermophile on agro-industrial residues medium and characterization of the enzyme

This study focused on the optimization of the production of a milk clotting protease by Aspergillus flavus, isolated from a thermal station. The fermentation was carried out on wheat bran as a medium, followed by a partial characterization of the enzyme. The fungal isolate was screened for protease production based on clear zone hydrolysis on skim milk agar, and then it was identified as belonging to the genus Aspergillus based on its morphological characteristics. This identification was confirmed by molecular analyzes with 100% similarity with the A. flavus strain. After 7 days of fermentation, the crude extract is recovered by simple filtration. The production and optimization of the milk coagulant protease using wheat bran and agro-industrial waste as a medium was carried out using a statistical method of experimental design based on the Plackett and Burman matrices. The results showed that the optimal fermentation medium consisted of wheat bran (4% w/w), incubated at a temperature of 30 °C, with a pH of 5.0 after 6 days of fermentation. The enzyme was partially purified by ammonium sulfate precipitation, yielding an estimated proteolytic activity of 2,985 U. The partially purified protease exhibits optimal activity at pH 5 with hemoglobin as a substrate and an optimal temperature of 50 °C. The Lineweaver-Burk plot showed a KM value of 4.83 g/L and a Vm of 1,560 U. This enzyme causes very rapid coagulation of fresh cow’s milk with an optimum at 45 °C, a pH 7 and a concentration of 50 mM of calcium.

Licuri (Syagrus coronata) Oil Cake as a Substrate for Collagenolytic Protease Production by Submerged Fermentation Using a Novel Strain of Penicillium citrinum Isolated from the Brazilian Caatinga

Licuri (Syagrus coronata) Oil Cake as a Substrate for Collagenolytic Protease Production by Submerged Fermentation Using a Novel Strain of Penicillium citrinum Isolated from the Brazilian Caatinga

Immune regulatory and anti-resorptive activities of tanshinone IIA sulfonate attenuates rheumatoid arthritis in mice.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and painful joint destruction. Current treatments are helpful in RA remission, but strong immunosuppressive activity and patient resistance are clinical issues. This study explores a dual-action inhibitor, possessing both anti-inflammatory and anti-resorptive properties, as a novel treatment for RA. Therapeutic efficacy and mechanisms of ectosteric (tanshinone IIA sulfonate [T06]) and active site-directed (odanacatib [ODN]) inhibitors of cathepsin K (CatK) were evaluated in RA mouse models. Pathology was assessed through biochemical analyses and histopathological examination. Flow cytometry analysis was performed to characterize immune cells. Anti-inflammatory effects of T06 on nuclear factor kappa beta (NF-κB) pathway were studied in macrophages. T06 effectively lowered the number of joint-resident immune cells, accompanied by significantly reduced production of inflammatory cytokines and collagenolytic proteases. This also included the suppression of Th17 cells and IL-17, resulting in the reduction of osteoclasts in arthritic joints and amplification of the overall anti-resorptive effect of T06, which has been attributed to its selective inhibition of the collagenolytic activity of CatK by preventing its oligomerization. The anti-inflammatory mechanism of T06 was based on blocking the phosphorylation of IκBα in the NF-κB pathway, resulting in reduced activation and expression of inflammatory cytokines. In contrast, ODN had no effect on inflammation and disease progression and was limited to the inhibition of CatK. The combined anti-resorptive and anti-inflammatory activities characterize T06 as a novel therapeutic agent for RA.

The Ecological Strategy Determines the Response of Fungi to Stress: A Study of the 2,4-diacetylphloroglucinol Activity Against Aspergillus and Fusarium Species.

Aspergillus and Fusarium are two economically important genera of fungi. They cause significant yield losses and contamination of crops with mycotoxins. In this study we aimed to evaluate the impact of 2,4-diacetylphloroglucinol (2,4-DAPG) on Aspergillus and Fusarium fungi. It is hypothesized that two fungal genera, which have different ecological strategies, react differently to stress caused by a secondary metabolite produced by rhizosphere Pseudomonas species. We found that 2,4-DAPG was able to reduce biofilm formation of Aspergillus and Fusarium, as reflected in biomass and its chemical composition. Furthermore, subinhibitory concentrations of 2,4-DAPG increased the levels of ergosterol and polysaccharides (α- and β-glucans, chitin) in the cell membrane and cell wall of Aspergillus, while decreasing them in Fusarium. 2,4-DAPG altered the production of secondary metabolites, especially mycotoxins and extracellular proteases. The production of ochratoxin A was decreased in A. ochraceus, and T-2 toxin and zearalenone, on the contrary, were increased in F. culmorum and F. sporotrichioides, respectively. Thus, using 2,4-DAPG we demonstrated that the ecological role of fungi determines their reaction to antibiotic substances produced by the plant microbiome. Our data contributes to understanding the molecular mechanisms behind symbiotic relationships in natural communities, which are mediated by the biosynthesis of antibiotics.

Isolation of Pathogenic Cronobacter Species as Bacteriological Risks Indicator in Powdered Infant Formula Available to Deprived Infants in Lagos Metropolis, Nigeria.

Powdered infant formula (PIF) is rich in nutrients that support the survival and growth of bacteria that trigger food safety disorders in deprived infants through life-threatening illnesses. The study aims to examine and identify the incidence of pathogenic bacteria of concern in PIF upon reconstitution in lukewarm water. A total of 172 samples consisting of 38 brands of PIF available in the Lagos metropolis were sampled, suspended in water (10g in 100mL), and bacteria strains were isolated using combinational enrichment and selective culture techniques. Pure bacterial strains were characterized and identified based on their physiology and 16S rRNA gene sequence homology. While 85 bacterial strains were isolated from the enriched culture system, 20 strains were selectively isolated based on tolerance to sodium deoxycholate. Approximately 13% of the selected bacteria were identified as Cronobacter spp., exhibiting virulence traits including extracellular protease production, coagulation and proteolysis of casein, haem-agglutination, and β-haemolysis of human blood. Approximately 82% of the Cronobacter strains tolerated NaCl (10%) and bile salt; and exhibited resistance to cefotaxime, ceftriaxone, gentamicin, and Amoxicillin-clavulanic acid antibiotics. The presence of Cronobacter spp. in 13% of the PIF brands available to infants calls for concern about the safety of deprived infants that might be fed with such PIF. Consequently, PIF safety alerts need to be activate while further studies on critical points at which the pathogens get introduced to the PIFs need to be identified.

Identification of Protease Producing Bacteria From Fish Gut.

Identification of Protease Producing Bacteria From Fish Gut.

Actinomycin D reduces virulence factors and biofilms against Aeromonas hydrophila.

Aeromonas hydrophila, a Gram-negative bacterium, is ubiquitously found in many aquatic habitats, causing septicemia in humans and fishes. Attributed to abuse or misuse of conventional antimicrobial drug usage, antimicrobial resistance is at an alarming rise. There is an available alternative strategy to bacterial resistance to antimicrobials, which is inhibition of virulence and pathogenicity employing quorum sensing inhibitors (QSIs). Hence, actinomycin D's effectiveness against A. hydrophila SHAe 115 as a QSI was investigated in decreasing virulence factors and preventing biofilm formation. Actinomycin D, belongs to the QSI combating Pseudomonas aeruginosa PAO1 originally isolated from an entophytic actinomycete (Streptomyces cyaneochromogenes RC1) in Areca catechu L. In the present work, further investigations were carried out to assess the effect of actinomycin D at subminimal inhibitory concentrations (sub-MICs), QS-regulated virulence factors, and biofilm inhibition strategies. Intrinsic properties encompassing inhibition of the production of protease and hemolysin and subsequent activities on biofilm formation and eradication of mature biofilm were established along with weakened swimming and swarming motilities in A. hydrophila SHAe 115. In the Tenebrio molitor survival assay, actinomycin D effectively reduced the virulence and pathogenicity of A. hydrophila, resulting in elimination of mortality. However, the hydrolysate of actinomycin D, 2-hydroxy-4,6-dimethyl-3-oxo-3H-phenoxazine-1,9-dicarboxylic acid (HDPD), had lost the QSI activity in A. hydrophila. Actinomycin D was proved as a viable QSI in lessening A. hydrophila's the virulence and pathogenicity, as evident from our research findings.

Evaluation of the potential probiotic Bacillus subtilis isolated from darkbarbel catfish (Pelteobagrus fulvidraco) on growth performance, serum immunity, and disease resistance of Aeromonas hydrophila.

This study aimed to identify potential probiotic strains of Bacillus subtilis from healthy fish gut microbiota for application in aquaculture. The effects of dietary B. subtilis administration on growth performance, serum enzyme activity, immune gene expression, and disease resistance in darkbarbel catfish (Pelteobagrus fulvidraco) were investigated. The isolate, identified through gene sequencing and biochemical tests, demonstrated resilience to pH 3.0% and 6.0% bile, and exhibited extracellular protease, cellulose, lipase, and amylase production. Darkbarbel catfish were fed diets with varying B. subtilis concentrations (0 CFU/kg [T0], 107 CFU/kg [T1], 108 CFU/kg [T2], and 109 CFU/kg [T3]). After 8 weeks, significant increases (p < 0.05) were observed in final body weight, weight gain rate, specific growth rate, serum lysozyme, serum superoxide dismutase, alkaline phosphatase, and total antioxidant capacity, whereas malondialdehyde levels significantly decreased. Feeding darkbarbel catfish with B. subtilis diets increased immunoglobulin M (IgM) and C3 gene expression (p < 0.05), indicating a positive impact on the fish's immune system. The strain upregulated interleukin 10 (IL-10) and transforming growth factor-β (TGF-β) expression and downregulated TNF-α and IL-1β, suggesting potential anti-inflammatory effects. Following a 7-day challenge with Aeromonas hydrophila, fish fed with B. subtilis exhibited lower mortality, with higher survival rates in the T2 and T3 groups. In conclusion, supplementing darkbarbel catfish diets with B. subtilis effectively enhances growth performance, immune response, and disease resistance.

Yarrowia lipolytica growth, lipids, and protease production in medium with higher alkanes and alkenes

Two strains of Yarrowia lipolytica (CBS 2075 and DSM 8218) were first studied in bioreactor batch cultures, under different controlled dissolved oxygen concentrations (DOC), to assess their ability to assimilate aliphatic hydrocarbons (HC) as a carbon source in a mixture containing 2 g·L−1 of each alkane (dodecane and hexadecane), and 2 g·L−1 hexadecene. Both strains grew in the HC mixture without a lag phase, and for both strains, 30 % DOC was sufficient to reach the maximum values of biomass and lipids. To enhance lipid-rich biomass and enzyme production, a pulse fed-batch strategy was tested, for the first time, with the addition of one or three pulses of concentrated HC medium. The addition of three pulses of the HC mixture (total of 24 g·L−1 HC) did not hinder cell proliferation, and high protease (> 3000 U·L−1) and lipids concentrations of 3.4 g·L−1 and 4.3 g·L−1 were achieved in Y. lipolytica CBS 2075 and DSM 8218 cultures, respectively. Lipids from the CBS 2075 strain are rich in C16:0 and C18:1, resembling the composition of palm oil, considered suitable for the biodiesel industry. Lipids from the DSM 8218 strain were predominantly composed of C16:0 and C16:1, the latter being a valuable monounsaturated fatty acid used in the pharmaceutical industry. Y. lipolytica cells exhibited high intrinsic surface hydrophobicity (> 69 %), which increased in the presence of HC. A reduction in surface tension was observed in both Y. lipolytica cultures, suggesting the production of extracellular biosurfactants, even at low amounts. This study marks a significant advancement in the valorization of HC for producing high-value products by exploring the hydrophobic compounds metabolism of Y. lipolytica.

Characterization of the extracellular proteases from Bacillus inaquosorum strain E1-8 and its application in the preparation of hydrolysates from plant and animal proteins with antioxidant, antifreeze and anti-browning properties.

Bacillus inaquosorum strains is widely recognized for their plant-growth-promoting and biocontrol capabilities, yet their roles in protease production remain unclear. The present study aimed to comprehensively assess the protease-producing performance of B. inaquosorum strain E1-8, at the same time as exploring the novel application of agricultural Bacillus proteases in the preparation of protein hydrolysates for fresh-cut fruits preservation. First, genomic sequencing revealed the diversity of E1-8 proteases, indicating 15 putative extracellular proteases. Subsequently, the fermentation conditions for E1-8 protease production were optimized, with sweet potato powder and soybean meal identified as the most suitable carbon and nitrogen sources, respectively, resulting in a maximum protease activity of 321.48 U mL-1. Upon culturing the strain under these optimized conditions, only an S8 family serine protease and an M48 family metalloprotease were revealed by secretomic analysis and protease inhibitor assays. Additionally, the optimal protease conditions for generating protein hydrolysates from soy, pea, fish and porcine proteins were determined. The molecular weight of the hydrolysates primarily ranged from 2000 to 180 Da, with a total of 17 amino acids identified. The application of these hydrolysates demonstrated a 2,2-diphenyl-1-picrylhydrazyl (i.e. DPPH) scavenging activity ranging from 58.64% to 84.12%, significantly reducing of the melting peaks and the freezing points. Furthermore, the browning index of apple slices stored at 4 °C decreased by 14.81% to 22.15% on the second day, and similar effects were observed in fresh-cut banana stored at 4 °C for 7 days. The protein hydrolysates obtained exhibit remarkable antioxidant, antifreeze and anti-browning properties for fresh-cut fruits. © 2024 Society of Chemical Industry.

Impeding microbial biofilm formation and Pseudomonas aeruginosa virulence genes using biologically synthesized silver Carthamus nanoparticles

Long-term antibiotic treatment results in the increasing resistance of bacteria to antimicrobials drugs, so it is necessary to search for effective alternatives to prevent and treat pathogens that cause diseases. This study is aimed for biological synthesis of silver Carthamus nanoparticles (Ag-Carth-NPs) to combat microbial biofilm formation and Pseudomonas aeruginosa virulence genes. Ag-Carth-NPs are synthesized using Carthamus tenuis aqueous extract as environmentally friendly method has no harmful effect on environment. General factorial design is used to optimize Ag-Carth-NPs synthesis using three variables in three levels are Carthamus extract concentration, silver nitrate concentration and gamma radiation doses. Analysis of response data indicates gamma radiation has a significant effect on Ag-Carth-NPs production. Ag-Carth-NPs have sharp peak at λ max 425 nm, small and spherical particles with size 20.0 ± 1.22 nm, high stability up to 240 day with zeta potential around − 43 ± 0.12 mV, face centered cubic crystalline structure and FT-IR spectroscopy shows peak around 620 cm−1 that corresponding to AgNPs that stabilized by C. tenuis extract functional moiety. The antibacterial activity of Ag-Carth-NPs against pathogenic bacteria and fungi was determined using well diffusion method. The MIC values of Ag-Carth-NPs were (6.25, 6.25, 3.126, 25, 12.5, 12.5, 25 and 12.5 µg/ml), MBC values were (12.5, 12.5, 6.25, 50, 25, 25, 50 and 25 µg/ml) and biofilm inhibition% were (62.12, 68.25, 90.12, 69.51, 70.61, 71.12, 75.51 and 77.71%) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Candida tropicalis and Candida albicans respectively. Ag-Carth-NPs has bactericidal efficacy and significantly reduced the swarming, swimming motility, pyocyanin and protease production of P. aeruginosa. Furthermore, P. aeruginosa ToxA gene expression was significantly down regulated by 81.5%, while exoU reduced by 78.1%, where lasR gene expression reduction was 68%, while the reduction in exoU was 66% and 60.1% decrease in lasB gene expression after treatment with Ag-Carth-NPs. This activity is attributed to effect of Ag-Carth-NPs on cell membrane integrity, down regulation of virulence gene expression, and induction of general and oxidative stress in P. aeruginosa. Ag-Carth-NPs have no significant cytotoxic effects on normal human cell (Hfb4) but have IC50 at 5.6µg/mL against of HepG-2 cells. Limitations of the study include studies with low risks of silver nanoparticles for in vitro antimicrobial effects and its toxicity.

Agricultural Biotechnological Potential of Bacillus velezensis C3-3 and Cytobacillus sp. T106 from Resource Islands of a Semi-arid Zone of La Guajira-Colombia

Resource islands are vegetative formations in arid and semi-arid ecosystems that harbor microorganisms facing extreme conditions. However, there is a limitation in the knowledge of the agricultural biotechnological potential of microorganisms present in these islands. This study aimed to determine the capacity of Bacillus velezensis C3-3 and Cytobacillus sp. T106 isolates from resource islands to promote plant growth and control the phytopathogen Rhizoctonia solani. The bacteria were sequenced, and both grew at 50 °C, resisted 5% NaCl, withstood UV exposure, and grew in extreme pH conditions. Sixty-six genes in C3-3 and 71 in T106 were identified associated with plant growth promotion, and C3-3 was shown to promote leaf growth in lettuce plants. This promotional effect was associated with the production of indole-3-acetic acid (IAA), phosphorus solubilization, and the presence of genes related to the assimilation of rhizosphere exudates. Both strains inhibited R. solani through the production of volatile compounds and antagonism. Forty-five and 40 of these genes in C3-3 and T106, respectively, were associated with the production of proteases, lipases, siderophores, antimicrobial compounds, degradation enzymes, and secretion systems. Notably, Cytobacillus sp. has not been previously reported as a biocontrol agent. This work contributes to the evidence of the biotechnological potential of semi-arid region bacteria, offering prospects for improving agricultural production in areas with limiting conditions.

Production of milk-coagulating protease by fungus Pleurotus djamor through solid state fermentation using wheat bran as the low-cost substrate

Proteases are enzymes that hydrolyze peptide bonds present in proteins and peptides. They are widely used for various industrial applications, such as in the detergent, food, and dairy industries. Cheese is one of the most important products of the dairy industry, and the coagulation stage is crucial during the cheese-making process. Enzymatic coagulation is the most common technique utilized for this purpose. Microbial enzymes are frequently used for coagulation due to their advantages in terms of availability, sustainability, quality control, product variety, and compliance with dietary and cultural/religious requirements. In the present study, we identified and subsequently characterized milk coagulant activity from the fungus Pleurotus djamor PLO13, obtained during a solid-state fermentation process, using the agro-industrial residue, wheat bran, as the fermentation medium. Maximum enzyme production and caseinolytic activity occurred 120 h after cultivation. When the enzyme activity against various protease-specific synthetic substrates and inhibitors was analyzed, the enzyme was found to be a serine protease, similar to elastase 2. This elastase-2-like serine protease was able to coagulate pasteurized whole and reconstituted skim milk highly efficiently in the presence and absence of calcium, even at room temperature. The coagulation process was influenced by factors such as temperature, time, and calcium concentration. We demonstrate here, for the first time, an elastase-2-like enzyme in a microorganism and its potential application in the food industry for cheese production.

Agro-industrial Waste Utilization, Medium Optimization, and Immobilization of Economically Feasible Halo-Alkaline Protease Produced by Nocardiopsis dassonvillei Strain VCS-4.

The oceanic actinobacteria have strong potential to secrete novel enzymes with unique properties useful for biotechnological applications. The Nocardiopsis dassonvillei strain VCS-4, associated with seaweed Caulerpa scalpeliformis, was a halo-alkaline protease producer. Further investigation focuses on medium optimization and the use of agro-industrial waste for economically feasible, high-yield protease production. A total of 12 experimental runs were designed using Minitab-20 software and Placket-Burman design. Among the 7 physicochemical parameters analyzed, incubation time and gelatin were detected as significant factors responsible for higher protease production. Incubation time and gelatin were further analyzed using OVATs. Optimal protease production was achieved with 2% gelatin, 0.1% yeast extract, 0.1% bacteriological peptone, 7% NaCl, pH 8, 5% inoculum, and a 7-day incubation period, resulting in a maximum protease activity (Pmax) of 363.97 U/mL, generation time of 11.9h, specific growth rate of 0.161g/mL/h, and protease productivity (Qp) of 61.65 U/mL/h. Moreover, utilizing groundnut cake as an agro-industrial waste led to enhanced production parameters: Pmax of 408.42 U/mL, generation time of 9.74h, specific growth rate of 0.361g/mL/h, and Qp of 68.07 U/mL/h. The immobilization of crude protease was achieved using Seralite SRC 120 as a support matrix resulting in 470.38 U/g immobilization, 88.20% immobilization yield, and 28.90% recovery activity. Characterization of both crude and immobilized proteases revealed optimal activity at pH 10 and 70°C. Immobilization enhanced the shelf-life, reusability, and stability of VCS-4 protease under extreme conditions.

Genomic insights and Taguchi-based optimization of culture conditions for enhanced alkaline protease production in Streptomyces barkulensis RC1831

This study presents a genome analysis identifying various alkaline protease genes and culture condition optimization to enhance the production of the enzyme in Streptomyces barkulensis RC1831. The analysis of genes using bioinformatics tools such as RAST, KEGG, KAAS, and BLASTx revealed the presence of 10 genes that are responsible for alkaline protease production belonging to different families. Multiple sequence alignment showed a high sequence identity with M24 family serine protease followed by subtilisin-like serine protease. Optimal conditions for enhanced protease production were determined to be at 37°C, pH 11, casein1 % (W/V), dextrose 0.5 % (W/V), urea 0.5 % (W/V), tryptophan 1 % (W/V), 1 mM Mn+2, 1 % (V/V) Tween-80 in LB medium and an incubation time of 72 hours. Out of the 10 alkaline protease genes, 2 genes expressed significantly according to the activity observed during optimization processes i.e., M24 family, Mn+2 dependent metalloprotease and subtilisin-like serine, ca+2 dependent metalloprotease. Furthermore, the AP enzyme was remarkably stable in the presence of various cofactors (Metal ions) and surfactants, indicating its potential for industrial applications under diverse conditions.

Assessing the Impact of Transport Phenomena on Modeling and Optimization of Solid-State Fermentation for the Revalorization of Agro-Industrial Residues in a Wall‐Cooled Packed‐Bed Bioreactor

This work assesses the influence of transport phenomena on the growth of Yarrowia lipolytica 2.2ab (Yl2.2ab) and protease production during Solid-State Fermentation (SSF) in a bench-scale wall-cooled tubular bioreactor packed with substrate-based pellets derived from agro-industrial residues. Our engineering methodology, in a novel manner, includes: (i) developing a new pseudo-heterogeneous model, (ii) identifying and quantifying the impact of all transport phenomena on microbial kinetics, (iii) elucidating how fluid dynamics enhances heat and mass transfer processes, and hence microbial kinetics, and (iv) determining the operational and geometric parameters for optimal bioreactor performance. As main results: (i) all transport phenomena occurring within the bench-scale bioreactor are fundamental for its reliable simulation and will be essential for its scale-up, and (ii) the maximum production of proteases, 420 U kgDS‐1−1, is achieved under the following operational conditions: a tube to particle diameter ratio of 5.6, a bath temperature of 45°C, an inlet airflow rate of 1 L min−1, and inlet fluid temperature of 43 °C. Finally, the pseudo-heterogeneous model is assessed by describing SSF-based observations from the literature, appropriately predicting the performance of Yl2.2ab in a bench-scale bioreactor. These results pave the way for future exploration of Yl2.2ab in SSF within larger-scale packed-bed bioreactors.