Protease Enzyme Research Articles

Screening of Casuarina equisetifolia rhizosphere-promoting bacteria and their effects on seed germination and seedling growth.

To promote the growth of Casuarina equisetifolia and address the abnormalities in the structure and function of rhizosphere soil microbial community, we isolated eight strains with multiple functions from the root nodules of C. equisetifolia, including nitrogen fixation (N), production of cell wall-degrading enzymes (protease and cellulase), auxin (IAA) production, siderophore production, ammonia (NH3) production, and phosphate solubilization. Among these strains, LB08, LB18, LB19, LB42, LB46, LB63, and LB69 were identified as Paenibacillus species, while LQ10 was identified as a Brucella sp. Results of seed soaking experiments showed that all the eight strains promoted the growth of C. equisetifolia seedlings. Strain LB69 significantly increased the germination rate and seedling vigor by 19.7% and 28.3%, respectively. Strain LQ10 significantly enhanced root length and root vigor by 48.2% and 334.4%, respectively. Strains LB18 and LB42 had the strongest effects on early shoot length and biomass accumulation, with increases of 22.4% and 32.8%, respectively. After seed soaking, the number of isozymes bands of polyphenol oxidase, superoxide dismutase, and peroxidase increased, with some bands showing enhanced intensity and increased diversity of enzyme isoforms, thereby enhancing stress resistance. In summary, the addition of these eight strains promoted plant growth and antioxidant enzyme activity, indicating their potential role as biofertilizers.

Exploring the extraction, functional properties, and industrial applications of papain from Carica papaya.

Papain a protease enzyme naturally present in the Carica papaya has gained significant interest across several industries due to its unique properties and versatility. The unique structure of papain imparts the functionality that assists in elucidating how papain enzyme works and making it beneficial for a variety of purposes. This review highlights recent advancements in papain extraction techniques to enhance production efficiency to meet market demand. The extraction of papain from the Carica papaya plant offers various advantages such as cost-effectiveness, biodegradability, safety, and the ability to withstand a wide range of pH and temperature conditions. Key findings reveal that non-conventional papain extraction techniques offer significant advantages in terms of efficiency, product quality, and environmental sustainability. Furthermore, papain treatment enhances the value of final products due to its anti-bacterial, anti-oxidant, and anti-obesity properties. The ability of papain to hydrolyze a wide range of proteins across various conditions makes it a suitable protease enzyme. While the study emphasizes the advantages of papain, the study also acknowledges limitations such as the continuous research and development to optimize extraction processes which will help unlock papain's potential and meet the growing demand. © 2024 Society of Chemical Industry.

Alginate-mediated immobilization of jackfruit (Artocarpus heterophyllus) latex serine protease enzyme exhibits improved catalytic properties

Alginate-mediated immobilization of jackfruit (Artocarpus heterophyllus) latex serine protease enzyme exhibits improved catalytic properties

Bioactivities of black soldier fly larvae protein hydrolysate

One of the potential insects for feed and food sources is the Black Soldier Fly (BSF) (Hermetia illucens). The BSF originated in South America and has now spread to many tropical countries. The BSF larvae (BSFL) had attracted much attention due to their good nutrient content and were recently reported as having useful bioactivities. BSFL has been widely used as feed and has been found to reduce bad smells associated with the growth of pathogenic microbes. The high protein content in BSFL is advantageous in the production of bioactive peptides. This research aimed to extract and hydrolyze the protein from BSFL with protease enzyme to produce protein hydrolysate or peptides with antioxidant and antibacterial activities. The antioxidant activity was measured using DPPH and FRAP, while the antibacterial was analyzed using the agar well diffusion method. BSFL was hydrolyzed with bromelain extracted from pineapple or commercial enzyme (Enzyplex). The IC50 shown by protein hydrolysate was 9.43±0.84% (v/v) or 0.72 mg/mL. The FRAP value was 521.21±264.53 µM for BSFL protein without enzyme addition, 792.14±291.02 for BSFL with the addition of bromelain, and 5,352.114±861.99 µM for BSFL with the addition of Enzyplex. The addition of bromelain increased the FRAP value 1.2 times, while the addition of Enzyplex increased the FRAP value 10.2 times. The antibacterial activity indicated that protein hydrolysate with phosphate buffer pH 8 at 50℃ and 24 hrs incubation showed the highest antibacterial activity to the lowest are Vibrio cholerae, Bacillus cereus and Staphylococcus aureus, respectively. The results suggest that BSFL has antibacterial and antioxidant bioactivities using bromelain extracted from pineapple and Enzyplex, a commercial enzyme that contains protease, lipase and amylase. Using Enzyplex as an enzyme to hydrolyze BFSL shows a very high antioxidant capacity compared to bromelain as an enzyme. This suggests that more protein and other macromolecules contribute to the antioxidant capacity. Using bromelain as an enzyme to hydrolyze BSFL, shows that BSFL peptide has higher antibacterial activity in gramnegative bacteria.

Cardioprotective role of long-term kefir and omega-3 fatty acid supplementation on myocardial apoptosis via oxidative stress-mediated lysosomal cathepsin release in isoproterenol-induced myocardial infarction rat model

Objective: The antiapoptotic and antioxidative role of long-term kefir and omega-3 fatty acids and their relationship with cysteine proteases on isoproterenol (ISO) induced myocardial infarction (MI) experimental model was investigated in our study. Material and Methods: Fifty male Sprague-Dawley rats were evenly divided into five distinct groups (n=10): Control, MI, kefir +MI, omega-3+MI, and kefir+omega 3+MI groups. Kefir 10% (with drinking water) and omega-3 fatty acid (30 mg/day per 100g body weight into the standard chow) were administrated during 30 days. ISO was subcutaneously injected into the rats (100 mg/ kg b.w.) on the 29th and 30th days. Myocardial tissue and blood samples were taken 12 hours after the last ISO dose. Creatine kinase MB (CK-MB) activities were measured in serum samples. Caspase-3, superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO), DNA fragmentation, cathepsin B and L levels, were measured in myocardial tissue. Results: Serum CK-MB (p<0.05) and cardiac tissue MDA (p>0.05), NO (p<0.01), caspase 3 (p<0.01), DNA fragmentation (p<0.001), cathepsin B (p<0.05) and L (p<0.05) activities were increased and SOD (p<0.001) activities were decreased in MI group compared to control group. The preventive effects of long-term therapy with kefir and omega-3 fatty acids have been demonstrated on apoptosis, oxidative stress markers, and cysteine protease enzymes. Conclusion: Our results showed that long-term administration of kefir and omega-3 fatty acids might be effective in reducing myocardial apoptosis through oxidative stress-mediated release of cysteine proteases in myocardial infarction, especially in the kefir and combined therapy groups.

Screening and characterization of lactic acid bacteria with antibacterial effect against heat-resistant spore outgrowth of Bacillus sporothermodurans

This study is the first to screen of lactic acid bacteria strains with antimicrobial effect against spore outgrowth of Bacillus sporothermodurans and characterize their technological properties. Eighty strains were isolated from Tunisia raw milk but only 8 isolates were selected based on their antagonistic activity against germinated spores of B. sporothermodurans and nine pathogenic bacteria. Phenotypic and genotypic characterization allowed us to distinguish three different species, namely Lactiplantibacillus plantarum, Levilactobacillus brevis and Lactococcus lactis. The lactic acid strains able to produce protease and lipase enzymes. The important antagonistic activity was observed in presence of L. lactis TLAB6 strain which produced a bacteriocin and lactic acid. The bacteriocin produced by the L. lactis TLAB6 is heat stable and resistant to trypsin. The results suggest that the selected LAB with antibacterial activity against germinated spore and technological properties may be used to preserve the food and formulate of bioactive functional foods.

Mechanisms of Cell Wall Degrading Enzymes from Bacillus methylotrophicus and Bacillus subtilis in Suppressing Foliar Blight Pathogens

Bacillus strains are potent Biocontrol agents (BCAs) and have been identified as an effective way to control the growth of phytopathogens of wheat. Through a direct inhibition mechanism which involves the production of cell wall degrading enzymes (proteases, glucanase, and chitinases) and siderophores they suppress the foliar blight disease. Both the strains of Bacillus P10 and UP11 shows growth inhibition Alternaria triticina (77.56%, 67.83%) and Bipolaris sorokiniana (73.97%, 62.16%) through a dual culture assay. These strains were subsequently examined for their ability to produce cell wall-degrading enzymes i.e., chitinase, protease, and β-1,3-glucanases and antifungal metabolites i.e., siderophores. It was found that when the antagonist’s bacteria were Co-cultured with fungal pathogens then maximum production of hydrolytic enzymes and siderophore was achieved at 96 hrs, but when both strains P10 and UP11 were alone maximum production was found at 48 hrs i.e., the exponential phase. The current study determined that Bacillus methylotrophicus (P10) and Bacillus subtilis (UP11) are highly effective strain for controlling Foliar Blight disease. Direct or Antibiosis is the main mechanism involved in this study. Further research on the interaction mechanisms between Bacillus-derived compounds and host plants is necessary.

Characterization of a thermostable protease from Bacillus subtilis BSP strain

This study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca2+ and Fe2+ increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H2O2). Characteristics, such as tolerance to high SDS and H2O2 concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.

Effect of casein bioactive peptides on performance, nutrient digestibility, enzyme activity and intestinal microbial population in broiler chickens.

This experiment was carried out to investigate the effect of bioactive peptides derived from casein on performance, nutrient digestibility, enzyme activity and intestinal microbial population in broiler chickens. In this study, 350 1-day-old male Ross 308 broiler chicks were distributed among 35 pens in a completely randomized design with seven treatments, five replicates and 10 chicks in each replicate. The experimental treatments included: basal diet without any additives (control), basal diet + Avilamycin antibiotic, basal diet + 200, 400, 600, 800 and 1000 mg of peptides per kg of diet. Results showed no significant effects of the experimental treatments on weight gain and feed conversion ratio during the starter period, but there was a significant improvement in weight gain in grower, finisher and whole periods in chicks fed with diet containing 1000 mg/kg peptides (p < 0.05). Adding peptides improved intestinal morphology (in duodenum and ileum) (p < 0.05). Supplementation of casein peptides significantly reduced thiobarbituric acid reactive substances concentration in breast and thigh meat. The activity of amylase, lipase and protease enzymes improved in treatments containing 800 and 1000 mg peptides in comparison to the control treatment. The use of casein-peptides increased intestinal Lactobacillus and decreased Coliform populations.

Extremozymes and compatible solute production potential of halophilic and halotolerant bacteria isolated from crop rhizospheric soils of Southwest Saurashtra Gujarat

Halophiles are one of the classes of extremophilic microorganisms that can flourish in environments with very high salt concentrations. In this study, fifteen bacterial strains isolated from various crop rhizospheric soils of agricultural fields along the Southwest coastline of Saurashtra, Gujarat, and identified by 16S rRNA gene sequencing as Halomonas pacifica, H. stenophila, H. salifodinae, H. binhaiensis, Oceanobacillus oncorhynchi, and Bacillus paralicheniformis were investigated for their potentiality to produce extremozymes and compatible solute. The isolates showed the production of halophilic protease, cellulase, and chitinase enzymes ranging from 6.90 to 35.38, 0.004–0.042, and 0.097–0.550 U ml−1, respectively. The production of ectoine-compatible solute ranged from 0.01 to 3.17 mg l−1. Furthermore, the investigation of the ectoine-compatible solute production at the molecular level by PCR showed the presence of the ectoine synthase gene responsible for its biosynthesis in the isolates. Besides, it also showed the presence of glycine betaine biosynthetic gene betaine aldehyde dehydrogenase in the isolates. The compatible solute production by these isolates may be linked to their ability to produce extremozymes under saline conditions, which could protect them from salt-induced denaturation, potentially enhancing their stability and activity. This correlation warrants further investigation.

Investigating the anti-inflammatory potential of Elaeagnus an-gustifolia L. through enzyme inhibition assays

Elaeagnaceae family, which is common in the steppe regions of Asia, Europe, and North America, contains about 45 species, including three genus: Hippophae, Shepherdia, and Elaeagnus. There are two species in the genus Hippophae rhamnoides and Elaeagnus angustifolia. The species of the Elaeagnaceae family is widely used in public and traditional medicine due to its widespread prevalence in the world and in Turkey. Ethnobotanical field studies have revealed that the fruits of E. angustifolia L. are used in Anatolia as an anti-inflammatory, antipyretic, urolithiatic, antidiarrheal, and anti-inflammatory agent in eye infections. Furthermore, the fruit of this plant is consumed as an appetizer and a source of nutrition. The study aims to evaluate the in vitro anti-inflammatory activity of extracts in different polarities from the leaves, branches, and fruits of E. angustifolia and to establish the content of polyphenolic compounds (flavones and polyphenolic carboxylic acids) in the ethyl acetate extract branches using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). In this context, the anti-inflammatory effects of n-hexane, ethyl acetate, and methanol extracts prepared from the plant have been evaluated through in vitro cyclooxygenase (COX-1 and COX-2) enzyme inhibition, inhibition of protein denaturation and proteinase enzyme, and anti-lipoxygenase activity tests. The results showed the highest activity of the ethyl acetate extract from branches. The HPLC-MS analysis revealed that the ethyl acetate extract prepared from the branches at a concentration of 100 mg/g contains p-coumaric acid (123.91 mg%), ferulic acid (62.07 mg%), kaempferol (43.11 mg%), sinapic acid (9.82 mg%), and quercetol (12.46 mg%). It was concluded that the data obtained in this context supported the use of the plant in folk medicine.

Isolation and Characterization of Aeromonas taiwanensis Strain for Simultaneous Production of Cellulase, Amylase, Pectinase, and Protease Enzymes

Isolation and Characterization of Aeromonas taiwanensis Strain for Simultaneous Production of Cellulase, Amylase, Pectinase, and Protease Enzymes

Enhancing Enzymatic Activity Through Co-Immobilization of Protease and Lipase Enzymes on Magnetic Nano-Iron Oxides Coated with Gum Arabic

Enhancing Enzymatic Activity Through Co-Immobilization of Protease and Lipase Enzymes on Magnetic Nano-Iron Oxides Coated with Gum Arabic

Field-scale assessment of vermicompost amendments for diuron-contaminated soil: Implications for soil quality and pesticide fate

This comprehensive study investigated the distribution, dissipation, and environmental impact of diuron and its metabolites (DPMU, DPU, and DCA), as well as its effects on enzyme activities, bacterial abundance, and community composition in response to different soil treatments using two vermicomposts. One vermicompost was derived from vine shoot waste, and the other from wet olive cake (alperujo). The study was conducted under field conditions in a semi-arid region. The results indicated that diuron concentrations decreased with soil depth, with the highest levels in the upper 0–5 cm layer. The application of both vermicomposts reduced diuron leaching into the soil, facilitating its dissipation compared to the organically unamended soil. DPMU was the main metabolite detected in the soil, especially at the end of the experiment. Enzyme activities, including dehydrogenase, β-glucosidase, protease, and acid phosphatase, showed variations with treatment and time, indicating microbial adaptation. In general, both vermicomposts increased soil enzyme activities, even when diuron was co-applied. Bacterial communities were affected by diuron and vermicomposts, with Actinobacteria and Alphaproteobacteria showing significant shifts in relative abundance. Principal Component Analysis (PCA) confirmed structural changes in bacterial communities due to diuron and vermicomposts. Warmer temperatures and evapotranspiration may contribute to the distribution of diuron in the different layers of soil and affect microbial activity. Overall, vermicomposts from wet olive cake proved effective in mitigating diuron in the different soil layers, promoting enzyme activities, and influencing bacterial communities. These findings underscore the complexity of interactions between diuron, organic amendments, and soil microorganisms, highlighting the potential for enhanced herbicide dissipation and microbial resilience and adaptation.

Microbial Biodiversity of Cryoconites in Palandöken Mountain and Culturing Cold-Adapted Enzyme Producers

Palandöken Mountain is among the highest and coldest ecosystems of Türkiye, and knowledge on its microbial diversity has not been adequately characterized. The aim of the present study is to expand the current understanding of microbial diversity in Palandöken Mountain. Sampling studies were carried out at in winter an altitude of 2400–2900 m. A next-generation sequencing (NGS) based culture-independent approach was employed for elucidating the community structure in the 8 cryoconite samples using 16S and 23S rRNA gene sequencing. In addition, biotechnologically important cold-adapted enzyme producers were isolated and identified by employing conventional culture-dependent studies. High-throughput sequencing analysis revealed the presence of 36 bacterial and 2 fungal phyla. The bacterial communities of 8 samples were dominated by Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Acidobacteria. The fungal communities were dominated by Ascomycota and Basidiomycota. 2900 different taxonomic group for bacterial diversity and 51 for fungal diversity were reported in the present study. Furthermore, 25 cold-adapted enzyme-producers were isolated and identified in the culture-dependent studies. Each selected isolate was capable of producing at least one of the enzymes amylase, protease, lipase, cellulase and xylanase isolates were assigned to Aspergillus, Bacillus, Cladosporium, Dioszegia, Flavobacterium, Fusarium, Holtermanniella, Naganishia, Polaromonas, Protomyces, Pseudomonas Rhodosporidiobolus, Vishniacozyma and Yarrowia genera. Consequently, this is the first geomicrobiological investigation that describes bacterial and fungal diversity of cryoconites from Palandöken Mountain using an NGS-based culture-independent approach. Additionally, cultured cold-adapted enzyme producer isolates of the present study have a valuable potential for the development of new biotechnological tools for various industrial applications.

Computational analysis to comprehend the structure-function properties of fibrinolytic enzymes from Bacillus spp for their efficient integration into industrial applications

BackgroundThe fibrinolytic enzymes from Bacillus sp. are proposed as therapeutics in preventing thrombosis. Computational-based analyses of these enzymes’ amino acid composition, basic physiological properties, presence of functional domain and motifs, and secondary and tertiary structure analyses can lead to developing a specific enzyme with improved catalytic activity and other properties that may increase their therapeutic potential. MethodsThe nucleotide sequences of fibrinolytic enzymes produced by the genus Bacillus and its corresponding protein sequences were retrieved from the NCBI database and aligned using the PRALINE programme. The varied physiochemical parameters and structural and functional analysis of the enzyme sequences were carried out with the ExPASy-ProtParam tool, MEME server, SOPMA, PDBsum tool, CYS-REC tool, SWISS-MODEL, SAVES servers, TMHMM program, GlobPlot, and peptide cutter software. The assessed in-silico data were compared with the published experimental results for validation. ResultsThe alignment of sixty fibrinolytic serine protease enzymes (molecular mass 12–86 kDa) sequences showed 49 enzymes possess a conserved domain with a catalytic triad of Asp196, His242, and Ser569. The predicted instability and aliphatic indexes were 1.94–37.77, and 68.9–93.41, respectively, indicating high thermostability. The random coil means value suggested the predominance of this secondary structure in these proteases. A set of 50 amino acid residues representing motif 3 signifies the Peptidase S8/S53 domain that was invariably observed in 56 sequences. Additionally, 28 sequences have transmembrane helices, with two having the most disordered areas, and they pose 25 enzyme cleavage sites. A comparative analysis of the experimental work with the results of in-silico study put forward the characteristics of the enzyme sequences JF739176.1 and MF677779.1 to be considered when creating a potential mutant enzyme as these sequences are stable at high pH with thermostability and to exhibit αβ-fibrinogenase activity in both experimental and in-silico studies.

Production of the Protease Enzyme from Bacillus cereus Bacteria Isolated from Some Cooked Foods and Determining the Factors Affecting the Production of the Enzyme

Production of the Protease Enzyme from Bacillus cereus Bacteria Isolated from Some Cooked Foods and Determining the Factors Affecting the Production of the Enzyme

Phytochemical analysis of Robinia pseudoacacia flowers and leaf: quantitative analysis of natural compounds and molecular docking application

Phenolic compounds are widely found and well-known secondary metabolites in plants. Identification, and quantification of phenolic compounds, and determination of their biological activities reveal the unknown secrets of plants. Robinia pseudoacacia (RP) is known as the white-flowered false acacia and is distributed in Northern Anatolia in Turkey. Spectrophotometric and chromatographic techniques are used to identify the presence and amount of phenolics. In this study, RP flowers and leaves were extracted with methanol and analyzed by LC-MS/MS to determine their phytochemical content. Salicylic acid and syringic acid were found as major products in leaves and flowers. RP extracts have been reported to have antibacterial activity and BChE inhibitory properties. Therefore, the BChE and DD peptidase enzyme inhibitory properties of the main components salicylic acid and syringic acid were investigated by molecule docking (MolDock). According to MolDock results, syringic acid interacted with BChE and DD peptidase and was calculated as a MolDock score of -79.38, and -71.25, with binding energies -5.90, and -5.40 kcal/mol respectively. Salicylic acid interacted with BChE and DD peptidase and was calculated as a MolDock score of -63.54, and -66.18, with binding energies of -6.10, and -5.70 kcal/mol respectively. As a result, salicylic acid had higher binding energy in its interactions with BChE and DD peptidase enzymes. In theory, salicylic acid can be used as a good BChE and DD peptidase inhibitor.

Exploring the potential of black fungus, Auricularia auricula, as a feed additive in African catfish, Clarias gariepinus, farming

This study explores the beneficial effects of Auricularia auricula (AA) as a feed additive in promoting growth, digestive enzyme activities, antioxidative responses, heat tolerance, and disease resistance against Edwardsiella tarda in African catfish (Clarias gariepinus) farming. The application of feed additives is a hot topic in recent aquaculture studies aimed at promoting the growth and health of aquaculture species. After 8 weeks of feeding trial, the results of the present study revealed that fish-fed AA diets performed significantly better (p < 0.05) compared to the control group in growth performances, including final weight, weight gain, and specific growth rate. The highest performances were observed in the fish-fed AA at 3 and 4 %. A similar trend was also observed in the values of feed conversion ratio, hepatosomatic index, and visceral somatic index, with the lowest values (p < 0.05) in the fish-fed AA at 3 and 4 %. AA diets enhanced the activities of all tested digestive enzymes (amylase, protease, and lipase) significantly (p < 0.05), with the highest activities in the fish-fed AA at 3 and 4 %. Meanwhile, fish-fed AA diets exhibited significantly higher (p < 0.05) catalase, superoxide dismutase, and glutathione peroxidase activities both before and after heat stress, with the highest activities in the fish that received AA at 3 and 4 %. Furthermore, AA diets stimulated disease resistance in African catfish, with the fish-fed AA at 4 % performing the highest cumulative survival rate (73.3 ± 5.77 %) post-infection with E. tarda in African catfish. The findings of the current study suggest that AA has huge potential as a feed additive in African catfish farming.

Mode of Action of Antimicrobial Potential Protease SH21 Derived from Bacillus siamensis.

Global public health is facing a major issue with emerging resistance to antimicrobial agents. Antimicrobial agents that are currently on the market are strong and efficient, but it has not been ruled out that these medications will eventually cause resistance to bacteria. Exploring novel bioactive compounds derived from natural sources is therefore, crucial to meet future demands. The present study evaluated the mode of action of the antimicrobial potential protease enzyme SH21. Protease SH21 exhibited antimicrobial activity, strong heat stability (up to 100 °C), and pH stability (pH 3.0 to 9.0). In terms of mode of action, we found that protease SH21 was able to disrupt the bacterial cell membrane as the results of the nucleotide leakage and cell membrane permeability assay. In addition, we also checked inner membrane permeability by PI uptake assay which suggested that protease SH21 has the ability to enter the bacterial cell membrane. Our results revealed that the antimicrobial protease SH21 might be a promising candidate for treating microbial infections.