Production Of Protease Enzyme Research Articles

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.

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.

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

Enhanced production of alkaline protease enzyme by mutated Aspergillus oryzae developed by random mutagenesis

Enhanced production of alkaline protease enzyme by mutated Aspergillus oryzae developed by random mutagenesis

PRODUCTION OF ALKALINE PROTEASE BY A NOVEL ANAEROBIC BACTERIUM ISOLATED FROM A MUNICIPAL ANAEROBIC TREATMENT SYSTEM

PRODUCTION OF ALKALINE PROTEASE BY A NOVEL ANAEROBIC BACTERIUM ISOLATED FROM A MUNICIPAL ANAEROBIC TREATMENT SYSTEM

MOLECULAR IDENTIFICATION OF NOVEL PROBIOTIC BACTERIA ISOLATED FROM COW MILK AND ITS ANTIBACTERIAL APPLICATIONS

The purpose of the study conducted in the year 2021 was to isolate and identify Lactobacillus species through biochemical characterization and to find antibacterial potential of the species. Researchers collected milk sample from local milk vendor, Eachanari in December, 2021. In an Enriching MRS broth the colonies were isolated and loop full of isolate were streaked on MRS agar plate. By morphological characteristics determined through gram staining method the isolated bacteria were identified as Lactobacillus species. Through different identification methods suchas casein digestion, acid and salt tolerance the isolate was characterized for probiotic properties. In a skim milk agar plate the isolated strain digested casein. This indicated production of protease enzyme which is a significant probiotic property. In MRS broth, an acid tolerance test was conducted at pH levels 3, 4, 5, 6, 7 and 8. The isolate was able to endure both an extremely acidic pH and alkaline media. NaCl tolerance test was done in MRS broth at concentrations of 3%, 4%, 5%, 6%, 7%, and 8% NaCl. The isolate survived well in solutions containing 3%, 4%, and 6% NaCl. After biochemical studies, molecular identification was carried out to identify the probiotic starin. The isolated probiotic was identified as Limosilactobacillus fermentum strain BTFSN. The isolated strain was then checked for antibacterial activity against three pathogens. Zone of inhibition were observed. The study indicated that Lactobacillus species from milk sample have potential probiotic properties and possess antimicrobial property.

Effect of Monosodium Glutamate in Artificial Food on Feed Utilization Efficiency, Growth, and Survival of Fingerling Tilapia (Oreochromis niloticus)

Tilapia (Oreochromis niloticus) is one of the leading freshwater fish commodities in Indonesia. The problem often faced by tilapia farmers is the low level of feed utilization, especially protein, which is still low, resulting in suboptimal growth. The glutamate content in monosodium glutamate (MSG) can act as a precursor for the formation of arginine and glutamine, which can increase the production of protease and lipase enzymes that are excreted into the intestines during the feed hydrolysis process, which is useful in increasing the absorption of nutrients in the form of protein and fat. This research was conducted using fingerling tilapia as a test fish with an average individual length of 6.59 ± 0.21 cm/fish and an average individual weight of 5.33 ± 0.27 g/fish. This research was carried out in an aquarium container measuring 40x30x30 cm with a water volume of 27 l and a density of 15 fish/aquarium. The aim of this research was to determine the effect of MSG in fish feed on total feed consumption (TKP), feed utilization efficiency (FUE), protein efficiency ratio (PER), protein retention, relative growth rate (RGR), and survival (SR) in fingerling tilapia. This research used an experimental method with a completely randomized design (CRD) of 4 treatments with 3 replications. Treatments A, B, C, and D are fed with the addition of 0 MSG in commercial feed; 0.75; 1.5; and 2.25 g/kg. Variables observed included TKP, FUE, PER, protein retention, RGR, and SR. The research results showed that MSG in feed had a significant effect (P < 0.05) on the FUE, PER, protein retention, and RGR values but had no effect (P < 0.05) on the TKP and SR values. FUE value; PER, protein retention; The highest RGR was obtained in treatment D (2.25 g/kg); FUE (87.21±2.28); PER(1.83±0.05); protein retention (50.47±0.76); The RGR (3.06 ± 0.03) and survival value for all treatments were found to be 100%. The water quality during the research was in optimal condition.

Production and characterization of protease enzyme from Acinetobacter pittii using peanut meal as substrate

ABSTRACT A significantly high protease enzyme yield of 617 U/ml was achieved with Acinetobacter pittii as the microorganism and peanut oil meal as the substrate. Peanut oil meal, which consists of proteins (40-60%) and carbohydrates (22-30%), serves as a sufficient source of nitrogen and carbon necessary for microbial growth and production of enzymes. Moreover, peanut meal offers the advantages of being affordable and available in large quantities, making the meal suitable for cost-effective enzyme production. In the present study, two bacterial strains and one fungal strain were selected to produce proteases utilizing peanut oil meal as the substrate. The experimental conditions during the enzyme production, including pH and temperature, were optimized. In addition, the substrate was enriched with various carbon and nitrogen sources. The microbial strains were streaked on nutritional agar (for bacteria) and potato dextrose agar (for fungus). Following an incubation period, the plates were stored at 4°C for further studies. The molecular weight of partially purified proteases of Acinetobacter pittii was found to be ≅ 95.5 kDa. Potassium nitrate was the most ideal nitrogen source (up to 411% increase in activity) and fructose was the best carbon source (425% increase). These enzymes exhibited excellent temperature tolerance and were capable of functioning over a wide pH range. Furthermore, the obtained proteases demonstrated ability to coagulate milk effectively, indicating their potential for various food-related applications.

Screening and characterization of endophytic bacteria isolated from celery with the potential to promote plant growth

Abstract. Nuriyanah, Widowati T, Masnang A, Nurjanah L, Novilasari D, Lekatompessy SJR, Simarmata R. 2023. Screening and characterization of endophytic bacteria isolated from celery with the potential to promote plant growth. Biodiversitas 24: 6897-6904. Endophytic bacteria that inhabit plant tissue contribute to the enhancement of host plant growth by producing secondary metabolites. They can improve plant growth by phytohormone modulation and nutrient uptake. In addition, endophytic bacteria can increase plant health and tolerance by antibiotic and hydrolytic enzyme production. This study aims to screen and characterize endophytic bacteria of celery as plant growth promoter agents. Thirty endophytic bacteria were isolated successfully from part of the celery plant using the spread plate and plant piece method which grew at Nutrient Agar media. Based on the characterization assay, 30 isolates may produce IAA with various concentrations of 0.21-7.18 mg/L. In addition, 3 of 30 isolates had phosphate solubilizing activity, in different indexes ranging from 0.71-1.43. Twenty-one isolates were able to grow in an N-free medium, indicating the capability of isolates to fix nitrogen qualitatively. The hydrolytic enzymes assay resulted in 14 amylolytic, 17 proteolytic, and 12 cellulolytic activities. In addition, nine of 30 isolates show multi-activity in amylase, protease, and cellulase enzyme production. Based on the screening results, there are four potential isolates (SLBg 1.2; SLBg 1.5; SLAg 1.1, and SLAg 1.5) as promising plant-growth promoters and were molecularly identified as members of the Bacillus genus. These isolates can be developed as biofertilizers for supporting the cultivation of celery plants.

Production of alkaline protease by Aspergillus niger in a new combinational paper waste culture medium

Enzymes derived from microbial sources have gained increasing popularity in industrial applications over the past decades. Despite the high production cost, alkaline proteases have wide applications in industries such as tanneries, food production, and detergents. In recent years, there has been a shift towards utilizing natural carbon sources for cultivating microorganisms and extracting proteases in order to reduce production costs. This study aimed to investigate the biochemical and kinetic properties of protease enzymes obtained from Aspergillus niger cultivated in a paper waste medium and compare with the enzyme produced in a basal medium. Glucose is a more favorable carbon source compared to cellulose, so paper waste was pretreated with cellulose-degrading bacteria to convert cellulose into smaller carbohydrates. After the growth of A.niger in basal and combinational media, the enzymatic properties were compared between the extracted enzymes by using casein as substrate. The results demonstrated that A.niger could produce protease enzymes in the paper waste medium similar to the basal medium with more than 5-fold cost saving. The specific activity of the enzymes isolated from the basal and paper waste media was calculated to be 184.95 ± 10.56 Uml-1 and 169.88 ± 11.05 Uml-1, respectively. Carbon sources did not affect the optimum pH and temperature of the protease enzyme, which were found to be 8 and 37 °C, respectively. This study provides valuable insights into the production of alkaline protease from A.niger using a combinational medium (paper waste pretreated by cellulose-degrading bacteria), offering a cost-effective approach for industrial applications.

Antagonistic Test of Bacteria Producing Siderophore and Protease Enzymes from The Rhizosfer of Peanut Plants on The Growth of Pathogenic Fungus Colletotrichum gloeosporioides

This study aims to determine the inhibitory effect of bacterial isolates producing siderophores and protease enzymes on the growth of the pathogenic fungus Colletotrichum gloeosporioides. The initial stage of research begins with the isolation of pathogenic bacteria and fungi, and is followed by testing the production of siderophores and protease enzymes. Bacteria were isolated from the rhizosphere of peanut plants, while pathogenic fungi were isolated from large chili fruit infected with anthracnose disease taken from Jelantik Village, Central Lombok Regency. Characterization of isolates for siderophore production used the Arnow method, while the protease enzyme production test used SKIM Milk Agar media. Next, the inhibition test of bacterial isolates against pathogenic fungi was carried out using the dual culture method. Characterization of potential isolates was carried out by observing bacterial colony morphology, gram staining and biochemical tests. The results of the siderophore production test showed that there was one isolate capable of producing siderophores with the isolate code RKT2. Meanwhile, the protease enzyme production test showed that all bacterial isolates produced protease enzymes, where isolate RKT9 had the highest Proteolytic Index, namely 1.57. The two isolates showed different inhibitory test results, namely isolate RKT2 had high inhibition, while RKT9 had low inhibition. The results of the research showed that a bacterial isolate (RKT2) from the rhizosphere of peanut plants was able to inhibit the growth of the pathogenic fungus Colletotrichum gloeosporioides in the high category.

Protease production and molecular characterization of a protease dipeptidyl-aminopeptidase gene from different strains of Sordaria fimicola.

The current research was designed to reach extracellular protease production potential in different strains of Sordaria fimicola which were previously obtained from Dr. Lamb (Imperial College, London) from North Facing Slope and South Facing Slope of Evolution Canyon. After initial and secondary screening, two hyper-producers strains S2 and N6 were selected for submerged fermentation and cultural conditions including temperature, pH, incubation period, inoculum size, substrate concentration, and different carbon and nitrogen sources were optimized for enzyme production. S2 strain showed maximum protease production of 3.291 U/mL after 14 days of incubation at 30 °C with 7 pH, 1% substrate concentration and 1 mL inoculum, While N6 strain showed maximum protease production of 1.929 U/mL under fermentation optimized conditions. Another aim of the present research was to underpin the biodiversity of genetics and post-translational modifications (PTMs) of protease DPAP (peptidyl-aminopeptidase) in Sordaria fimicola. Five polymorphic sites were observed in amino acid sequence of S. fimicola strains with reference to Neurospora crassa. PTMs prediction from bioinformatics tools predicted 38 phosphorylation sites on serine residues for protease peptidyl-aminopeptidase in S1 strain of S. fimicola while 45 phosphorylation sites on serine in N7 strain and 47 serine phosphorylation modifications were predicted in N. crassa. Current research gave an insight that change in genetic makeup effected PTMs which ultimately affected the production of protease enzyme in different strains of same organism (S. fimicola). The production and molecular data of the research revealed that environmental stress has strong effects on the specific genes through mutations which may cause genetic diversity. S. fimicola is non- pathogenic fungus and has a short life cycle. This fungus can be chosen to produce protease enzyme on a commercial scale.

Biodisintegration of human mucin protein by protease produced from Escherichia coli AJ55 isolated from Urinary Tract Infection of Iraqi pa-tients

Proteases have various applications in the food, pharmaceutical, medicine, pathogenicity of some pathogenic bacteria, and detergent sectors as well as meeting the needs of approximately 60% of the global enzyme industry, whereas they catalyze the breakdown of protein molecules into peptides and amino acids. Production and purification of protease enzyme by the isolate Escherichia coli AJ55 was scrutinized in the present study. Cultivation optimum conditions, were various complex medium, carbon source, nitrogen source, temperature, pH of the medium, and time of incubation were optimized to enhance the total protease production in shake flask culture of E.coli AJ55. The nutrient broth supplemented with 2% glucose and 2% yeast extract, with a pH of 7.0 and incubated at 37 °C for 24 hours, better conditioned for producing the maximum production of protease. Escherichia coli AJ55 proteolytic enzyme was separated and purified using ion-exchange chromatography on a DEAE-Cellulose column and Sephadex G-150 gel after being precipitated with 0-70% saturated ammonium sulfate. Protease that had been partially purified had a yield of 34%, a purification fold of 13.4, an activity of 12.16 U/ml, a protein concentration of 0.005 mg/ml, and a specific activity of 2432 U/mg. By using gel filtration chromatography on a Sephadex G-150 gel, partially purified protease was examined for its ability to cleave the mucin protein. The findings of mucin biodegradation showed that the five fractions of the small peptides were produced after treatment of mucin with partially purified protease.

Molecular Characterization and Optimization of Alkaline Protease Production by Bacillus cereus LS23B

The study aimed to determine the conditions leading to maximum protease production using submerged fermentation and detecting the presence of protease genes in the bacteria. This is necessary to meet the increasing demand for protease enzymes in the industrial market. The ability of the bacterial isolate to produce protease enzymes was evaluated through primary screening. After that, morphological characterization, biochemical tests, and 16S rRNA analysis were done to identify the bacterial strain. To confirm the presence of the gene-encoding enzyme, the protease (npr) gene primer was amplified using a polymerase chain reaction. The pH, incubation duration, temperature, carbon, and nitrogen sources were studied. Others include metal ions, substrate concentration, and agitation speed. The bacterial strain has 100% similarity to Bacillus cereus A9, while the protease encoding gene was confirmed with positive bands of 951bp. The enzyme was optimally produced at 40 oC with a pH of 9 after 72 h incubation. Starch, gelatin, 1 % substrate concentration, 1mM K+, and agitation speed of 160 rpm fully supported protease production. The presence of the npr gene in the isolate was confirmed. Also, the optimization study reveals that Bacillus cereus LS23B can be used in large-scale protease production, which may be used in different biotechnological applications.

Heavy metal-resistant rhizobacteria fosters to alleviate the cadmium toxicity in Arabidopsis by upregulating the plant physiological responses

This study was designed to investigate the role of Morganella morganii strains in alleviating Cd stress in Arabidopsis seedlings under controlled conditions. Both M. morganii strains ABT3 (ON316873) and ABT9 (ON316874) strains isolated from salt-affected areas showed higher resistance against Cd and possess plant growth-promoting traits such as nitrogen fixation, indole-acetic acid production, ammonia production, phosphate solubilization, and, catalase, gelatinase and protease enzyme production. Plant inoculation assay showed that varying concentration of Cd (1.5 mM and 2.5 mM) significantly reduced Arabidopsis growth, quantum yield (56.70%-66.49%), and chlorophyll content (31.90%-42.70%). Cd toxicity also triggered different associations between lipid peroxidation (43.61%-69.77%) and enzymatic antioxidant mechanisms. However, when both strains were applied to the Arabidopsis seedlings, the shoot and root length and fresh and dry weights were improved in the control and Cd-stressed plants. Moreover, both strains enhanced the resistance against Cd stress by increasing antioxidant enzyme activities [catalase (19.47%-27.39%) and peroxidase (37.50%-48.07%)]that ultimately cause a substantial reduction in lipid peroxidation (27.71%-41.90%). Both strains particularly ABT3 also showed positive results in improving quantum yield (73.84%-98.64%) and chlorophyll content (41.13%-48.63%), thus increasing the growth of Arabidopsis seedlings. The study suggests that PGPR can protect plants from Cd toxicity, and Cd-tolerant rhizobacterial strains can remediate heavy metal polluted sites and improve plant growth.

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

Isolation and Characterization of Proteolytic Enzyme Produced from Fungi

Protease is an enzyme that conducts proteolysis and receives high attention as the most important enzyme in many industries. The enzymes find applications in waste treatment, bioremediation processes, detergents, and the leather industry. However, the search for microbial sources of novel proteases in nature is a promising area of interest for researchers. In the present research work, protease-producing microorganisms were isolated from soil samples collected from two different locations in UNIZIK, from the surface and at depths of 15 cm. The isolated organisms were screened for their protease-generating abilities by using the skim milk agar experiment. The isolates that gave high positive results in screening were identified as Aspergillus sp. and Penicillium sp. by analysing the colonies morphology and using the lacto-phenol cotton blue (LPCB) staining technique. Of these isolates, Aspergillus sp. that was isolated from the soil surface demonstrated the highest protease activity, as indicated by the clear zone around the colony that was incubated at room temperature for 96 hours. The results showed that among the isolates, two fungi had great potential to be used for the production of protease enzymes and upscaled for industrial production.

Production and Optimization Studies of Protease by Soil Isolate Actinomycetes Using Mixed Substrate of Pomegranate Peel Powder and Banana Peel Powder under Solid State Fermentation

The Actinomycetes are gram-positive, unicellular bacteria that grow in a typical filamentous manner on solid substrates and may synthesis a wide range of secondary metabolites, including enzymes. Proteases are universally present enzymes that catalyze hydrolytic processes that break down protein molecules into peptides and amino acids. The protease enzyme is used in food and detergent industries. The goal of the current investigation is to identify and isolate a prospective isolate of Actinomycetes capable of solid state fermentation for the protease enzyme production. The soil samples yielded a total of 30 isolates. On starch casein nutrition agar medium, B5 isolate was observed to be a potential strain for production of protease enzyme. The optimum conditions for the maximum protease enzyme production were observed to be at 30°C with a pH of 7 when incubated for 5 days with a 6th day old culture and 2mL of the inoculum volume for protease with substrate concentration (pomegranate and banana peel powder of each 2.5 g) of 5 g. The addition of Carbon source starch and Nitrogen source casein to the production medium has enhanced the enzyme activity to 442.8 U/gds.

Identification of Sponge-Associated Bacteria From the Coast of Kuwait and Their Potential Biotechnological Applications.

Sponges are among the most ancient animals harboring complex microbial communities with potential applications in biotechnology. The Arabian Gulf is a thermally stressed enclosed body of water located in an arid region where sponges and their halobionts are understudied. This study combined 16S rRNA next-generation gene amplicon sequencing and cultivation techniques to explore the abundance and diversity of sponge-associated bacteria. Culture-independent techniques showed the associations of more than 25 bacterial phyla with Amphimedon sp., Chondrilla australiensis, Haliclona sp., and Niphates spp. Regarding cultivable bacteria, 315 bacterial isolates associated with the sponge Haliclona sp. were cultivated; these isolates were affiliated with the phyla Proteobacteria and Firmicutes and were distributed among six bacterial genera. Selected strains of Bacillus, Ferrimonas, Pseudovibrio, Shewanella, Spongiobacter, and Vibrio were tested for antimicrobial activity against indicator microorganisms and protease enzyme production. Seven Bacillus strains exhibited weak to moderate growth inhibition against Bacillus subtilis, Staphylococcus aureus, and Candida albicans. Furthermore, 29 different strains of Bacillus, Ferrimonas, Shewanella, and Vibrio exhibited different degrees of positive protease activity. In addition, cultivated strains of Bacillus, Shewanella, Pseudovibrio, and Vibrio were tested for their biomineralization abilities. Herein we report for the first time the isolation of biomineralizing bacteria from sponge tissue where eleven bacterial isolates produced different shapes of calcium carbonate crystals on agar. Our observations shed light on the diversity and biotechnological potentials of sponges-associated bacteria inhabiting one of the world’s hottest seas.

Effective Enzyme-Producing Bacteria Isolated from Diversified Thai Rice and Native Thai Bees (Cavity Nesting Honey Bees) and their Potential for Production of Protease Enzymes

Effective Enzyme-Producing Bacteria Isolated from Diversified Thai Rice and Native Thai Bees (Cavity Nesting Honey Bees) and their Potential for Production of Protease Enzymes