Bacillus Sps Research Articles

Approach towards sustainable leather: Characterization and effective industrial application of proteases from Bacillus sps. for ecofriendly dehairing of leather hide

Tanneries are one of the most polluted industries known for production of massive amount of solid and liquid wastes without proper management and disposal. In this project we demonstrated the ecofriendly single step dehairing of leather hides with minimum pollution load. In this study, Bacillus species (Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P) capable of producing proteases was successfully isolated by employing the new optimized selective media named M9-PEA as confirmed by 16sRNA genes sequencing. Sequence of 1493 bp long 16S rRNA genes of Bacillus paralicheniformis strain BL.HK and Bacillus cereus strain BS. P was submitted to GenBank under the accession number OP612692.1, OP612721.1 respectively The Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P produced extracellur proteases of 28 and 37 KDa as resolved by SDS-PAGE respectively. The enzymes showed temperature optima at 50 °C and 55 °C and pH optima at 8.5, 9.5 respectively. The Proteases of Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P were employed for dehairing of animal hides. The process resulted in significant removal of interfibriller substances without damage to collagen layer after one hour treatment, which was confirmed by histology, scanning electron microscopy. The quantification of various skin constituents (collagen, uronic acid, hexosamines, and GAGs) and pollution load parameters revealed that enzymatic treatment are more reliable. The results of skin application trials at industrial level with complete elimination of chemicals remark the biotechnological potential of these proteases for ecofriendly dehairing of animal hides without affecting the quality of the leathers produced.

Predominance and bioactive potential studies of endophytic Bacillus Sps. from marine algae of Palkbay costal region, India

The genus Bacillus is a widely distributed and prominent species that has been extensively studied for its numerous benefits in various industries, medical applications, and agriculture. In this study, endophytic Bacillus species were isolated from seven different algal species using various growth media such as Nutrient agar, starch casein agar, ISP 2, and ISP 4. Throughout the study period, a total of 36 bacterial strains with similar morphological characteristics were identified. Among the isolates, five bacterial strains were found to be predominant when grown on ISP 2 media. These five strains were further subjected to 16s RNA Sequencing to determine their taxonomic classification. The results revealed that all of these strains belonged to the genus Bacillus. However, the other endophytic strains could not be identified due to limited sequence homology with existing entries in the NCBI database. To gain further insights into the isolated Bacillus cultures, analytical studies were conducted. Firstly, the functional groups present in the cultures were identified using Fourier-transform infrared spectroscopy (FTIR). Additionally, the compounds present in the crude extracts of the cultures were analyzed using gas chromatography-mass spectrometry (GC-MS). The findings of this current research demonstrate the presence of Bacillus species among green and brown algae in the Palk Bay coastal range. Moreover, these Bacillus strains exhibit potential for various biotechnological applications. The study aims to establish the predominance of Bacillus species in the algae of the Palk Bay region in South India, thereby contributing to our understanding of the microbial ecology in this specific geographical area

Efficacy of Bacillus sps. and Pseudomonas sps on Growth and Yield of Cherry Tomato in the sub-tropical climate of Prayagraj, U.P., India

Prayagraj is situated at the bank of river Ganga and Yamuna, having humid, hot weather during summer and severe winter that comes under sub-tropical climatic zone. Cherry tomato is a climate-sensitive crop grown successfully in India’s hilly and foothill region. In climatic zone such as Prayagraj, it experiences natural stresses which hamper its growth and development finally affecting yield and quality. PGPRs are the natural source growth regulator which has been found effective in increasing growth and is also known for reducing stresses. Bacillus and Pseudomonas are well-known PGPR that are effective in mitigating stress effects and improving growth and yield. This study is designed to understand Bacillus and Pseudomonas’ efficacy in improving cherry tomato growth and yield in sub-tropical conditions like Prayagraj. Our finding states that Bacillus + Pseudomonas soil application effectively increases plant height (117.33cm), Number of Leaves (106.60), Number of fruits/plant (220), Fruit weight (8.78g), chlorophyll a(0.68 mg/g F/w), chlorophyll b(0.36 mg/g F/w), Carotenoids (0.55 mg/g F/w), Lycopene (156.33µg/g) than compared to control. We also find that soil application was more successful than foliar application.

The use of microbial fuel cell for efficient treatment of cauliflower waste and generation of electricity

ABSTRACT Microbial fuel cell (MFC) is an alternative way for household organic waste treatment as it produces sustainable clean energy. Chemical parameters of the cauliflower leaf waste before treatment were 143.5 mg/g COD, 0.90 mg/g ammoniacal nitrogen, 67.8 mg/g phosphorous, 214.8 mg/g total reducing sugar and 0.53 mg/g soluble reducing sugar. After eight days of treatment in MFC at the optimised condition of multiwalled carbon nanotubules (MWCNT)’ coated graphite as anode and potassium ferricyanide added phosphate buffer as catholyte with an external resistance of 1000 Ω showed a reduction in COD, ammoniacal nitrogen, phosphorous and total reducing sugar by 24.7, 76.9, 22.5 and 53.4%, respectively, along with a maximum power density of 10.1 W/m3. Scanning electron microscopy (SEM) showed bacterial adherence in the graphite electrode and its molecular characterisation using 16S rRNA sequencing confirmed as Bacillus sps.

The Effects of Silicon Dioxide Nanoparticles and Zinc Oxide Nanoparticles on Waste Land soil Bacterial and Fungal Isolates

Objective: Different bacterial and fungal isolates were collected from the wasteland municipality site, Tambaram. The antimicrobial activity of two types of nanoparticles ZnO & SiO2 [Zinc oxide and Silicon dioxide] against several types of Gram-negative bacteria and fungi was investigated in this work.
 Methods: P. aeruginosa, B. subtilis, Penicillium oxalicum and Aspergillus fumigatus were isolated from 5 soil samples taken from three sites of Tambaram Municipality wasteland (Chennai). After collecting the samples, we used culturing and biochemical tests to identify the microbes and then used a chemical approach to make ZnO and SiO2 nanoparticles with altered structure and morphological features. Minimum inhibitory concentration (MIC) was used to assess the antibacterial activity of these nanoparticles against various microorganisms.
 Results: The best inhibition zone was found in Pseudomonas sps and Bacillus sps growth at concentrations of 10 µg/ml and 5 µg /ml of nano-ZnO, respectively, whereas the lower inhibition zone was found in Penicillium oxalicum and Aspergillus fumigatus at a dosage of 2.5 µg /ml of the same nanoparticle. It was also discovered that no inhibitory zone existed in any of the bacteria and fungi at a concentration of 10 µg /ml nano-SiO2. We found that all of the bacteria and fungi we tested were completely inhibited at a concentration of 1.25 g/ml nano-ZnO (MIC), with no antibacterial activity below this concentration. When compared to data that showed that all tested bacteria were not completely inhibited even at a concentration of 0.625 g/ml of nano-SiO2.
 Conclusion: In comparison to the two nanoparticles (ZnO and SiO2), nano-ZnO outperformed nano-SiO2 in inhibiting most bacteria and fungi at the quantities tested in wasteland soil.

Exraction of Secondary Metabolites Using Bacillus endopyticus and Its Applications

Bacillus Species was isolated from a soil sample, the isolate was cultivated, identified and its culture medium was extracted. Specific media called Gibbon’s media used for the cultivation and confirmation of the bacteria. 16S rRNA study was done for the morphological identification of the isolate. The crude which was collected from Bacillus endopyticus was purified by column chromatography. The antibacterial activity of the crude extract was identified by agar diffusion test by measuring the minimum inhibitory concentration by dilution assay. The purified extract was active against Staphylococcus aureus, Bacillus cerues, Escherichia coli. The study suggest that Bacillus Sps have the potential to produce antibiotics and can be used to control the growth in future. The further work was able to bring the microbial strains under control. The percentage of cell death was calculated the cell death was 97.95%.

Isolation and characterization of a protease from Bacillus sps

Abstract Proteases are highlighted group of enzymes which corresponds to approximately 60% of enzymes used globally in commercial applications. Present study describes isolation of a new protease from TSA5 strain. Sequencing of 16SrRNA revealed identity to Bacillus sps with common five restriction sites (Acc651, Btscl, Fok1, Plel, Kpnl). The enzyme from TSA5 strain partial purified by, salting and dialysis which resulted a homogeneity of 6-fold with the yield of 34%. Enzyme activity was characterised for a broad range of pH (4–10.5) and temperature (0–120 °C). Optimum activity was observed at pH 8.5 and 80 °C. The enzyme showed substrate specificity to casein. The effect of different solvents and chemicals were assayed. It is found to be more active in presence of polar solvents. EDTA (Ethylenediaminetetraacetic acid) and β-mercaptoethanol shows potent inhibition. The purified fraction showed silver recovery from X-ray films. The protease also exhibited detergent activity and antibacterial property suggesting the use of enzymes for enzyme hydrolysis in industries for commercial applications.

Versatile and Valuable Utilization of Amidohydrolase L-glutaminase in Pharma and Food industries: A Review.

L-glutaminase has versatile applications in pharma and food industries. In pharmaceutical industry, L-glutaminase can be used as anti-oxidant and anti-cancer agent to treat Acute Lymphocytic Leukaemia (ALL). Whereas, in the food industry, L-glutaminase is used for acrylamide degradation, theanine production, flavour enhancer, soy sauce and many. The other applications include nitrogen metabolism and its use as biosensor in hybridoma technology. Both intra-cellular and extra-cellular L-glutaminases from wide range of sources were identified. Because of its diverse applications, there is a need to improve the production of L-glutaminase by enzyme engineering technology. Effect of recombination on L-glutaminase production was also reported. Researchers also confirmed the antitumor properties of L-glutaminase by conducting in vitro, in vivo and in silico studies. Bacillus sps. and Aspergillus sps. are the commercial producers of L-glutaminase. In this review, the applications, different sources of Lglutaminase, anti-cancer properties were discussed.

Molecular Characterization of Bacillus sps. having Ability to utilize Leachate

Molecular Characterization of Bacillus sps. having Ability to utilize Leachate

In Silico Appraisal, Synthesis, Antibacterial Screening and DNA Cleavage for 1,2,5-thiadiazole Derivative.

Thiadiazole not only acts as "hydrogen binding domain" and "two-electron donor system" but also as constrained pharmacophore. The maleate salt of 2-((2-hydroxy-3-((4-morpholino-1, 2,5-thiadiazol-3-yl) oxy) propyl) amino)- 2-methylpropan-1-ol (TML-Hydroxy)(4) has been synthesized. This methodology involves preparation of 4-morpholino-1, 2,5-thiadiazol-3-ol by hydroxylation of 4-(4-chloro-1, 2,5-thiadiazol-3-yl) morpholine followed by condensation with 2-(chloromethyl) oxirane to afford 4-(4-(oxiran-2-ylmethoxy)-1,2,5-thiadiazol- 3-yl) morpholine. Oxirane ring of this compound was opened by treating with 2-amino-2-methyl propan-1- ol to afford the target compound TML-Hydroxy. Structures of the synthesized compounds have been elucidated by NMR, MASS, FTIR spectroscopy. The DSC study clearly showed that the compound 4-maleate salt is crystalline in nature. In vitro antibacterial inhibition and little potential for DNA cleavage of the compound 4 were explored. We extended our study to explore the inhibition mechanism by conducting molecular docking, ADMET and molecular dynamics analysis by using Schrödinger. The molecular docking for compound 4 showed better interactions with target 3IVX with docking score of -8.508 kcal/mol with respect to standard ciprofloxacin (docking score= -3.879 kcal/mol). TML-Hydroxy was obtained in silico as non-carcinogenic and non-AMES toxic with good percent human oral absorption profile (69.639%). TML-Hydroxy showed the moderate inhibition against Mycobacteria tuberculosis with MIC 25.00 μg/mL as well as moderate inhibition against S. aureus, Bacillus sps, K. Pneumoniae and E. coli species. In view of the importance of the 1,2,5-thiadiazole moiety involved, this study would pave the way for future development of more effective analogs for applications in medicinal field.

Biodegradation of diuron by endophytic Bacillus licheniformis strain SDS12 and its application in reducing diuron toxicity for green algae.

The endophytic bacteria live in close nuptial relationship with the host plant. The stress experienced by the plant is expected to be transferred to the endophytes. Thus, plants thriving at polluted sites are likely to harbor pollutant-degrading endophytes. The present study reports the isolation of phenylurea herbicides assimilating Bacillus sps. from Parthenium weed growing at diuron-contaminated site. The isolated endophytes exhibited plant growth-promoting (PGP) activities. Among five isolated diuron-degrading endophytes, the most efficient isolate Bacillus licheniformis strain SDS12 degraded 85.60 ± 1.36% of 50 ppm diuron to benign form via formation of degradation intermediate 3, 4-dichloroaniline (3,4-DCA). Cell-free supernatant (CFS) obtained after diuron degradation by strain SDS12 supported algal growth comparable with the pond water. The chlorophyll content and photosynthetic efficiency of green algae decreased significantly in the presence of diuron-contaminated water; however, no such change was observed in CFS of strain SDS12, thus, suggesting that strain SDS12 can be applied in aquatic bodies for degrading diuron and reducing diuron toxicity for primary producers. Further, the use of PGP and diuron-degrading bacteria in agriculture fields will not only help in remediating the soil but also support plant growth.

Schiff base clubbed benzothiazole: synthesis, potent antimicrobial and MCF-7 anticancer activity, DNA cleavage and computational study

Schiff bases containing benzothiazole unit are synthesized and characterized by 1H NMR, 13C NMR, LC–MS, UV–visible and fluorescence spectroscopy methods. Synthesized compounds were evaluated for in vitro antibacterial activities on four different strains (Staphylococcus aureus, Bacillus sps, Escherichia. coli and Klebsiella pneumoniae) and antifungal activity on one strain (Candida albicans) by using ‘micro-broth dilution method’ (MIC in µg/mL). All compounds showed excellent antibacterial activity for S. aureus, E. coli and K. pneumoniae strains, as well as an antifungal activity for C. albicans, compared to standard Ciprofloxacin and Fluconazole, respectively. Molecular docking studies were carried out to get more insight into the binding mechanism. Reactivity of molecules was assessed using molecular electrostatic potential (MEP). HOMO–LUMO gap, chemical hardness and global softness were calculated and correlated with antimicrobial activity. VS5-e exhibits excellent antimicrobial activities and also shows more chemical reactivity and less toxicity which were confirmed by smaller HOMO–LUMO gap, lesser chemical hardness, higher global softness and lesser electrophilicity index. Henceforth, it was further assessed for DNA cleavage and against MCF-7 breast cancer cell. VS5-e reveals 85.82% inhibition of cancer cells at a concentration of 200 µg/mL. The compound VS5-e showed less toxicity to normal cells at the concentration required to produce an anticancer effect (with high IC50 = 973 µg/mL).Communicated by Ramaswamy H. Sarma

Screening, optimization of culture conditions and scale-up for production of the L-Glutaminase by novel isolated Bacillus sps. mutant endophyte using response surface methodology

Abstract L-Glutaminase is known as the anti-cancer drug to treat anti-leukemic cancer besides its use in food industry for the acrylamide degradation of fried foods. In the present study, submerged fermentation was carried out for the production; statistical based experimental designs were employed to optimize the culture conditions and to maximize the L -Glutaminase activity from novel Bacillus sps., mutant endophyte isolated from Ocimum tenuiflorum. Eleven process variables of the medium were examined for their significance using Plackett-Burman design on the production of L-Glutaminase i.e., Galactose, L -Glutamine, Na2HPO4, KH2PO4, NaCl, MgSO4·H2O, Yeast extract, pH, Temperature, RPM, Time were screened and it was observed that activity was 22.49 IU/ml. After Plackett-Burman design experiments, the activity was found to be 2.34 folds increase compared to one variable at a time method. Further, the production was optimized with Response Surface Methodology (RSM) using Central Composite Design (CCD) for significant variables i.e., Galactose (g/L), Time (h), Temperature (T°C) and pH obtained from Plackett-Burman design and the activity was found to be 24.585 IU/ml i.e., 2.56 fold increment in the production compared to unoptimized medium. Experimentation revealed that the isolated novel Bacillus sps., mutants as the potential bacterial source for L -Glutaminase production.

Synthesis, Antitubercular and Antimicrobial Activity of 1,2,4‐Triazolidine‐3‐thione Functionalized Coumarin and Phenyl Derivatives and Molecular Docking Studies

AbstractTuberculosis (TB) is still a demanding worldwide health problem and mycobacterium tuberculosis (MTB) remains one of the most toxic human pathogens. In pursuit of searching new antitubercular and antimicrobial agents, substituted coumarin and phenyl‐1,2,4‐triazolidine‐3‐thiones 4a‐i and 5a‐e have been synthesized and evaluated for their antitubercular and antimicrobial activities. Most of the Substituted coumarin and phenyl‐1,2,4‐triazolidine‐3‐thiones showed promising activity against Mycobacterium tuberculosis (H37Rv). The title compounds have exhibited excellent in vitro antibacterial activity against the S.aureus, Bacillus sps and E.coli with the values of low MIC ranging from of 0.4 to 1.6 μg/mL. In vitro antifungal activity shown that the compounds 4a‐i and 5a‐e are excellent antifungal agents against Candida albicans, Aspergillus flavus, Aspergillus niger and Aspergillus fumigate fungal stains with the values of low minimum inhibitory concentrations (MIC) ranging from 0.4 to 6.25 μg/mL. Molecular docking study was performed for all the synthesized compounds with E.coli as antibacterial and Mycobacterium tuberculosis DprE1 as antituberculosis.

Specificity studies on proteases for dehairing in leather processing using decorin as model conjugated protein

Enzyme based dehairing of animal skins in leather processing is considered a greener alternative to conventional chemical based dehairing process. The specificity based screening of proteases would be an important criterion as it would help in removing hairs by acting particularly on the proteoglycans holding the hair to the bulb without damaging the other proteins. The present paper describes a comparative study of the specificity of a dehairing protease with non-dehairing one isolated from Bacillus sps. Dehairing protease was found to be trypsin-like, non-collagenolytic and non-keratinolytic enzyme whereas non-dehairing one was a metalloprotease with non-collagenolytic activity but specificity for soluble keratin. Substrate specificity was studied using decorin as model proteoglycan. The dehairing enzyme was effective in digesting the core protein of the decorin as confirmed by high performance liquid chromatography analysis and SDS-polyacrylamide gel electrophoresis. The histochemical studies on dehaired skins also confirmed preservation of skin matrix with no visible damage to collagen. This study emphasizes the importance of screening proteases based on their specificity characteristics for effective and safer enzyme-only dehairing process. The acquired knowledge can be employed in formulating bacterial growth media to produce dehairing proteases that would help in commercial exploitation of this greener option.

Emergence of multi drug resistance among soil bacteria exposing to insecticides.

Impacts of pesticide exposure on the soil microbial flora and cross resistance to antibiotics have not been well documented. Development of antibiotic resistance is a common issue among soil bacteria which are exposing to pesticides continuously at sub-lethal concentration. The present study was focused to evaluate the correlation between pesticide exposures and evolution of multi drug resistance among isolates collected from soil applied with insecticides. Twenty five insecticide (Monochrotophos) degrading bacteria were isolated from contaminated agricultural soil. The bacterial isolates Bacillus Sps, Bacillus cereus, Bacillus firmus and Bacillus thuringiensis were found to be resistant against chloramphenical, monochrotophos, ampicillin, cefotaxime, streptomycin and tetracycline antibiotics used. Involvement of plasmid in drug as well as insecticide resistant was confirmed through plasmid curing among selected bacterial strains. Bacillus Sps (MK-07), Bacillus cereus (MK-11), Bacillus firmus (MK-13) and Bacillus thuringiensis (MK-24) lost their resistant against insecticides and antibiotics once after removal of plasmid by exposing to 2% sodium dodecyl sulphate. The plasmid was transformed back to bacteria which produced similar derivatives when cultured in Minimal Salt medium (pH 7.0) supplemented with 0.4% of insecticide. Homology modeling was used to prove that organophosphorus hydrolase and able to metabolize all the antibiotics showed positive interaction with high docking score. The present study revealed that persistent of insecticides in the agricultural soil may lead to increasing development of multidrug resistance among soil bacteria.

Phytochemical analysis, anti-oxidant and anti-microbial activity of <em>Acalypha indica</em> leaf extracts in different organic solvents

The herb <em>Acalypha indica </em>which belongs to Euphorbiaceae family has multiple medicinal properties which include anti-oxidant, anti-bacterial, anti-fungal, anti-inflammatory, antiulcer, anti-helmintic, anti-cancerous, anti-venom, and neuro-protective activity. The present study was designed to evaluate the phytochemical, antimicrobial and anti-oxidant activity of <em>Acalypha indica </em>leaves extracts in different solvent extractions like methanol, hexane, ethyl acetate and petroleum ether. Fresh leaves of the plant were collected and shade dried. Dried leaves were milled to obtain powder. Powder was subjected to soxhlet extraction using solvents and extracts were successively obtained. Phytochemical analysis was conducted following standard methods. Phytochemical analysis showed the presence of Alkaloids, Phenols, Saponins, Flavanoids and Amino acids. Leaf extract of methonal have shown the highest anti-oxidation capacity than hexane, ethyl acetate and petroleum ether. Anti-microbial activity has been performed on microbes like <em>Bacillus sps</em>, <em>E.coli,</em> <em>Psuedomonas sps</em> and <em>Streptococcus sps.</em> A highest value of zone of inhibition was found in methanol extract against <em>E.coli.</em> These results provide evidence that <em>Acalypha indica </em>leaf extract possesses vital phytochemicals, antimicrobial and antioxidant properties. Hence this plant can be studied further for drug analysis for finding potent medicines for diseases.

Study of Heavy metal uptake by Bacillus sp and E. coli

Background: Over the past century, unrestricted mining, extensive industrialization, modern agricultural practices and faultywaste disposal methods have resulted in the release of unprecedented levels of toxic heavy metals like Cd, Hg, Ag, Sn, Pb, Cu, Co,Mn, Zn, etc into the environment. Many metals are essential for microbial growth in less concentration, yet are toxic in higherconcentrations. Various methods are available for the removal and management of heavy metals: Conventional physico-chemicalmethods and emerging biosorption methods. Biosorption is an attractive alternative approach which involves the binding oradsorption of heavy metals to living or dead cells. Many microbes have the ability to selectively accumulate metals. Aim: Thepresent study was intended to analyze the uptake systems of Bacillus and E. coli against different concentration of heavy metalslike Zn, Cu, Cd, and Hg in their salt form incorporated into nutrient broth medium observed over a regular interval of time.Methodology: Analysis was based on how much of the metal from the original concentration used was left behind in the mediaafter the rest being up taken by the organism. This was done using Atomic Absorption Spectrophotometry (AAS) which wasindirectly the representation of percent uptake of heavy metal by the respective organism. Results: The study showed that Gram –ve organisms like E.coli exhibited more resistance to metals like Zn, Cu and Hg in relative comparison with Gram +ve organismslike Bacillus. Conclusion: Bacillus sps was less sensitive to effect of Cd than in E. coli

STUDY OF LIGNINOLYTIC BACTERIA ISOLATION AND CHARACTERIZATION FROM DHAMDHA AGRO FIELD OF BHILAI-DURG REGION

Lignin is a complex, three-dimensional aromatic polymer consisting of dimethoxylated, monomethoxylated and non-methoxylated phenylpropanoid subunits. It is the most abundant renewable carbon source on Earth. The majority of plant biomass, including stems and leaves, is composed of lignocellulose. In present study isolation, identification and characterization of ligninolytic bacterial flora were done from the agro-field, using a lignin residue. 2 types of soil samples black and mixed soil were selected from agro fields of Dhamdha of Bhilai-Durg for isolation of ligninolyitc bacterial colony. Microbes from both mixed and black soil samples were grown in solid media by Hungate method and result shown that three types of bacterial colonies (1-MS, 2-BS), were isolated from Dhandha agro field and used to check the activity of lignolytic capability. Out of 3 types colonies only 1types of colony (j-MS), was shown potential of lignin degradation. The Morphological, gram’s reaction and endospores staining reaction, biochemical characteristics of the isolate obtained from this agro-field soil samples identified with reference to Bergey’s Manual of Determinative Bacteriology. These identified isolate (j) - Bacillus species was shown presence of Laccase, Manganese peroxidase (MnP) and Lignin peroxidase (LiP), etc. lignin degrading enzymes. This results concluded that Bacillus sps. strain was able to degrade lignin substrate which was second abundant and waste material in the world. It is also concluded that to expand on the range of products which can be obtained from lignocellulosic biomass. In this study, soil bacteria was isolated by enrichment on Kraft lignin and evaluated for their ligninolytic potential as a source of novel enzymes use to generate 2 nd

Macrobrachium rosenbergii mannose binding lectin: Synthesis of MrMBL-N20 and MrMBL-C16 peptides and their antimicrobial characterization, bioinformatics and relative gene expression analysis

Mannose-binding lectin (MBL), an antimicrobial protein, is an important component of innate immune system which recognizes repetitive sugar groups on the surface of bacteria and viruses leading to activation of the complement system. In this study, we reported a complete molecular characterization of cDNA encoded for MBL from freshwater prawn Macrobrachium rosenbergii (Mr). Two short peptides (MrMBL-N20: 20AWNTYDYMKREHSLVKPYQG39 and MrMBL-C16: 307GGLFYVKHKEQQRKRF322) were synthesized from the MrMBL polypeptide. The purity of the MrMBL-N20 (89%) and MrMBL-C16 (93%) peptides were confirmed by MS analysis (MALDI-ToF). The purified peptides were used for further antimicrobial characterization including minimum inhibitory concentration (MIC) assay, kinetics of bactericidal efficiency and analysis of hemolytic capacity. The peptides exhibited antimicrobial activity towards all the Gram-negative bacteria taken for analysis, whereas they showed the activity towards only a few selected Gram-positive bacteria. MrMBL-C16 peptides produced the highest inhibition towards both the Gram-negative and Gram-positive bacteria compared to the MrMBL-N20. Both peptides do not produce any inhibition against Bacillus sps. The kinetics of bactericidal efficiency showed that the peptides drastically reduced the number of surviving bacterial colonies after 24 h incubation. The results of hemolytic activity showed that both peptides produced strong activity at higher concentration. However, MrMBL-C16 peptide produced the highest activity compared to the MrMBL-N20 peptide. Overall, the results indicated that the peptides can be used as bactericidal agents. The MrMBL protein sequence was characterized using various bioinformatics tools including phylogenetic analysis and structure prediction. We also reported the MrMBL gene expression pattern upon viral and bacterial infection in M. rosenbergii gills. It could be concluded that the prawn MBL may be one of the important molecule which is involved in antimicrobial mechanism. Moreover, MrMBL derived MrMBL-N20 and MrMBL-C16 peptides are important antimicrobial peptides for the recognition and eradication of viral and bacterial pathogens.