Isolation, Extraction, Purification, and Characterization of Fibrinolytic Enzyme from Pseudomonas aeruginosa and Estimation of the Molecular Weight of the Enzyme.

Pseudomonas aeruginosa was isolated from injuries of patients' wounds and burns, and to ensure that the isolate was belonging to P. aeruginosa, several tests were performed, such as staining techniques, a biochemical test, morphological test, Vitek 2 system, and sensitivity test. The results of the gram stain test showed rod pink gram-negative bacteria, demonstrating that the isolate belonged to P. aeruginosa. Growth optimization of bacterial was performed by assessing different combinations of pH and temperatures. It is revealed that the best conditions for increasing the number of bacteria were achieved at 37°C with the bacterial number of 5.53×108 and pH 6 with the bacterial number of 5.87×108. Fibrinolytic enzyme is an agent that lysis fibrin clots. This fibrinolytic factor has prospective use to treat cardiovascular diseases, such as stroke and heart attack. Cardiovascular diseases have attracted worldwide attention for their elevation morbidity and mortality. Fibrinolytic enzyme was extracted by centrifugation at 10000 × g at 4°C for 10 min, the supernatant was kept and the pellet having bacterial cells was discarded. Purification of the fibrinolytic enzyme was achieved using salt precipitation, ion exchange, and gel filtration chromatographic techniques. The results showed that the gel filtration chromatography had optimal specific activity and purification fold at 562.6 U/ml, and the final specific activity of the purified enzyme increased 4.1 times. The molecular weight of the fibrinolytic enzyme was determined at26 kDa by gel filtration chromatography. The purified fibrinolytic enzyme had optimum activity atpH 7 and40°C.The pH stability for the enzyme activity was found in pH 6-7 and the range of 10-40°C.

NADPH oxidase 1 mediates caerulein-induced pancreatic fibrosis in chronic pancreatitis.

Inflammatory disorders of the pancreas are divided into acute (AP) and chronic (CP) forms. Both states of pancreatitis are a result of pro-inflammatory mediators, including reactive oxygen species (ROS). One of the sources of ROS is NADPH oxidase (Nox). The rodent genome encodes Nox1-4, Duox1 and Duox2. Our purpose was to assess the extent to which Nox enzymes contribute to the pathogenesis of both AP and CP using Nox-deficient mice. Using RT-PCR, Nox1 was found in both isolated mouse pancreatic acini and pancreatic stellate cells (PaSCs). Subsequently, mice with genetically deleted Nox1 were further studied and showed that the histo-morphologic characteristics of caerulein-induced CP, but not caerulein-induced AP, was ameliorated in Nox1 KO mice. We also found that the lack of Nox1 impaired caerulein-induced ROS generation in PaSCs. Using Western blotting, we found that AKT mediates the fibrotic effect of Nox1 in a mouse model of CP. We also found a decrease in phospho-ERK and p38MAPK levels in Nox1 KO mice with CP, but not with AP. Both CP-induced TGF-ß up-regulation and NF-ĸB activation were impaired in pancreas from Nox1 KO mice. Western blotting indicated increases in proteins involved in fibrosis and acinar-to-ductal metaplasia in WT mice with CP. No change in those proteins were observed in Nox1 KO mice. The lack of Nox1 lowered mRNA levels of CP-induced matrix metalloproteinase MMP-9 and E-cadherin repressor Twist in PaSCs. CONCLUSION: Nox1-derived ROS in PaSCs mediate the fibrotic process of CP by activating the downstream redox-sensitive signaling pathways AKT and NF-ĸB, up-regulating MMP-9 and Twist, and producing α-smooth muscle actin and collagen I and III.

Bacopaside N1 biosynthetic potential of endophytic Aspergillus sp. BmF 16 isolated from Bacopa monnieri.

Chemically unique environment of endophytes makes them to have various adaptive mechanisms for survival. One of such mechanisms involves the production of pharmacologically significant plant-specific metabolites. In the present study, 26 endophytic fungi were isolated from stem of Bacopa monnieri (L.) Wettst. plants. All the isolates were screened for bacopaside production property by HPLC. Among these, the fungal isolate BmF 16 which was identified as Aspergillus sp. was confirmed for bacopaside N1 production (m/z 796) by LC-MS/MS analysis. As the extract of BMF16 used in the study was prepared from the fifth generation of culture, the obtained result can be confirmed as due to fungal production of bacopaside. In addition, this property was identified only for one among the 26 fungi screened. As bacopaside N1 production in fungi has not yet been reported, the results of the study are novel.

Plant growth and diosgenin enhancement effect of silver nanoparticles in Fenugreek (Trigonella foenum-graecum L.).

Various methods have been used to enhance production of chemically diverse phytochemicals especially medicinal natural products. With the advancement in nanotechnology, nanoparticles have been reported to have varying impact in plant growth and inducibility of phytochemical composition. Major objective of the study was to study the secondary metabolite modulatory effect of silver nanoparticles. In the current study, treatment of fenugreek seedlings with biosynthesized silver nanoparticles (Ag-NPs) was found to have significant impact on its growth parameters such as leaf number, root length, shoot length and wet weight. On HPLC based analysis, Ag-NPs treated seedlings showed an enhancement in the production of major phytochemical diosgenin to a level of 214.06 ± 17.07 µg/mL. An untreated control gave an yield of only 164.44 ± 7.67 µg/mL of diosgenin, and the observed phytochemical enhancement effect induced by Ag-NP was very significant. Most remarkably, the Ag-NP used in the study was found to play dual role of enhancement of both plant growth and diosgenin synthesis. Hence the study is of immense application as it opens up development of new methods based on nanoelicitors to enhance the biosynthesis of medicinal natural products in plants.

Identification of a novel endophytic Bacillus sp. from Capsicum annuum with highly efficient and broad spectrum plant probiotic effect.

AIM: The study mainly aimed the isolation and characterization of plant probiotic endophytic bacteria from Capsicum annuum to explore its multipotent agricultural applications. MATERIALS AND METHODS: Endophytic bacteria were isolated from the surface sterilized fruit tissue. The isolates were then subjected to PCR-based screening for the presence of potential biosynthetic gene clusters. The PCR positive isolate was then analysed for its inhibitory effect towards fungal and bacterial pathogens. The compounds responsible for the antimicrobial activity was purified from large scale culture and subjected to identification by LC-MS/MS. The ability of the selected isolate in plant growth enhancement was also done using Vigna radiata seedlings. CONCLUSION: In this study, an endophytic bacterium isolated from C. annuum was found to have the phenotypic and genetic basis for broad antimicrobial property. PCR-based sequence analysis has resulted in the identification of nonribosomal peptide synthases, PKS Type I, Iturin, surfactin, DAPG and gacA genes in the selected isolate CaB 5. The bioactivity-guided fractionation using column and HPLC purification of active fraction followed by LC-MS/MS analysis has proved the presence of surfactin derivatives (M+H(+) - 1008 & 1036) and iturin (M+H(+) - 1058) as the basis of antimicrobial activity of CaB 5. The isolate was identified as a novel Bacillus sp. because of its low (76%) identity to the reported sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytes are considered to have the genetic basis for a diverse array of bioactive metabolites which can have significant applications in both pharmaceutical industry and agriculture. The identification of CaB 5 with broad bioactivity and excellent plant growth enhancement on taxonomically distinct plant species as explained in current study and our previous reports highlights its plant probiotic applicability. This proves the potential of the isolate obtained in the study to be an excellent plant probiotic.

Surfactin, Iturin, and Fengycin Biosynthesis by Endophytic Bacillus sp. from Bacopa monnieri.

Endophytic microorganisms which are ubiquitously present in plants may colonize intracellularly or intercellularly without causing any diseases. By living within the unique chemical environment of a host plant, they produce a vast array of compounds with a wide range of biological activities. Because of this, natural products of endophytic origin have been exploited for antimicrobial, antiviral, anticancer, and antioxidant properties. Also, they can be considered to function as an efficient microbial barrier to protect plants from various pathogens. In the present study, endophytic bacterium BmB 9 with antifungal and antibacterial activity isolated from the stem tissue of Bacopa monnieri was studied for the molecular and chemical basis of its activity. PCR-based genome mining for various biosynthetic gene clusters proved the presence of surfactin, iturin, and type I polyketide synthase (PKS) genes in the isolate. The LC-MS/MS based analysis of the extract further confirmed the production of surfactin derivatives (M + H(+)-1008.6602, 1022.6755), iturin (M + H(+)-1043.5697), and fengycin (M + H(+)-1491.8195, 1477.8055) by the selected bacterial isolate. The 16S rDNA sequence similarity based analysis identified the isolate BmB 9 as Bacillus sp. with 100 % identity to Bacillus sp. LCF1 (KP257289).

Metabolite and Mechanistic Basis of Antifungal Property Exhibited by Endophytic Bacillus amyloliquefaciens BmB 1.

Plants are ubiquitously colonized by endophytic microorganisms which contribute significantly to plant health through production of plant growth regulators or disease suppression. In the present study, an endophytic bacterial isolate designated as BmB 1 with significant antifungal and plant growth promoting properties was isolated from the stem tissue of Bacopa monnieri (L.) Pennell. The isolate was studied in detail for the molecular and chemical basis of its bioactivity which proved it to have the presence of surfactin, iturin, and type I polyketide synthase (PKS) genes. For the analysis of the chemical basis of antifungal property, extract of the isolate was initially checked for its activity on test pathogens and LC-MS/MS based analysis further confirmed the presence of bacillomycin (m/z (M+H(+)) 1031.8) and surfactin (m/z (M+H(+)) 1008.6 and 1022.6) in the extract prepared. The light microscopic and SEM analysis of the treated and untreated mycelia of the pathogens clearly revealed the hypal destruction caused by the compounds produced by the selected isolate. This confirms the ability of the organism to directly inhibit the growth of the tested pathogens. The GC-MS analysis also confirmed the isolate to have the presence of volatile compounds with the expected role to induce induced systemic resistance (ISR) of the plant. Because of the multitargeted antifungal property, the isolate which was identified as Bacillus amyloliquefaciens can have potential biocontrol applications.

Identification of endophytic Bacillus mojavensis with highly specialized broad spectrum antibacterial activity.

Biosynthetic adaptation of endophytic bacteria to chemically support host plant is very remarkable. Hence these organisms from medicinal plants are considered as highly valuable sources for natural products with diverse bioactivity. Their metabolite diversity and biosynthetic versatility have been increasingly explored for drug discovery. In this study, an endophytic Bacillus mojavensis with broad spectrum antibacterial properties has been analyzed for the chemical basis of its activity. By LC-MS/MS the organism was identified to have the biosynthetic ability to produce lipopeptides surfactin and fengycin. The impressive antibacterial activity of B. mojavensis as reported in the study indicates its broad antimicrobial applications.

Studies on Plant Growth Promoting Properties of Fruit-Associated Bacteria from Elettaria cardamomum and Molecular Analysis of ACC Deaminase Gene.

Endophytic microorganisms have been reported to have diverse plant growth promoting mechanisms including phosphate solubilization, N2 fixation, production of phyto-hormones and ACC (1-aminocyclopropane-1-carboxylate) deaminase and antiphyto-pathogenic properties. Among these, ACC deaminase production is very important because of its regulatory effect on ethylene which is a stress hormone with precise role in the control of fruit development and ripening. However, distribution of these properties among various endophytic bacteria associated with fruit tissue and its genetic basis is least investigated. In the current study, 11 endophytic bacteria were isolated and identified from the fruit tissue of Elettaria cardamomum and were studied in detail for various plant growth promoting properties especially ACC deaminase activity using both culture-based and PCR-based methods. PCR-based screening identified the isolates EcB 2 (Pantoea sp.), EcB 7 (Polaromonas sp.), EcB 9 (Pseudomonas sp.), EcB 10 (Pseudomonas sp.) and EcB 11 (Ralstonia sp.) as positive for ACC deaminase. The PCR products were further subjected to sequence analysis which proved the similarity of the sequences identified in the study with ACC deaminase sequences reported from other sources. The detailed bioinformatic analysis of the sequence including homology-based modelling and molecular docking confirmed the sequences to have ACC deaminase activity. The docking of the modelled proteins was done using patch dock, and the detailed scrutiny of the protein ligand interaction revealed conservation of key amino acids like Lys51, Ser78, Tyr268 and Tyr294 which play important role in the enzyme activity. These suggest the possible regulatory effect of these isolates on fruit physiology.

Studies on the factors modulating indole-3-acetic acid production in endophytic bacterial isolates from Piper nigrum and molecular analysis of ipdc gene.

AIMS: The study mainly aimed quantitative analysis of IAA produced by endophytic bacteria under various conditions including the presence of extract from Piper nigrum. Analysis of genetic basis of IAA production was also conducted by studying the presence and diversity of the ipdc gene among the selected isolates. MATERIALS AND METHODS: Five endophytic bacteria isolated previously from P. nigrum were used for the study. The effect of temperature, pH, agitation, tryptophan concentration and plant extract on modulating IAA production of selected isolates was analysed by colorimetric method. Comparative and quantitative analysis of IAA production by colorimetric isolates under optimal culture condition was analysed by HPTLC method. Presence of ipdc gene and thereby biosynthetic basis of IAA production among the selected isolates were studied by PCR-based amplification and subsequent insilico analysis of sequence obtained. CONCLUSIONS: Among the selected bacterial isolates from P. nigrum, isolate PnB 8 (Klebsiella pneumoniae) was found to have the maximum yield of IAA under various conditions optimized and was confirmed by colorimetric, HPLC and HPTLC analysis. Very interestingly, the study showed stimulating effect of phytochemicals from P. nigrum on IAA production by endophytic bacteria isolated from same plant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is unique because of the selection of endophytes from same source for comparative and quantitative analysis of IAA production under various conditions. Study on stimulatory effect of phytochemicals on bacterial IAA production as explained in the study is a novel approach. Studies on molecular basis of IAA production which was confirmed by sequence analysis of ipdc gene make the study scientifically attractive. Even though microbial production of IAA is well known, current report on detailed optimization, effect of plant extract and molecular confirmation of IAA biosynthesis is comparatively novel in its approach.

LC-MS/MS based identification of piperine production by endophytic Mycosphaerella sp. PF13 from Piper nigrum.

Piper nigrum is very remarkable for its medicinal properties due to the presence of metabolites like piperine. Emerging understanding on the biosynthetic potential of endophytic fungi suggests the possibility to have piperine producing fungi in P. nigrum. In the current study, endophytic fungi isolated from P. nigrum were screened for the presence of piperine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This resulted in the identification of a Mycosphaerella sp. with the ability to produce piperine extracellularly. The biosynthesis of piperine (C17H19NO3) by the endophytic fungal isolate was confirmed by the presence of m/z 286.1 (M + H(+)) in the LC-MS/MS analysis using positive mode ionization. This was further supported by the presence of specific fragment ions with masses 135, 143, 171 and 201 formed due to the fragmentation of piperine present in the fungal extract.

Phenazine carboxylic acid production and rhizome protective effect of endophytic Pseudomonas aeruginosa isolated from Zingiber officinale.

Ginger (Zingiber officinale) is cultivated commercially in most parts of the world especially in India for its culinary and medicinal applications. One of the major challenges that limit the yield of ginger is rhizome rot disease caused by organisms including Pythium myriotylum. A feasible ecofriendly method is yet to be devised to prevent the plant from this threatening disease. Recent studies on plant microbiome show the possibility of having endophytic organisms with plant protective characteristics associated with the plants. Because of the uniquely evolved underground nature of the ginger rhizome and its peculiar survival in soil for a long time, many interesting endophytic microbes with plant protective characters can be well expected from it. In the current study, previously isolated endophytic Pseudomonas aeruginosa from ginger was investigated in detail for its effect on Pythium myriotylum. The rhizome protective effect of the organism was also studied by co-inoculation studies, which confirmed that Pseudomonas aeruginosa has very potent inhibitory effect on Pythium myriotylum. On further studies, the active antifungal compound was identified as phenazine 1-carboxylic acid.

Isolation and characterization of plant growth promoting endophytic bacteria from the rhizome of Zingiber officinale.

Endophytes, by residing within the specific chemical environment of host plants, form unique group of microorganisms. Microbially unexplored medicinal plants can have diverse and potential microbial association. The rhizome of ginger is very remarkable because of its metabolite richness, but the physiological processes in these tissues and the functional role of associated microorganisms remain totally unexplored. Through the current study, the presence of four different endophytic bacterial strains were identified from ginger rhizome. Among the various isolates, ZoB2 which is identified as Pseudomonas sp. was found to have the ability to produce IAA, ACC deaminase and siderophore. By considering these plant growth promoting properties, ZoB5 can expect to have considerable effect on the growth of ginger.

Piperine production by endophytic fungus Colletotrichum gloeosporioides isolated from Piper nigrum.

Many endophytic fungi have been reported with the biosynthetic potential to produce same or similar metabolites present in host plants. The adaptations that might have acquired by these fungi as a result of the long-term association with their host plants can be the possible basis of their biosynthetic potential. The bioactive compounds originated from endophytes are currently explored for their potential applications in pharmaceutical, agriculture and food industries. Piper nigrum, a plant of the Piperaceae is very remarkable because of the presence of the alkaloid piperine. Piperine has been reported to have broad bioactive properties ranging from antimicrobial, antidepressant, anti-inflammatory, antioxidative to anticancer activities. Interestingly, piperine also plays a vital role in increasing the bioavailability of many drugs which again is a promising property. The current study was carried out to identify piperine producing endophytic fungus from Piper nigrum L. By screening various endophytic fungi, the isolate which was identified as member of Colletotrichum gloeosporioides was found to have the ability to form piperine and was confirmed by HPLC and LCMS. Considering the broad bioactive potential of piperine, the piperine producing fungi identified in the study can expect to have much industrial potential.

Identification of two strains of Paenibacillus sp. as indole 3 acetic acid-producing rhizome-associated endophytic bacteria from Curcuma longa.

Curcuma longa is well known for its use as spice and medicine. The remarkable feature of the plant is the presence of rhizome, which provides an interesting habitat for association by various groups of bacteria. Some of these associated endophytic bacteria can have growth-promoting effects. In the current study, two species of endophytic Paenibacillus has been identified from the rhizome as indole 3 acetic acid producers. These isolates can thus have potential growth-regulating effect in rhizomes.