Quorum sensing is a mechanism of cell to cell communication that requires the production and dete... more Quorum sensing is a mechanism of cell to cell communication that requires the production and detection of signaling molecules called autoinducers. Although mesophilic bacteria is known to utilize this for synchronization of physiological processes such as bioluminescence, virulence, biofilm formation, motility and cell competency through signaling molecules (acyl homoserine lactones, AI-1; oligopeptides, peptide based system and furanosyl borate diester, AI-2), the phenomenon of quorum sensing in thermophiles is largely unknown. In this study, proteomes of 106 thermophilic eubacteria and 21 thermophilic archaea have been investigated for the above three major quorum sensing systems to find the existence of quorum sensing in these thermophiles as there are evidences for the formation of biofilms in hot environments. Our investigation demonstrated that AI-1 system is absent in thermophiles. Further, complete peptide based two component systems for quorum sensing was also not found in ...
Acinetobacter baumannii is an opportunistic pathogen that has acquired resistance to all availabl... more Acinetobacter baumannii is an opportunistic pathogen that has acquired resistance to all available drugs. The rise in multi-drug resistance in A. baumannii has been exacerbated by its ability to tolerate antibiotics due to the persister cells, which are phenotypic variants of normal cells that can survive various stress conditions, resulting in chronicity of infection. In the present study we observed that A. baumannii formed persister cells against lethal concentration of ciprofloxacin in exponential phase. The transcriptome of A. baumannii was analyzed after exposure to high concentration of ciprofloxacin (50X MIC) to determine the possible mechanisms of survival. Transcriptome analysis showed differential expression of 146 genes, of which 101 were up-regulated and 45 were down-regulated under ciprofloxacin stress. Differentially expressed genes that might be important for persistence against ciprofloxacin were involved in DNA repair, phenylacetic acid degradation, leucine catabol...
Mannan is the main polysaccharide component of coffee extract and is responsible for its high vis... more Mannan is the main polysaccharide component of coffee extract and is responsible for its high viscosity, which in turn negatively affects the technological processing involved in making instant coffee. In our study, we isolated mannan from coffee beans and extract of commercial coffee and it was enzymatically hydrolyzed using alkali-thermostable mannanase obtained from Bacillus nealsonii PN-11. As mannan is found to be more soluble under alkaline conditions, an alkali-thermostable mannanase is well suited for its hydrolysis. The process of enzymatic hydrolysis was optimized by response surface methodology. Under the following optimized conditions viz enzyme dose of 11.50 U mannanase g−1 coffee extract, temperature of 44.50 °C and time of 35.80 min, significant twofold decrease in viscosity (50 mPas to 26.00 ± 1.56 mPas) was achieved. The application of this process in large-scale industrial production of coffee will help in reduction of energy consumption used during freeze-drying. ...
TspMI, a thermostable isoschizomer of XmaI from a Thermus sp., has been characterized. The enzyme... more TspMI, a thermostable isoschizomer of XmaI from a Thermus sp., has been characterized. The enzyme was purified to homogeneity using Cibacron-Blue 3GA agarose, Heparin agarose, SP sephadex C50, and Mono-Q fast protein liquid chromatography and was found to be a homodimer of 40 kDa. Restriction mapping and run-off sequencing of TspMI-cleaved DNA ends depicted that it cleaved at 5′C/CCGGG3′ to generate a four-base, 5′-CCGG overhang. The enzyme was sensitive to methylation of second and third cytosines in its recognition sequence. TspMI worked optimally at 60°C with 6 mM Mg2+, no Na+/K+, and showed no star activity in the presence of 25% glycerol. The enzyme could efficiently digest the DNA labeled with a higher concentration of YOYO-I (one dye molecule to one nucleotide), making it a useful candidate for real-time imaging experiments. Single molecule interaction between TspMI and λ DNA was studied using total internal reflection fluorescence microscopy. The enzyme survived 30 polymeras...
The two factors important when optimization of enzyme immobilization for the fabrication of biose... more The two factors important when optimization of enzyme immobilization for the fabrication of biosensor are: activity and stability. The present study investigates the 2 factors when laccase was immobilized on various supports by different methods. Immobilization of partially purified laccase showed that enzyme expressed 100% activity when immobilized on the nitrocellulose membrane. pH and temperature optimum of immobilized laccase was 6.5 and 55°C respectively, when syringaldazine was used as a substrate. Immobilized laccase on nitrocellulose membrane was 100% stable at 4°C-30°C for three months. At 60ºC enzyme showed 50% stability after 30 min. Immobilized laccase showed best response with syringaldazine which gave reaction even at 1 to 5µM concentration. Immobilized laccase gave response to catechol, catechin and L-methyl DOPA in the range of 40 to 90, 40 to 60, 30 to 70 µM respectively. ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate)) also showed response to the laccase from 0.1 to 0.2 mM.
International Journal of Biological Macromolecules
A bacterial laccase having potential to work in industry without mediator is of special interest.... more A bacterial laccase having potential to work in industry without mediator is of special interest. In this work, gene (1.83kb) encoding a novel laccase from Rheinheimera sp., having potential to deink waste paper without mediator, was cloned, over-expressed and the induced 69kDa protein (RhLacc) was purified and characterized. rhlacc gene was mutated by error prone PCR and mutants were sequenced. One mutant showed protein truncation resulting in the absence of domain 3 that contains T1 copper center. It is known that redox potential of T1 is the key parameter for substrate oxidation. Overexpression of this mutant gene showed induced 41.1kDa protein (∆RhLacc) that exhibited laccase activity but in the presence of added copper, compared to RhLacc which showed activity without added copper ions. Optimum temperature for both was 55°C. However, optimum pH varied with substrates. Kinetic studies showed ∆RhLacc had lower affinity for substrates except for guaiacol and reduced kcat in comparison to RhLacc. Both were able to deink old newspaper and degrade indigo carmine without mediator. The study suggests that the novel property to deink waste paper without mediator may not depend on the redox potential of T1 but other mechanisms using domains 1 and 2 may be involved.
Persisters are phenotypic variants of normal susceptible bacterial populations that survive prolo... more Persisters are phenotypic variants of normal susceptible bacterial populations that survive prolonged exposure to high doses of antibiotics and are responsible for pertinacious infections and post-treatment relapses. Out of the three antibiotics, Acinetobacter baumannii formed the highest percentage of persister cells against rifampicin followed by amikacin and the least against colistin. Colistin-treated cells formed the high levels of reactive oxygen species (ROS) whose quenching with bipyridyl and thiourea led to an increased persister population. Curcumin, a polyphenolic pro-oxidant, significantly decreased persistence against colistin. The quenching of ROS generated by curcumin-colistin combination and the use of resveratrol, an anti-oxidant, with colistin increased the persister population, supporting the significance of ROS in decreased persistence against this combination. The down-regulation of repair genes by this combination in comparison to colistin alone supported the m...
Bacterial infections have always been an unrestrained challenge to the medical community due to r... more Bacterial infections have always been an unrestrained challenge to the medical community due to rise of multi-drug tolerant and resistant strains. Pioneering work on Escherichia coli polyphosphate kinase (PPK) by Arthur Kornberg has generated great interest in this polyphosphate (PolyP) synthesizing enzyme. PPK has wide distribution among pathogens and is involved in promoting pathogenesis, stress management and susceptibility to antibiotics. Further, the absence of PPK orthologue in humans makes it a potential drug target. This review covers the functional and structural aspects of polyphosphate kinases in bacterial pathogens. A description on molecules being designed against PPKs has been provided, challenges associated with PPK inhibitor design are highlighted and strategies to enable development of efficient drug against this enzyme have also been assessed.
The Journal of general and applied microbiology, Jan 28, 2018
Laccases are unable to oxidize the non-phenolic components of complex lignin polymer due to their... more Laccases are unable to oxidize the non-phenolic components of complex lignin polymer due to their less redox potential (E0). Catalytic efficiency of laccases relies on the mediators that potentiates their oxidative strength; for breaking the recalcitrant lignin. Laccase from Bacillus sp. SS4 was evaluated for its compatibility with natural and synthetic mediators. (2 mM). It was found that acetosyringone, vanillin, orcinol and veratraldehyde have no adverse effect on the laccase activity up to 3 h. Syringaldehyde, p-coumaric acid, ferulic acid and hydroquinone reduced the enzyme activity ≥50% after 1.0 h, but laccase activity remained 100 to ~120% in the presence of synthetic mediators HBT (1-Hydroxylbenzotrizole) and ABTS. (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) after 3 h. MgSO and MnSO (40 mM) increased the enzyme activity 3.5 fold and the enzyme possessed ≥70% activity at a very high concentration. (2 M) of NaCl. The enzyme retained 40-110% activity in the p...
In the present study, an extracellular alkali stable laccase (Lac DS) fromDS which has pH optima ... more In the present study, an extracellular alkali stable laccase (Lac DS) fromDS which has pH optima at 8.5 using-phenylenediamine (PPD) as substrate has been reported. Lac DS retained 70% activity for 4 h at pH 8.5 and 90% activity for 24 h at 55 °C. The enzyme yield was enhanced by optimization of fermentation conditions. A 746-fold increase in yield was observed under optimized conditions using 150 µM MgSO, 1.2% yeast extract, 0.35% tryptone, and 150 µM vanillic acid. Lac DS was used to polymerize natural dye precursor catechol, pyrogallol, syringaldehyde, syringic acid, ferulic acid and gallic acid to develop a range of natural hair colors such as black, golden yellow, and reddish brown. The results indicate that alkaline Lac DS is a suitable candidate to develop a user-friendly and commercially applicable hair dyeing process in the area of cosmetic industry.
International journal of biological macromolecules, Jan 12, 2017
Disposal of chitinaceous waste is a major problem of seafood industry. Most of the known chitinol... more Disposal of chitinaceous waste is a major problem of seafood industry. Most of the known chitinolytic organisms have been studied with respect to pure chitin as substrate. Use of these organisms for degradation of seafood waste has not been explored much. In present study a marine bacterium capable of proficiently degrading shrimp waste with co-production of value added products like chitinase and chitin oligosaccharides was isolated from seafood waste dumping sites. On 16s rRNA and biochemical analysis bacterium was found to be a novel species of genus Paenibacillus.Under optimized condition complete shrimp waste degradation (99%) was achieved along with chitinase yield of 20.01IUml-1. SEM and FTIR showed the structural changes and breakage of bonds typical to that of chitin, which indicated that this process can be used for the degradation of other chitinaceous material also. Thin layer chromatography revealed the presence of chitin oligosaccharides of various degree of polymeriza...
Acinetobacter baumannii infections are responsible for major health problems in immunocompromised... more Acinetobacter baumannii infections are responsible for major health problems in immunocompromised patients particularly in intensive care units. Due to rapid acquisition of and also inherent drug resistance, a vaccine is an effective treatment option against this pathogen. BamA, an outer membrane β-barrel assembly protein, was identified in A. baumannii as potential vaccine candidate by in silico analysis. The immunoprotective efficacy of this highly conserved protein was investigated against a virulent multidrug resistant clinical isolate using murine pneumonia model. Recombinant BamA elicited a high IgG antibody titer (160000) in mice. Opsonophagocytic killing assay showed non-neutrilizing, opsonizing antibodies with combinatorial bactericidal activity of antibodies and complement components. Active and passive immunization protected 80 and 60% mice respectively against intranasal challenge with lethal dose (10 9 CFU) of virulent A. baumannii along with efficient clearance of bacteria in mice lungs and reduction in levels of pro-inflammatory cytokines viz. TNF-α, IL-6 and IL-1β in sera and lung tissue homogenate. Increase in levels of IL-10, an anti-inflammatory cytokine and reduction of neutrophils in lungs facilitated the control of infection. This study demonstrates the potential of BamA as effective vaccine candidate and a promising target for antibody-based therapy to protect against MDR A. baumannii infections. A. baumannii is an opportunistic nosocomial pathogen that causes pneumonia, sepsis and soft tissue infections. Due to its ability to survive on dry surfaces and to form biofilms on biotic/abiotic surfaces, it has emerged as a critical pathogen. Currently, antibiotics are the only treatment option against these infections but the frequent use of antibiotics has led to emergence of multidrug resistant strains particularly against cephalosporins, fluoroquinolones, aminoglycosides and even carbapenems that has made treatment of A. baumannii infections complicated 1-5. bla-NDM harboring strains are resistant to all broad spectrum β-lactams due to enzymatic degradation by β-lactamases whereas resistance to broad spectrum cephalosporins usually results from over-expression of the AmpC-type cephalosporinase gene and from acquisition of extended-spectrum β-lactamases (ESBLs) 2,6,7. Due to failure of these antibiotics, use of colistin against A. baumannii has been increased that has unfortunately led to the emergence of colistin-resistant strains 4,8. Recently, WHO has released a list of antibiotic resistant priority pathogens and mentioned A. baumannii as the critically dangerous pathogen 9. Emergence of multi-, extensiveand pan-drug resistant strains worldwide is a serious concern and novel approaches are direly needed to prevent A. baumannii infections in which effective vaccination could become efficient and economical method to prevent the bacterial infections. Numerous vaccine candidates have been tested against this ubiquitous opportunistic pathogen. Immunization with whole cell organism 10 , outer membrane vesicles 11 , outer membrane complex 12 , capsule components 13 or poly-N-acetyl-β-(1-6)-glucosamine 14 has been suggested as efficient vaccination approach due to abundance of immunogenic components in them. Current immunization strategies target a single or multiple outer membrane proteins and such antigens are easy to prepare and are safe as there is no risk of pathogen reverting back to its virulent form and there are few adverse effects as compared to live attenuated or killed whole cell vaccination. Outer membrane proteins Ata 15 , OmpA 16 , OmpK, Ompp1 and FKIB 17 provided partial to complete mice protection
Bacterial diversity of hot springs of northern Himalayan region of India was studied and explored... more Bacterial diversity of hot springs of northern Himalayan region of India was studied and explored for laccases, the multicopper enzymes applicable in a large number of industries due to their ability to utilize a wide range of substrates. 220 operational taxonomic units (OTUs) out of 5551 sequence reads for bacterial diversity and 3 OTUs out of 19 sequence reads for Laccase like multicopper oxidases (LMCOs) diversity were generated. Bacteroidetes (74.28%) was the most abundant phylum including genus Paludibacter (66.96%), followed by phylum Proteobacteria (24.53%) including genera Chitinilyticum (7.55%) and Cellvibrio (6.14%). In case of laccase diversity, three LMCO sequences showed affiliation with proteobacteria and one with two domain laccase from uncultivable bacteroidetes. LMCO sequences belonged to H and N families.
Activated sludge is an artificial ecosystem known to harbor complex microbial communities. Bacter... more Activated sludge is an artificial ecosystem known to harbor complex microbial communities. Bacterial diversity in activated sludge from pulp and paper industry was studied to bioprospect for laccase, the multicopper oxidase applicable in a large number of industries due to its ability to utilize a wide range of substrates. Bacterial diversity using 454 pyrosequencing and laccase diversity using degenerate primers specific to conserved copper binding domain of laccase like multicopper oxidase (LMCO) genes were investigated. 1231 OTUs out of 11,425 sequence reads for bacterial diversity and 11 OTUs out of 15 reads for LMCO diversity were formed. Phylum Proteobacteria (64.95 %) with genus Thauera (13.65 %) was most abundant followed by phylum Bacteriodetes (11.46 %) that included the dominant genera Paludibacter (1.93 %) and Lacibacter (1.32 %). In case of LMCOs, 40 % sequences showed affiliation with Proteobacteria and 46.6 % with unculturable bacteria, indicating considerable novelty, and 13.3 % with Bacteroidetes. LMCOs belonged to H and J families.
Bacillus sp. MK716, a thermophilic and amylolytic soil isolate, produced a thermostable a-amylase... more Bacillus sp. MK716, a thermophilic and amylolytic soil isolate, produced a thermostable a-amylase active even at 100°C. The enzyme yield was increased 40 times through mutagenesis with ethylmethane sulfonate. The mutated gene was cloned in Escherichia coli-pBR322 system, and the 4.8 kb cloned fragment was mapped with restriction enzymes. Subcloning in Bacillus subtilis of a 2.0 kb portion of the cloned fragment resulted in 107 times increased enzyme yield. The recombinant plasmid was completely stable in B. subtilis.
ABSTRACT Restriction enzymes are basic tools in recombinant DNA technology. To shape the molecula... more ABSTRACT Restriction enzymes are basic tools in recombinant DNA technology. To shape the molecular biology experiments, the students must know how to work with these molecular scissors. Here, we describe an integrated set of experiments, introduced in the “Advances in Molecular Biology and Biotechnology” postgraduate course, which covers the important aspects about restriction endonucleases: preparation of cell-lysate, one-column partial-purification, assay, effect of temperature, buffers, Mg2+ ions and glycerol on activity, and quality control of two-column partially purified enzyme. Examples of study questions, which help students understand the theoretical basis of the experiments, have been given.
Quorum sensing is a mechanism of cell to cell communication that requires the production and dete... more Quorum sensing is a mechanism of cell to cell communication that requires the production and detection of signaling molecules called autoinducers. Although mesophilic bacteria is known to utilize this for synchronization of physiological processes such as bioluminescence, virulence, biofilm formation, motility and cell competency through signaling molecules (acyl homoserine lactones, AI-1; oligopeptides, peptide based system and furanosyl borate diester, AI-2), the phenomenon of quorum sensing in thermophiles is largely unknown. In this study, proteomes of 106 thermophilic eubacteria and 21 thermophilic archaea have been investigated for the above three major quorum sensing systems to find the existence of quorum sensing in these thermophiles as there are evidences for the formation of biofilms in hot environments. Our investigation demonstrated that AI-1 system is absent in thermophiles. Further, complete peptide based two component systems for quorum sensing was also not found in ...
Acinetobacter baumannii is an opportunistic pathogen that has acquired resistance to all availabl... more Acinetobacter baumannii is an opportunistic pathogen that has acquired resistance to all available drugs. The rise in multi-drug resistance in A. baumannii has been exacerbated by its ability to tolerate antibiotics due to the persister cells, which are phenotypic variants of normal cells that can survive various stress conditions, resulting in chronicity of infection. In the present study we observed that A. baumannii formed persister cells against lethal concentration of ciprofloxacin in exponential phase. The transcriptome of A. baumannii was analyzed after exposure to high concentration of ciprofloxacin (50X MIC) to determine the possible mechanisms of survival. Transcriptome analysis showed differential expression of 146 genes, of which 101 were up-regulated and 45 were down-regulated under ciprofloxacin stress. Differentially expressed genes that might be important for persistence against ciprofloxacin were involved in DNA repair, phenylacetic acid degradation, leucine catabol...
Mannan is the main polysaccharide component of coffee extract and is responsible for its high vis... more Mannan is the main polysaccharide component of coffee extract and is responsible for its high viscosity, which in turn negatively affects the technological processing involved in making instant coffee. In our study, we isolated mannan from coffee beans and extract of commercial coffee and it was enzymatically hydrolyzed using alkali-thermostable mannanase obtained from Bacillus nealsonii PN-11. As mannan is found to be more soluble under alkaline conditions, an alkali-thermostable mannanase is well suited for its hydrolysis. The process of enzymatic hydrolysis was optimized by response surface methodology. Under the following optimized conditions viz enzyme dose of 11.50 U mannanase g−1 coffee extract, temperature of 44.50 °C and time of 35.80 min, significant twofold decrease in viscosity (50 mPas to 26.00 ± 1.56 mPas) was achieved. The application of this process in large-scale industrial production of coffee will help in reduction of energy consumption used during freeze-drying. ...
TspMI, a thermostable isoschizomer of XmaI from a Thermus sp., has been characterized. The enzyme... more TspMI, a thermostable isoschizomer of XmaI from a Thermus sp., has been characterized. The enzyme was purified to homogeneity using Cibacron-Blue 3GA agarose, Heparin agarose, SP sephadex C50, and Mono-Q fast protein liquid chromatography and was found to be a homodimer of 40 kDa. Restriction mapping and run-off sequencing of TspMI-cleaved DNA ends depicted that it cleaved at 5′C/CCGGG3′ to generate a four-base, 5′-CCGG overhang. The enzyme was sensitive to methylation of second and third cytosines in its recognition sequence. TspMI worked optimally at 60°C with 6 mM Mg2+, no Na+/K+, and showed no star activity in the presence of 25% glycerol. The enzyme could efficiently digest the DNA labeled with a higher concentration of YOYO-I (one dye molecule to one nucleotide), making it a useful candidate for real-time imaging experiments. Single molecule interaction between TspMI and λ DNA was studied using total internal reflection fluorescence microscopy. The enzyme survived 30 polymeras...
The two factors important when optimization of enzyme immobilization for the fabrication of biose... more The two factors important when optimization of enzyme immobilization for the fabrication of biosensor are: activity and stability. The present study investigates the 2 factors when laccase was immobilized on various supports by different methods. Immobilization of partially purified laccase showed that enzyme expressed 100% activity when immobilized on the nitrocellulose membrane. pH and temperature optimum of immobilized laccase was 6.5 and 55°C respectively, when syringaldazine was used as a substrate. Immobilized laccase on nitrocellulose membrane was 100% stable at 4°C-30°C for three months. At 60ºC enzyme showed 50% stability after 30 min. Immobilized laccase showed best response with syringaldazine which gave reaction even at 1 to 5µM concentration. Immobilized laccase gave response to catechol, catechin and L-methyl DOPA in the range of 40 to 90, 40 to 60, 30 to 70 µM respectively. ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate)) also showed response to the laccase from 0.1 to 0.2 mM.
International Journal of Biological Macromolecules
A bacterial laccase having potential to work in industry without mediator is of special interest.... more A bacterial laccase having potential to work in industry without mediator is of special interest. In this work, gene (1.83kb) encoding a novel laccase from Rheinheimera sp., having potential to deink waste paper without mediator, was cloned, over-expressed and the induced 69kDa protein (RhLacc) was purified and characterized. rhlacc gene was mutated by error prone PCR and mutants were sequenced. One mutant showed protein truncation resulting in the absence of domain 3 that contains T1 copper center. It is known that redox potential of T1 is the key parameter for substrate oxidation. Overexpression of this mutant gene showed induced 41.1kDa protein (∆RhLacc) that exhibited laccase activity but in the presence of added copper, compared to RhLacc which showed activity without added copper ions. Optimum temperature for both was 55°C. However, optimum pH varied with substrates. Kinetic studies showed ∆RhLacc had lower affinity for substrates except for guaiacol and reduced kcat in comparison to RhLacc. Both were able to deink old newspaper and degrade indigo carmine without mediator. The study suggests that the novel property to deink waste paper without mediator may not depend on the redox potential of T1 but other mechanisms using domains 1 and 2 may be involved.
Persisters are phenotypic variants of normal susceptible bacterial populations that survive prolo... more Persisters are phenotypic variants of normal susceptible bacterial populations that survive prolonged exposure to high doses of antibiotics and are responsible for pertinacious infections and post-treatment relapses. Out of the three antibiotics, Acinetobacter baumannii formed the highest percentage of persister cells against rifampicin followed by amikacin and the least against colistin. Colistin-treated cells formed the high levels of reactive oxygen species (ROS) whose quenching with bipyridyl and thiourea led to an increased persister population. Curcumin, a polyphenolic pro-oxidant, significantly decreased persistence against colistin. The quenching of ROS generated by curcumin-colistin combination and the use of resveratrol, an anti-oxidant, with colistin increased the persister population, supporting the significance of ROS in decreased persistence against this combination. The down-regulation of repair genes by this combination in comparison to colistin alone supported the m...
Bacterial infections have always been an unrestrained challenge to the medical community due to r... more Bacterial infections have always been an unrestrained challenge to the medical community due to rise of multi-drug tolerant and resistant strains. Pioneering work on Escherichia coli polyphosphate kinase (PPK) by Arthur Kornberg has generated great interest in this polyphosphate (PolyP) synthesizing enzyme. PPK has wide distribution among pathogens and is involved in promoting pathogenesis, stress management and susceptibility to antibiotics. Further, the absence of PPK orthologue in humans makes it a potential drug target. This review covers the functional and structural aspects of polyphosphate kinases in bacterial pathogens. A description on molecules being designed against PPKs has been provided, challenges associated with PPK inhibitor design are highlighted and strategies to enable development of efficient drug against this enzyme have also been assessed.
The Journal of general and applied microbiology, Jan 28, 2018
Laccases are unable to oxidize the non-phenolic components of complex lignin polymer due to their... more Laccases are unable to oxidize the non-phenolic components of complex lignin polymer due to their less redox potential (E0). Catalytic efficiency of laccases relies on the mediators that potentiates their oxidative strength; for breaking the recalcitrant lignin. Laccase from Bacillus sp. SS4 was evaluated for its compatibility with natural and synthetic mediators. (2 mM). It was found that acetosyringone, vanillin, orcinol and veratraldehyde have no adverse effect on the laccase activity up to 3 h. Syringaldehyde, p-coumaric acid, ferulic acid and hydroquinone reduced the enzyme activity ≥50% after 1.0 h, but laccase activity remained 100 to ~120% in the presence of synthetic mediators HBT (1-Hydroxylbenzotrizole) and ABTS. (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) after 3 h. MgSO and MnSO (40 mM) increased the enzyme activity 3.5 fold and the enzyme possessed ≥70% activity at a very high concentration. (2 M) of NaCl. The enzyme retained 40-110% activity in the p...
In the present study, an extracellular alkali stable laccase (Lac DS) fromDS which has pH optima ... more In the present study, an extracellular alkali stable laccase (Lac DS) fromDS which has pH optima at 8.5 using-phenylenediamine (PPD) as substrate has been reported. Lac DS retained 70% activity for 4 h at pH 8.5 and 90% activity for 24 h at 55 °C. The enzyme yield was enhanced by optimization of fermentation conditions. A 746-fold increase in yield was observed under optimized conditions using 150 µM MgSO, 1.2% yeast extract, 0.35% tryptone, and 150 µM vanillic acid. Lac DS was used to polymerize natural dye precursor catechol, pyrogallol, syringaldehyde, syringic acid, ferulic acid and gallic acid to develop a range of natural hair colors such as black, golden yellow, and reddish brown. The results indicate that alkaline Lac DS is a suitable candidate to develop a user-friendly and commercially applicable hair dyeing process in the area of cosmetic industry.
International journal of biological macromolecules, Jan 12, 2017
Disposal of chitinaceous waste is a major problem of seafood industry. Most of the known chitinol... more Disposal of chitinaceous waste is a major problem of seafood industry. Most of the known chitinolytic organisms have been studied with respect to pure chitin as substrate. Use of these organisms for degradation of seafood waste has not been explored much. In present study a marine bacterium capable of proficiently degrading shrimp waste with co-production of value added products like chitinase and chitin oligosaccharides was isolated from seafood waste dumping sites. On 16s rRNA and biochemical analysis bacterium was found to be a novel species of genus Paenibacillus.Under optimized condition complete shrimp waste degradation (99%) was achieved along with chitinase yield of 20.01IUml-1. SEM and FTIR showed the structural changes and breakage of bonds typical to that of chitin, which indicated that this process can be used for the degradation of other chitinaceous material also. Thin layer chromatography revealed the presence of chitin oligosaccharides of various degree of polymeriza...
Acinetobacter baumannii infections are responsible for major health problems in immunocompromised... more Acinetobacter baumannii infections are responsible for major health problems in immunocompromised patients particularly in intensive care units. Due to rapid acquisition of and also inherent drug resistance, a vaccine is an effective treatment option against this pathogen. BamA, an outer membrane β-barrel assembly protein, was identified in A. baumannii as potential vaccine candidate by in silico analysis. The immunoprotective efficacy of this highly conserved protein was investigated against a virulent multidrug resistant clinical isolate using murine pneumonia model. Recombinant BamA elicited a high IgG antibody titer (160000) in mice. Opsonophagocytic killing assay showed non-neutrilizing, opsonizing antibodies with combinatorial bactericidal activity of antibodies and complement components. Active and passive immunization protected 80 and 60% mice respectively against intranasal challenge with lethal dose (10 9 CFU) of virulent A. baumannii along with efficient clearance of bacteria in mice lungs and reduction in levels of pro-inflammatory cytokines viz. TNF-α, IL-6 and IL-1β in sera and lung tissue homogenate. Increase in levels of IL-10, an anti-inflammatory cytokine and reduction of neutrophils in lungs facilitated the control of infection. This study demonstrates the potential of BamA as effective vaccine candidate and a promising target for antibody-based therapy to protect against MDR A. baumannii infections. A. baumannii is an opportunistic nosocomial pathogen that causes pneumonia, sepsis and soft tissue infections. Due to its ability to survive on dry surfaces and to form biofilms on biotic/abiotic surfaces, it has emerged as a critical pathogen. Currently, antibiotics are the only treatment option against these infections but the frequent use of antibiotics has led to emergence of multidrug resistant strains particularly against cephalosporins, fluoroquinolones, aminoglycosides and even carbapenems that has made treatment of A. baumannii infections complicated 1-5. bla-NDM harboring strains are resistant to all broad spectrum β-lactams due to enzymatic degradation by β-lactamases whereas resistance to broad spectrum cephalosporins usually results from over-expression of the AmpC-type cephalosporinase gene and from acquisition of extended-spectrum β-lactamases (ESBLs) 2,6,7. Due to failure of these antibiotics, use of colistin against A. baumannii has been increased that has unfortunately led to the emergence of colistin-resistant strains 4,8. Recently, WHO has released a list of antibiotic resistant priority pathogens and mentioned A. baumannii as the critically dangerous pathogen 9. Emergence of multi-, extensiveand pan-drug resistant strains worldwide is a serious concern and novel approaches are direly needed to prevent A. baumannii infections in which effective vaccination could become efficient and economical method to prevent the bacterial infections. Numerous vaccine candidates have been tested against this ubiquitous opportunistic pathogen. Immunization with whole cell organism 10 , outer membrane vesicles 11 , outer membrane complex 12 , capsule components 13 or poly-N-acetyl-β-(1-6)-glucosamine 14 has been suggested as efficient vaccination approach due to abundance of immunogenic components in them. Current immunization strategies target a single or multiple outer membrane proteins and such antigens are easy to prepare and are safe as there is no risk of pathogen reverting back to its virulent form and there are few adverse effects as compared to live attenuated or killed whole cell vaccination. Outer membrane proteins Ata 15 , OmpA 16 , OmpK, Ompp1 and FKIB 17 provided partial to complete mice protection
Bacterial diversity of hot springs of northern Himalayan region of India was studied and explored... more Bacterial diversity of hot springs of northern Himalayan region of India was studied and explored for laccases, the multicopper enzymes applicable in a large number of industries due to their ability to utilize a wide range of substrates. 220 operational taxonomic units (OTUs) out of 5551 sequence reads for bacterial diversity and 3 OTUs out of 19 sequence reads for Laccase like multicopper oxidases (LMCOs) diversity were generated. Bacteroidetes (74.28%) was the most abundant phylum including genus Paludibacter (66.96%), followed by phylum Proteobacteria (24.53%) including genera Chitinilyticum (7.55%) and Cellvibrio (6.14%). In case of laccase diversity, three LMCO sequences showed affiliation with proteobacteria and one with two domain laccase from uncultivable bacteroidetes. LMCO sequences belonged to H and N families.
Activated sludge is an artificial ecosystem known to harbor complex microbial communities. Bacter... more Activated sludge is an artificial ecosystem known to harbor complex microbial communities. Bacterial diversity in activated sludge from pulp and paper industry was studied to bioprospect for laccase, the multicopper oxidase applicable in a large number of industries due to its ability to utilize a wide range of substrates. Bacterial diversity using 454 pyrosequencing and laccase diversity using degenerate primers specific to conserved copper binding domain of laccase like multicopper oxidase (LMCO) genes were investigated. 1231 OTUs out of 11,425 sequence reads for bacterial diversity and 11 OTUs out of 15 reads for LMCO diversity were formed. Phylum Proteobacteria (64.95 %) with genus Thauera (13.65 %) was most abundant followed by phylum Bacteriodetes (11.46 %) that included the dominant genera Paludibacter (1.93 %) and Lacibacter (1.32 %). In case of LMCOs, 40 % sequences showed affiliation with Proteobacteria and 46.6 % with unculturable bacteria, indicating considerable novelty, and 13.3 % with Bacteroidetes. LMCOs belonged to H and J families.
Bacillus sp. MK716, a thermophilic and amylolytic soil isolate, produced a thermostable a-amylase... more Bacillus sp. MK716, a thermophilic and amylolytic soil isolate, produced a thermostable a-amylase active even at 100°C. The enzyme yield was increased 40 times through mutagenesis with ethylmethane sulfonate. The mutated gene was cloned in Escherichia coli-pBR322 system, and the 4.8 kb cloned fragment was mapped with restriction enzymes. Subcloning in Bacillus subtilis of a 2.0 kb portion of the cloned fragment resulted in 107 times increased enzyme yield. The recombinant plasmid was completely stable in B. subtilis.
ABSTRACT Restriction enzymes are basic tools in recombinant DNA technology. To shape the molecula... more ABSTRACT Restriction enzymes are basic tools in recombinant DNA technology. To shape the molecular biology experiments, the students must know how to work with these molecular scissors. Here, we describe an integrated set of experiments, introduced in the “Advances in Molecular Biology and Biotechnology” postgraduate course, which covers the important aspects about restriction endonucleases: preparation of cell-lysate, one-column partial-purification, assay, effect of temperature, buffers, Mg2+ ions and glycerol on activity, and quality control of two-column partially purified enzyme. Examples of study questions, which help students understand the theoretical basis of the experiments, have been given.
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