BACKGROUND Hormone-sensitive lipase (HSL) is a neutral lipase capable of hydrolysing various kind... more BACKGROUND Hormone-sensitive lipase (HSL) is a neutral lipase capable of hydrolysing various kinds of lipids. In comparison to single human Hormone Sensitive Lipase (hHSL), that is induced under nutritional stress, twelve serine hydrolases are annotated as HSL in Mycobacterium tuberculosis (mHSL). Mycobacterium is exposed to multiple stresses inside the host. Therefore, the present study was carried out to investigate if mHSL are also expressed under stress condition and if there is any correlation between various stress conditions and expression pattern of mHSL. METHODS AND RESULTS The expression pattern of mHSL under different environmental conditions (in-vitro and ex-vivo) were studied using qRT-PCR in M. tuberculosis H37Ra strain with 16 S rRNA as internal control. Out of 12, only two genes (lipU and lipY) were expressed at very low level in mid log phase culture under aerobic conditions, while 9 genes were expressed at stationary phase of growth. Ten mHSLs were expressed post-infection under ex-vivo conditions in time dependent manner. LipH and lipQ did not express at any time point under ex-vivo condition. The relative expression of most of the genes under individual stress was much higher than observed in ex-vivo conditions. The expression pattern of genes varied with change in stress condition. CONCLUSIONS Different sets of mHSL genes were expressed under different individual stress conditions pointing towards the requirement of different mHSL to combat different stress conditions. Overall, most of the mHSLs have demonstrated stress dependent expression pointing towards their role in intracellular survival of mycobacteria.
Journal of biomolecular structure & dynamics, Jan 19, 2018
To study the effect of conserved cysteins on biochemical properties of a previously cloned metage... more To study the effect of conserved cysteins on biochemical properties of a previously cloned metagenomic polygalacturonase (PecJKR01), single point variants A42C, M283C, and double variants M283C + F24C, M283C + A42C were constructed. Mutations resulted in shifting the pH toward lower range and enhanced thermostability. The mutants were optimally active at pH 5.0 as compared to pH 7.0 for wild type. Point variants demonstrated slightly higher enzyme activity at 60o C than that of the wild type. In addition, the A42C/M283C + A42C variants displayed nearly 28-40% enhanced thermostability, while M283C + 24C was least thermostable among all variants/ wild type. Cys (pKa 8.18) possibly interfered in the ionization state resulting in change in pH optima of variants. Structure function analysis suggested that the increased activity in A42C could be due to van der Waals interactions in S···Ar with Phe29 and formation of an additional hydrogen bond between Cys42-S....HN-Ala31. Higher thermosta...
The Rv1497 (LipL) of the Mycobacterium tuberculosis H37Rv was predicted to be similar to hypothet... more The Rv1497 (LipL) of the Mycobacterium tuberculosis H37Rv was predicted to be similar to hypothetical esterases and penicillin binding proteins ofM. tuberculosis as well as to be involved in lipid metabolism. Sequence alignment revealed that Rv1497 protein contains characteristic consensus -lactamase motif 'SXXK' in addition to a conserve pentapeptide-GXSXG-, characteristic of lipolytic enzymes, at the Cterminus of protein in contrast to its usual N-terminus location. For detailed characterization of protein, the rv1497 gene was cloned, expressed with N-terminal His-tag and purified to homogeneity on Ni-NTA column. Rv1497 demonstrated both esterase and -lactamase activities. A serine located within consensus -lactamase motif 'SXXK' was identified as catalytic residue in both esterase and -lactamase enzymatic activities whereas serine residue located within conserved pentapeptide did not show any effect on both enzyme activities. The catalytic residues of Rv1497 for -lactamase activity were determined to be Ser88, Tyr-175 and His355 residues by site-directed mutagenesis. The enzyme demonstrated preference for short chain esters (pNP-butyrate). The expression of lipL gene was significantly upregulated during acidic stress as compared to normal conditions in in vitro culture of M. tuberculosis H37Ra. This is perhaps the first report demonstrating an esterase of mycobacterium showing -lactamase activity.
Bacillus lipolytic enzymes of subfamily 1.4 are industrially attractive because of its alkaline o... more Bacillus lipolytic enzymes of subfamily 1.4 are industrially attractive because of its alkaline optimum pH and broad substrate specificity. The activity and stability of these enzymes for a limited temperature range (30-50 °C) need attention for its industrial application. In the present study, Bacillus subtilis LipJ was rationally designed for low-temperature adaptation. Small amino acids with lower volume and without side chain branches have high occurrence among psychrophilic proteins. Met residue is reported to be preferred for cold adaptation as it is thermolabile in nature and undergoes oxidation at high temperature. Therefore, the Ile137 residue, three residues downstream the catalytic residue Asp133, was substituted by Met. Biochemical study demonstrated that variant Ile137Met was optimally active at 20 °C whereas parent enzyme was most active at 37 °C. The variant retained 70-80 % relative activity at 10 °C where parent enzyme demonstrated low activity. Ile137Met was observed to be unstable at and above 30 °C. Kinetic study demonstrated increased K m and k cat values for variant referring improved catalytic efficiency but poor substrate affinity. Homolog modelling predicted lowered number of weak interactions by substituted Met137 as molecular basis of increased flexibility of variant. Hence, increased structure flexibility might be responsible for poor substrate affinity but increased molecular motion for higher catalysis at cold.
Cold adapted enzymes have applications in detergent, textile, food, bioremediation and biotechnol... more Cold adapted enzymes have applications in detergent, textile, food, bioremediation and biotechnology processes. Bacillus lipases are 'generally recognized as safe' (GRAS) and hence are industrially attractive. Bacillus lipase of 1.4 subfamily are of lowest molecular weight and are reversibly unfolded due to absence of disulphide bonds. Therefore these are largely used to study energetic of protein stability that represents unfolding of native protein to fully unfolded state. In present study, metagenomically isolated Bacillus LipJ was laboratory evolved for cold adaptation by error Prone PCR. Library of variants were screened for high relative activity at low temperature of 10°C compared to native protein LipJ. Point mutant sequenced as Phe19→Leu was determined to be active at cold and was selected for extensive biochemical, biophysical characterization. Variant F19L showed its maximum activity at 10°C where parent protein LipJ had 20% relative activity. Psychrophilic nature...
The genome of Mycobacterium is rich in GC content and poses problem in amplification of some gene... more The genome of Mycobacterium is rich in GC content and poses problem in amplification of some genes, especially those rich in the GC content in terminal regions, by standard/routine PCR procedures. Attempts have been made to amplify three GC rich genes of Mycobacterium sp. (Rv0519c and Rv0774c from M. tuberculosis and ML0314c from M. leprae). Out of these three genes, Rv0774c gene was amplified with normal primers under standard PCR conditions, while no amplification was observed in case of Rv0519c and ML0314c genes. In the present investigation a modified primer based approach was successfully used for amplification of GC rich sequence of Rv0519c through codon optimization without changing the native amino acid sequence. The strategy was successfully confirmed by redesigning the standard primers with similar modifications followed by amplification of ML0314c gene.
7. Snell AM. The effects of chronic disease of the liver on the composition and physicochemical p... more 7. Snell AM. The effects of chronic disease of the liver on the composition and physicochemical properties of blood: changes in proteins; reduction in oxygen saturation of the arterial blood.
To the Editor: Katz et al (Digestive Diseases and Sciences 32: 285: 288, 1987) wonder if abnormal... more To the Editor: Katz et al (Digestive Diseases and Sciences 32: 285: 288, 1987) wonder if abnormal intestinal permeability occurs in the elderly. The evidence presently available is that this is not the case in humans. Saweirs et al (1) found normal intestinal permeability in ...
The synthesis of chemically anchored adenosine with biocompatible poly(2-hydroxylethyl methacryla... more The synthesis of chemically anchored adenosine with biocompatible poly(2-hydroxylethyl methacrylate) grafted gold nanoparticles (Ado-i-PHEMA-g-AuNPs) was realized by employing a simple strategy. Disulfide-containing poly(2-hydroxylethyl methacrylate) (DT-PHEMA) was initially synthesized by atom transfer radical polymerization (ATRP). The formation of DT-PHEMA was confirmed by 1 H-NMR and FT-IR. The molecular weight and molecular weight distribution were found to be 9.6 kg/mol and 1.40 from GPC analysis. DT-PHEMA was subsequently used for the synthesis of PHEMA-g-AuNPs by a grafting to protocol. The grafting of DT-PHEMA on the surface of AuNPs was confirmed by FT-IR, TGA, XPS, and EDX analyses. The particle size of the PHEMA-g-AuNPs was found to be ca. 5.0 nm from HR-TEM analysis. Boronic acid was used for functionalization of PHEMA-g-AuNPs, which was then subjected for covalent immobilization with adenosine via strong interaction between free hydroxyl groups of adenosine and boronic acid. Characterization and properties of the Ado-i-PHEMA-g-AuNPs were investigated by taking advantage from FT-IR, XPS, EDX, and UV-visible spectroscopy. The Ado-i-PHEMA-g-AuNPs nanocomposite exhibits a surface plasmon resonance peak at 586 nm which is red shifted from AuNPs (521 nm), indicating significant changes of surface property upon PHEMA-adenosine immobilization onto the surface of AuNPs.
BACKGROUND Hormone-sensitive lipase (HSL) is a neutral lipase capable of hydrolysing various kind... more BACKGROUND Hormone-sensitive lipase (HSL) is a neutral lipase capable of hydrolysing various kinds of lipids. In comparison to single human Hormone Sensitive Lipase (hHSL), that is induced under nutritional stress, twelve serine hydrolases are annotated as HSL in Mycobacterium tuberculosis (mHSL). Mycobacterium is exposed to multiple stresses inside the host. Therefore, the present study was carried out to investigate if mHSL are also expressed under stress condition and if there is any correlation between various stress conditions and expression pattern of mHSL. METHODS AND RESULTS The expression pattern of mHSL under different environmental conditions (in-vitro and ex-vivo) were studied using qRT-PCR in M. tuberculosis H37Ra strain with 16 S rRNA as internal control. Out of 12, only two genes (lipU and lipY) were expressed at very low level in mid log phase culture under aerobic conditions, while 9 genes were expressed at stationary phase of growth. Ten mHSLs were expressed post-infection under ex-vivo conditions in time dependent manner. LipH and lipQ did not express at any time point under ex-vivo condition. The relative expression of most of the genes under individual stress was much higher than observed in ex-vivo conditions. The expression pattern of genes varied with change in stress condition. CONCLUSIONS Different sets of mHSL genes were expressed under different individual stress conditions pointing towards the requirement of different mHSL to combat different stress conditions. Overall, most of the mHSLs have demonstrated stress dependent expression pointing towards their role in intracellular survival of mycobacteria.
Journal of biomolecular structure & dynamics, Jan 19, 2018
To study the effect of conserved cysteins on biochemical properties of a previously cloned metage... more To study the effect of conserved cysteins on biochemical properties of a previously cloned metagenomic polygalacturonase (PecJKR01), single point variants A42C, M283C, and double variants M283C + F24C, M283C + A42C were constructed. Mutations resulted in shifting the pH toward lower range and enhanced thermostability. The mutants were optimally active at pH 5.0 as compared to pH 7.0 for wild type. Point variants demonstrated slightly higher enzyme activity at 60o C than that of the wild type. In addition, the A42C/M283C + A42C variants displayed nearly 28-40% enhanced thermostability, while M283C + 24C was least thermostable among all variants/ wild type. Cys (pKa 8.18) possibly interfered in the ionization state resulting in change in pH optima of variants. Structure function analysis suggested that the increased activity in A42C could be due to van der Waals interactions in S···Ar with Phe29 and formation of an additional hydrogen bond between Cys42-S....HN-Ala31. Higher thermosta...
The Rv1497 (LipL) of the Mycobacterium tuberculosis H37Rv was predicted to be similar to hypothet... more The Rv1497 (LipL) of the Mycobacterium tuberculosis H37Rv was predicted to be similar to hypothetical esterases and penicillin binding proteins ofM. tuberculosis as well as to be involved in lipid metabolism. Sequence alignment revealed that Rv1497 protein contains characteristic consensus -lactamase motif 'SXXK' in addition to a conserve pentapeptide-GXSXG-, characteristic of lipolytic enzymes, at the Cterminus of protein in contrast to its usual N-terminus location. For detailed characterization of protein, the rv1497 gene was cloned, expressed with N-terminal His-tag and purified to homogeneity on Ni-NTA column. Rv1497 demonstrated both esterase and -lactamase activities. A serine located within consensus -lactamase motif 'SXXK' was identified as catalytic residue in both esterase and -lactamase enzymatic activities whereas serine residue located within conserved pentapeptide did not show any effect on both enzyme activities. The catalytic residues of Rv1497 for -lactamase activity were determined to be Ser88, Tyr-175 and His355 residues by site-directed mutagenesis. The enzyme demonstrated preference for short chain esters (pNP-butyrate). The expression of lipL gene was significantly upregulated during acidic stress as compared to normal conditions in in vitro culture of M. tuberculosis H37Ra. This is perhaps the first report demonstrating an esterase of mycobacterium showing -lactamase activity.
Bacillus lipolytic enzymes of subfamily 1.4 are industrially attractive because of its alkaline o... more Bacillus lipolytic enzymes of subfamily 1.4 are industrially attractive because of its alkaline optimum pH and broad substrate specificity. The activity and stability of these enzymes for a limited temperature range (30-50 °C) need attention for its industrial application. In the present study, Bacillus subtilis LipJ was rationally designed for low-temperature adaptation. Small amino acids with lower volume and without side chain branches have high occurrence among psychrophilic proteins. Met residue is reported to be preferred for cold adaptation as it is thermolabile in nature and undergoes oxidation at high temperature. Therefore, the Ile137 residue, three residues downstream the catalytic residue Asp133, was substituted by Met. Biochemical study demonstrated that variant Ile137Met was optimally active at 20 °C whereas parent enzyme was most active at 37 °C. The variant retained 70-80 % relative activity at 10 °C where parent enzyme demonstrated low activity. Ile137Met was observed to be unstable at and above 30 °C. Kinetic study demonstrated increased K m and k cat values for variant referring improved catalytic efficiency but poor substrate affinity. Homolog modelling predicted lowered number of weak interactions by substituted Met137 as molecular basis of increased flexibility of variant. Hence, increased structure flexibility might be responsible for poor substrate affinity but increased molecular motion for higher catalysis at cold.
Cold adapted enzymes have applications in detergent, textile, food, bioremediation and biotechnol... more Cold adapted enzymes have applications in detergent, textile, food, bioremediation and biotechnology processes. Bacillus lipases are 'generally recognized as safe' (GRAS) and hence are industrially attractive. Bacillus lipase of 1.4 subfamily are of lowest molecular weight and are reversibly unfolded due to absence of disulphide bonds. Therefore these are largely used to study energetic of protein stability that represents unfolding of native protein to fully unfolded state. In present study, metagenomically isolated Bacillus LipJ was laboratory evolved for cold adaptation by error Prone PCR. Library of variants were screened for high relative activity at low temperature of 10°C compared to native protein LipJ. Point mutant sequenced as Phe19→Leu was determined to be active at cold and was selected for extensive biochemical, biophysical characterization. Variant F19L showed its maximum activity at 10°C where parent protein LipJ had 20% relative activity. Psychrophilic nature...
The genome of Mycobacterium is rich in GC content and poses problem in amplification of some gene... more The genome of Mycobacterium is rich in GC content and poses problem in amplification of some genes, especially those rich in the GC content in terminal regions, by standard/routine PCR procedures. Attempts have been made to amplify three GC rich genes of Mycobacterium sp. (Rv0519c and Rv0774c from M. tuberculosis and ML0314c from M. leprae). Out of these three genes, Rv0774c gene was amplified with normal primers under standard PCR conditions, while no amplification was observed in case of Rv0519c and ML0314c genes. In the present investigation a modified primer based approach was successfully used for amplification of GC rich sequence of Rv0519c through codon optimization without changing the native amino acid sequence. The strategy was successfully confirmed by redesigning the standard primers with similar modifications followed by amplification of ML0314c gene.
7. Snell AM. The effects of chronic disease of the liver on the composition and physicochemical p... more 7. Snell AM. The effects of chronic disease of the liver on the composition and physicochemical properties of blood: changes in proteins; reduction in oxygen saturation of the arterial blood.
To the Editor: Katz et al (Digestive Diseases and Sciences 32: 285: 288, 1987) wonder if abnormal... more To the Editor: Katz et al (Digestive Diseases and Sciences 32: 285: 288, 1987) wonder if abnormal intestinal permeability occurs in the elderly. The evidence presently available is that this is not the case in humans. Saweirs et al (1) found normal intestinal permeability in ...
The synthesis of chemically anchored adenosine with biocompatible poly(2-hydroxylethyl methacryla... more The synthesis of chemically anchored adenosine with biocompatible poly(2-hydroxylethyl methacrylate) grafted gold nanoparticles (Ado-i-PHEMA-g-AuNPs) was realized by employing a simple strategy. Disulfide-containing poly(2-hydroxylethyl methacrylate) (DT-PHEMA) was initially synthesized by atom transfer radical polymerization (ATRP). The formation of DT-PHEMA was confirmed by 1 H-NMR and FT-IR. The molecular weight and molecular weight distribution were found to be 9.6 kg/mol and 1.40 from GPC analysis. DT-PHEMA was subsequently used for the synthesis of PHEMA-g-AuNPs by a grafting to protocol. The grafting of DT-PHEMA on the surface of AuNPs was confirmed by FT-IR, TGA, XPS, and EDX analyses. The particle size of the PHEMA-g-AuNPs was found to be ca. 5.0 nm from HR-TEM analysis. Boronic acid was used for functionalization of PHEMA-g-AuNPs, which was then subjected for covalent immobilization with adenosine via strong interaction between free hydroxyl groups of adenosine and boronic acid. Characterization and properties of the Ado-i-PHEMA-g-AuNPs were investigated by taking advantage from FT-IR, XPS, EDX, and UV-visible spectroscopy. The Ado-i-PHEMA-g-AuNPs nanocomposite exhibits a surface plasmon resonance peak at 586 nm which is red shifted from AuNPs (521 nm), indicating significant changes of surface property upon PHEMA-adenosine immobilization onto the surface of AuNPs.
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