a b s t r a c t ␣-l-Rhamnosidase [E. C. 3.2.1.40] cleaves terminal ␣-l-rhamnose specifically from... more a b s t r a c t ␣-l-Rhamnosidase [E. C. 3.2.1.40] cleaves terminal ␣-l-rhamnose specifically from a large number of natural products. The enzyme has wide occurrence in nature and is reported from animal tissues, plants, yeasts, fungi and bacteria. It is a biotechnologically important enzyme due to its applications in debittering and clearance of citrus fruit juices, enhancement of wine aromas and derhamnosylation of many natural products containing terminal ␣-l-rhamnose to compounds of pharmaceutical interests. Though ␣-l-rhamnosidases have been investigated actively during recent years, there is no recent review on ␣-l-rhamnosidases. An attempt has been made to fill up this gap in this review. It consists of a brief introduction of ␣-l-rhamnosidase which is followed by a critical description of the methods used for assaying the enzyme activity. Purifications, characterizations and properties of ␣-l-rhamnosidases from different sources have been discussed and the available structural and molecular biological studies on the enzyme have been given. Biotechnological applications of this enzyme in different processes have been briefly described. The review concludes with the identification of areas which needs further extensive studies.
Lignin peroxidase from the liquid culture filtrate of Gloeophyllum sepiarium MTCC-1170 has been p... more Lignin peroxidase from the liquid culture filtrate of Gloeophyllum sepiarium MTCC-1170 has been purified to homogeneity. The molecular weight of the purified enzyme was 42 kDa as determined by SDS-PAGE. The K m values were 54 and 76 µM for veratryl alcohol and H2O2, respectively. The pH and temperature optima were 2.5 and 25°C, respectively. Depolymerization of coal by the fungal strain has been demonstrated using humic acid as a model of coal. Depolymerization of humic acid by the purified lignin peroxidase has been shown by the decrease in absorbance at 450 nm and increase in absorbance at 360 nm in presence of H2O2. Depolymerization of humic acid by the purified enzyme has also been demonstrated by the decrease in the viscosity with time of the reaction solution containing humic acid, H2O2, and the purified lignin peroxidase. The influence of NaCl and NaN3 and inhibitory effects of various metal chelating agents on the lignin peroxidase activity were studied.
Lignin peroxidase from the culture filtrate of Loweporus lividus MTCC-1178 has been purified to h... more Lignin peroxidase from the culture filtrate of Loweporus lividus MTCC-1178 has been purified to homogeneity using Amicon concentration and DEAE cellulose chromatography. The molecular weight of the purified lignin peroxidase using SDS-PAGE analysis has been found to be 40 kDa. The Km values for veratryl alcohol and H2O2 for the purified enzyme were 58 and 83 μM, respectively. The calculated kcat value of the purified enzyme using veratryl alcohol as the substrate was 2.5 s−1. The pH and temperature optima of lignin peroxidase have been found to be 2.6 and 24°C, respectively.
N-Oxidation of arylamines to their corresponding nitrosobenzenes using a new chloroperoxidase pur... more N-Oxidation of arylamines to their corresponding nitrosobenzenes using a new chloroperoxidase purified from Musa paradisiaca stem juice has been examined. The enzymatic characteristics of the stem chloroperoxidase using 4-chloroaniline as substrate were determined. The K ...
A total of 48 full-length protein sequences of pectin lyases from different source organisms avai... more A total of 48 full-length protein sequences of pectin lyases from different source organisms available in NCBI were subjected to multiple sequence alignment, domain analysis, and phylogenetic tree construction. A phylogenetic tree constructed on the basis of the protein sequences revealed two distinct clusters representing pectin lyases from bacterial and fungal sources. Similarly, the multiple accessions of different source organisms representing bacterial and fungal pectin lyases also formed distinct clusters, showing sequence level homology. The sequence level similarities among different groups of pectinase enzymes, viz. pectin lyase, pectate lyase, polygalacturonase, and pectin esterase, were also analyzed by subjecting a single protein sequence from each group with common source organism to tree construction. Four distinct clusters representing different groups of pectinases with common source organisms were observed, indicating the existing sequence level similarity among them. Multiple sequence alignment of pectin lyase protein sequence of different source organisms along with pectinases with common source organisms revealed a conserved region, indicating homology at sequence level. A conserved domain Pec_Lyase_C was frequently observed in the protein sequences of pectin lyases and pectate lyases, while Glyco_hydro_28 domains and Pectate lyase-like β-helix clan domain are frequently observed in polygalacturonases and pectin esterases, respectively. The signature amino acid sequence of 41 amino acids, i.e. TYDNAGVLPITVN-SNKSLIGEGSKGVIKGKGLRIVSGAKNI, related with the Pec_Lyase_C is frequently observed in pectin lyase protein sequences and might be related with the structure and enzymatic function.
An indigenously isolated fungal strain identified as Aspergillus terricola with assigned fungal s... more An indigenously isolated fungal strain identified as Aspergillus terricola with assigned fungal strain number MTCC 7588 has been used as source for pectin lyase production. The extracellular pectin lyase was purified to homogeneity from the culture filtrate of A. terricola by ion exchange and gel filtration chromatography. The determined molecular weight was 35 ± 01 kDa. The K m and k cat (turnover) values of the purified enzyme at 37 °C using citrus pectin as the substrate were found to be 1.0 mg/ml and 110.0 s−1, respectively. The pH and temperature optima of the enzyme were 8.0 and 50 °C, respectively. The retting ability of the purified pectin lyase for natural fibers viz. Cannabis sativa and Linum usitatissimum has been demonstrated for the first time.
An acidic pectin lyase (E.C. 4.2.2.10) produced byAspergillus ficuum MTCC 7591 of molecular weigh... more An acidic pectin lyase (E.C. 4.2.2.10) produced byAspergillus ficuum MTCC 7591 of molecular weight 31.6 kD was purified to apparent homogeneity by ion exchange and gel filtration chromatography. Eighty-six fold purification with 60% yield and a specific activity of 7.8 U/mg protein was obtained. The Km and calculated turnover number (kcat) of the purified enzyme were found to be 0.60 mg/ml and 74 s−1 respectively using citrus pectin as the substrate. The pH and temperature optima were 5.0 and 50°C respectively. Exposed to 24 hours at a particular pH the enzyme was found to be relatively stable in the pH range 2.0–9.0. Exposed to a particular temperature for 1 hour, the enzyme retains full activity up to 40°C. Metal ions and protein inhibitors did not have significant effects on the activity of the enzyme. The enzyme has been found to be very effective in the clarification of sweet lime and orange juices.
The importance of various parameters such as sugarcane juice concentration, pH of the medium, and... more The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K m and k cat values were found to be 1 mg/ml and 76 sec−1, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea.
The mycelia of Aspergillus niger MTCC-404 have been shown to act as a biocatalyst for the transfo... more The mycelia of Aspergillus niger MTCC-404 have been shown to act as a biocatalyst for the transformation of ethylbenzene to (R)-1-phenylethanol in 99% enantiomeric excess. 72% of the products are (R)-1-phenylethanol. The conversion yield is dependent on pH, temperature and mycelia concentration in suspension. A. niger MTCC-404 facilitates methylbenzene transformation to benzylalcohol, and propylbenzene to 1-phenylpropanol. Therefore, A. niger serves
Secretion and enzymatic characteristics of lignin peroxidases from Gloeophyllum sepiarium MTCC 11... more Secretion and enzymatic characteristics of lignin peroxidases from Gloeophyllum sepiarium MTCC 1170, Cladosporium herbarum MTCC 346, Lenzites betulina MTCC 1183, Daedalea flavida MTCC 145, Hexagonia teruis MTCC 1119 and Coirolopsis floccosa MTCC 1177 ligninolytic fungal strains have been reported. Secretion of lignin peroxidase by these ligninolytic fungal strains have been found to be in the range of 0.86 to 3.0 enzyme unit per ml of the culture medium. The enzymatic characteristics like Km, pH and temperature optima of all the lignin peroxidases of the above fungal strains have been determined using veratryl alcohol and H2O2 as the variable substrates. The Km values using veratryl alcohol as the substrate were found to be 65.0 μM, 58.5 μM, 63.0 μM, 54.5 μM, 54.6 μM and 61.0 μM respectively. The Km values using H2O2 as the substrate were found to be 88.0 μM, 86.0 μM, 71.0 μM, 67.0 μM, 80.0 μM and 78.0 μM respectively. The pH optima values for lignin peroxidases of the above ligninolytic fungal strains were found to be 2.5, 2.4, 2.4, 2.25, 2.5 and 2.8 respectively, where as the temperature optima values were 25°C, 24°C, 25°C, 23°C, 24°C and 25°C respectively.
The culture conditions for maximum secretion of laccase by Loweporus lividus MTCC-1178 have been ... more The culture conditions for maximum secretion of laccase by Loweporus lividus MTCC-1178 have been optimized. The laccase from the culture filtrate of L. lividus MTCC-1178 has been purified to homogeneity. The molecular weight of the purified laccase is 64.8 kDa. The enzymatic characteristics like K m, pH, and temperature optimum using 2,6-dimethoxyphenol have been determined and found to be 480 μM, 5.0, and 60 °C, respectively. The K m values for other substrates like catechol, m-cresol, pyrogallol, and syringaldazine have also been determined and found to be 230, 210, 320, and 350 μM, respectively.
Solanum melongena fruit juice contains peroxidase activity of the order of 0⋅125 IU/mL. A method ... more Solanum melongena fruit juice contains peroxidase activity of the order of 0⋅125 IU/mL. A method for the 11-fold purification of the enzyme was developed. The K m values of the peroxidase for the substrates guaiacol and hydrogen peroxide were 6⋅5 mM and 0⋅33 mM, respectively. The pH and temperature optima were 5⋅5 and 84°C, respectively using guaiacol as the substrate. Sodium azide and phenyl hydrazine inhibited the enzyme competitively.
a b s t r a c t ␣-l-Rhamnosidase [E. C. 3.2.1.40] cleaves terminal ␣-l-rhamnose specifically from... more a b s t r a c t ␣-l-Rhamnosidase [E. C. 3.2.1.40] cleaves terminal ␣-l-rhamnose specifically from a large number of natural products. The enzyme has wide occurrence in nature and is reported from animal tissues, plants, yeasts, fungi and bacteria. It is a biotechnologically important enzyme due to its applications in debittering and clearance of citrus fruit juices, enhancement of wine aromas and derhamnosylation of many natural products containing terminal ␣-l-rhamnose to compounds of pharmaceutical interests. Though ␣-l-rhamnosidases have been investigated actively during recent years, there is no recent review on ␣-l-rhamnosidases. An attempt has been made to fill up this gap in this review. It consists of a brief introduction of ␣-l-rhamnosidase which is followed by a critical description of the methods used for assaying the enzyme activity. Purifications, characterizations and properties of ␣-l-rhamnosidases from different sources have been discussed and the available structural and molecular biological studies on the enzyme have been given. Biotechnological applications of this enzyme in different processes have been briefly described. The review concludes with the identification of areas which needs further extensive studies.
Lignin peroxidase from the liquid culture filtrate of Gloeophyllum sepiarium MTCC-1170 has been p... more Lignin peroxidase from the liquid culture filtrate of Gloeophyllum sepiarium MTCC-1170 has been purified to homogeneity. The molecular weight of the purified enzyme was 42 kDa as determined by SDS-PAGE. The K m values were 54 and 76 µM for veratryl alcohol and H2O2, respectively. The pH and temperature optima were 2.5 and 25°C, respectively. Depolymerization of coal by the fungal strain has been demonstrated using humic acid as a model of coal. Depolymerization of humic acid by the purified lignin peroxidase has been shown by the decrease in absorbance at 450 nm and increase in absorbance at 360 nm in presence of H2O2. Depolymerization of humic acid by the purified enzyme has also been demonstrated by the decrease in the viscosity with time of the reaction solution containing humic acid, H2O2, and the purified lignin peroxidase. The influence of NaCl and NaN3 and inhibitory effects of various metal chelating agents on the lignin peroxidase activity were studied.
Lignin peroxidase from the culture filtrate of Loweporus lividus MTCC-1178 has been purified to h... more Lignin peroxidase from the culture filtrate of Loweporus lividus MTCC-1178 has been purified to homogeneity using Amicon concentration and DEAE cellulose chromatography. The molecular weight of the purified lignin peroxidase using SDS-PAGE analysis has been found to be 40 kDa. The Km values for veratryl alcohol and H2O2 for the purified enzyme were 58 and 83 μM, respectively. The calculated kcat value of the purified enzyme using veratryl alcohol as the substrate was 2.5 s−1. The pH and temperature optima of lignin peroxidase have been found to be 2.6 and 24°C, respectively.
N-Oxidation of arylamines to their corresponding nitrosobenzenes using a new chloroperoxidase pur... more N-Oxidation of arylamines to their corresponding nitrosobenzenes using a new chloroperoxidase purified from Musa paradisiaca stem juice has been examined. The enzymatic characteristics of the stem chloroperoxidase using 4-chloroaniline as substrate were determined. The K ...
A total of 48 full-length protein sequences of pectin lyases from different source organisms avai... more A total of 48 full-length protein sequences of pectin lyases from different source organisms available in NCBI were subjected to multiple sequence alignment, domain analysis, and phylogenetic tree construction. A phylogenetic tree constructed on the basis of the protein sequences revealed two distinct clusters representing pectin lyases from bacterial and fungal sources. Similarly, the multiple accessions of different source organisms representing bacterial and fungal pectin lyases also formed distinct clusters, showing sequence level homology. The sequence level similarities among different groups of pectinase enzymes, viz. pectin lyase, pectate lyase, polygalacturonase, and pectin esterase, were also analyzed by subjecting a single protein sequence from each group with common source organism to tree construction. Four distinct clusters representing different groups of pectinases with common source organisms were observed, indicating the existing sequence level similarity among them. Multiple sequence alignment of pectin lyase protein sequence of different source organisms along with pectinases with common source organisms revealed a conserved region, indicating homology at sequence level. A conserved domain Pec_Lyase_C was frequently observed in the protein sequences of pectin lyases and pectate lyases, while Glyco_hydro_28 domains and Pectate lyase-like β-helix clan domain are frequently observed in polygalacturonases and pectin esterases, respectively. The signature amino acid sequence of 41 amino acids, i.e. TYDNAGVLPITVN-SNKSLIGEGSKGVIKGKGLRIVSGAKNI, related with the Pec_Lyase_C is frequently observed in pectin lyase protein sequences and might be related with the structure and enzymatic function.
An indigenously isolated fungal strain identified as Aspergillus terricola with assigned fungal s... more An indigenously isolated fungal strain identified as Aspergillus terricola with assigned fungal strain number MTCC 7588 has been used as source for pectin lyase production. The extracellular pectin lyase was purified to homogeneity from the culture filtrate of A. terricola by ion exchange and gel filtration chromatography. The determined molecular weight was 35 ± 01 kDa. The K m and k cat (turnover) values of the purified enzyme at 37 °C using citrus pectin as the substrate were found to be 1.0 mg/ml and 110.0 s−1, respectively. The pH and temperature optima of the enzyme were 8.0 and 50 °C, respectively. The retting ability of the purified pectin lyase for natural fibers viz. Cannabis sativa and Linum usitatissimum has been demonstrated for the first time.
An acidic pectin lyase (E.C. 4.2.2.10) produced byAspergillus ficuum MTCC 7591 of molecular weigh... more An acidic pectin lyase (E.C. 4.2.2.10) produced byAspergillus ficuum MTCC 7591 of molecular weight 31.6 kD was purified to apparent homogeneity by ion exchange and gel filtration chromatography. Eighty-six fold purification with 60% yield and a specific activity of 7.8 U/mg protein was obtained. The Km and calculated turnover number (kcat) of the purified enzyme were found to be 0.60 mg/ml and 74 s−1 respectively using citrus pectin as the substrate. The pH and temperature optima were 5.0 and 50°C respectively. Exposed to 24 hours at a particular pH the enzyme was found to be relatively stable in the pH range 2.0–9.0. Exposed to a particular temperature for 1 hour, the enzyme retains full activity up to 40°C. Metal ions and protein inhibitors did not have significant effects on the activity of the enzyme. The enzyme has been found to be very effective in the clarification of sweet lime and orange juices.
The importance of various parameters such as sugarcane juice concentration, pH of the medium, and... more The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K m and k cat values were found to be 1 mg/ml and 76 sec−1, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea.
The mycelia of Aspergillus niger MTCC-404 have been shown to act as a biocatalyst for the transfo... more The mycelia of Aspergillus niger MTCC-404 have been shown to act as a biocatalyst for the transformation of ethylbenzene to (R)-1-phenylethanol in 99% enantiomeric excess. 72% of the products are (R)-1-phenylethanol. The conversion yield is dependent on pH, temperature and mycelia concentration in suspension. A. niger MTCC-404 facilitates methylbenzene transformation to benzylalcohol, and propylbenzene to 1-phenylpropanol. Therefore, A. niger serves
Secretion and enzymatic characteristics of lignin peroxidases from Gloeophyllum sepiarium MTCC 11... more Secretion and enzymatic characteristics of lignin peroxidases from Gloeophyllum sepiarium MTCC 1170, Cladosporium herbarum MTCC 346, Lenzites betulina MTCC 1183, Daedalea flavida MTCC 145, Hexagonia teruis MTCC 1119 and Coirolopsis floccosa MTCC 1177 ligninolytic fungal strains have been reported. Secretion of lignin peroxidase by these ligninolytic fungal strains have been found to be in the range of 0.86 to 3.0 enzyme unit per ml of the culture medium. The enzymatic characteristics like Km, pH and temperature optima of all the lignin peroxidases of the above fungal strains have been determined using veratryl alcohol and H2O2 as the variable substrates. The Km values using veratryl alcohol as the substrate were found to be 65.0 μM, 58.5 μM, 63.0 μM, 54.5 μM, 54.6 μM and 61.0 μM respectively. The Km values using H2O2 as the substrate were found to be 88.0 μM, 86.0 μM, 71.0 μM, 67.0 μM, 80.0 μM and 78.0 μM respectively. The pH optima values for lignin peroxidases of the above ligninolytic fungal strains were found to be 2.5, 2.4, 2.4, 2.25, 2.5 and 2.8 respectively, where as the temperature optima values were 25°C, 24°C, 25°C, 23°C, 24°C and 25°C respectively.
The culture conditions for maximum secretion of laccase by Loweporus lividus MTCC-1178 have been ... more The culture conditions for maximum secretion of laccase by Loweporus lividus MTCC-1178 have been optimized. The laccase from the culture filtrate of L. lividus MTCC-1178 has been purified to homogeneity. The molecular weight of the purified laccase is 64.8 kDa. The enzymatic characteristics like K m, pH, and temperature optimum using 2,6-dimethoxyphenol have been determined and found to be 480 μM, 5.0, and 60 °C, respectively. The K m values for other substrates like catechol, m-cresol, pyrogallol, and syringaldazine have also been determined and found to be 230, 210, 320, and 350 μM, respectively.
Solanum melongena fruit juice contains peroxidase activity of the order of 0⋅125 IU/mL. A method ... more Solanum melongena fruit juice contains peroxidase activity of the order of 0⋅125 IU/mL. A method for the 11-fold purification of the enzyme was developed. The K m values of the peroxidase for the substrates guaiacol and hydrogen peroxide were 6⋅5 mM and 0⋅33 mM, respectively. The pH and temperature optima were 5⋅5 and 84°C, respectively using guaiacol as the substrate. Sodium azide and phenyl hydrazine inhibited the enzyme competitively.
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