The proteolytic extract obtained from the latex of Funastrum clausum (Jacq.) Schlechter (Asclepia... more The proteolytic extract obtained from the latex of Funastrum clausum (Jacq.) Schlechter (Asclepiadaceae), a South American climbing plant, was assayed as a novel catalyst for peptide synthesis and compared with commercial papain under the same conditions. After ...
Http Dx Doi Org 10 1080 10826068 2011 544230, Mar 26, 2011
Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption/ionization... more Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry homogeneity by salt precipitation from two different crude extract sources: a refined preparation obtained in our laboratory and a commercial one. Sodium tetrathionate was tested in the purification process to preserve the enzymatic activity of the peptidase. Purification was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and cation exchange chromatography, using commercial pure papain as standard for a rapid comparison. The best purification yields (3.4%) were obtained in presence of 30 mM sodium tetrathionate for the crude extract prepared in our laboratory. The described purification method proved to be robust and reliable to obtain pure papain on a preparative scale.
Two novel arginine-based cationic surfactants were synthesized using as biocatalyst papain, an en... more Two novel arginine-based cationic surfactants were synthesized using as biocatalyst papain, an endopeptidase from Carica papaya latex, adsorbed onto polyamide. The classical substrate N (α)-benzoyl-arginine ethyl ester hydrochloride for the determination of cysteine and serine proteases activity was used as the arginine donor, whereas decyl- and dodecylamine were used as nucleophiles for the condensation reaction. Yields higher than 90 and 80 % were achieved for the synthesis of N (α)-benzoyl-arginine decyl amide (Bz-Arg-NHC10) and N (α)-benzoyl-arginine dodecyl amide (Bz-Arg-NHC12), respectively. The purification process was developed in order to make it more sustainable, by using water and ethanol as the main separation solvents in a single cationic exchange chromatographic separation step. Bz-Arg-NHC10 and Bz-Arg-NHC12 proved antimicrobial activity against both Gram-positive and Gram-negative bacteria, revealing their potential use as effective disinfectants as they reduced 99 % ...
Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by ... more Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by a simple purification procedure involving ultracentrifugation and ion exchange chromatography. Molecular weights of araujiain h II and araujiain h III were 23,718 and 23546 (mass spectrometry), respectively. The isoelectric point of araujiain h II was 8.9, whereas araujiain h III had a pI higher than 9.3. Maximum proteolytic activity on caseine was reached at pH 8.0-9.0 for both endopeptidases, which were irreversibly inhibited by iodoacetate and E-64, suggesting they belong to the cysteine protease family. Esterolytic activity was determined on N-alpha-CBZ-amino acid-p-nitrophenyl esters, and the highest kcat/Km values for the both enzymes were obtained with the glutamine derivative. The N-terminal sequences of araujiain h II and araujiain h III showed a high degree of homology with other plant cysteine endopeptidases.
ABSTRACT Lipase activity found in the insoluble fraction of Araujia sericifera Brot. (Apocynaceae... more ABSTRACT Lipase activity found in the insoluble fraction of Araujia sericifera Brot. (Apocynaceae) (ASL) latex, a native South American milkweed, was characterized by use of different test reactions and under different reaction conditions. In this context, hydrolytic lipase activity towards both natural and synthetic substrates and towards fatty acid esterifications was assayed at different temperatures, pH values, and biocatalyst loads. In the case of natural substrates (cottonseed oil), highest lipase activity was found at pH 8.5 and 60 °C. In the hydrolysis of synthetic substrates (p-nitrophenyl esters) the lipase showed preference for the lowest molecular-weight p-nitrophenyl ester assayed (butyrate). Results of the direct esterification of fatty acids of different chain length in organic media showed that esterification levels of up to 45% could be obtained in one hour of reaction. Activity results were compared with the activity shown by the commercial immobilized lipase Novozym 435 under defined reaction conditions. The high activity exhibited by ASL in the hydrolysis of natural substrates and particularly in the direct esterification of different fatty acids in organic medium, together with its high storage stability, suggests that this plant lipase is a promising biocatalyst for various biotechnological applications.
ABSTRACT This paper presents a rational strategy to purify the lipase B of Candida antarctica of ... more ABSTRACT This paper presents a rational strategy to purify the lipase B of Candida antarctica of the commercial extract Lipozyme® through size exclusion coupled with anionic exchange chromatography using a non conventional, easy to remove buffer system such as ammonia-ammonium. In this context, each step of the purification was followed through the determination of the protein content, esterase activity measurements, SDS-PAGE, agarose electrophoresis, UVspectroscopy and isoelectric focusing. The purification of the commercial extract afforded a sample that retains 47% of the proteins (being CALB the major enzymatic component of the purified sample) with a hydrolytic activity higher than the starting crude extract.
Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption/ionization... more Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry homogeneity by salt precipitation from two different crude extract sources: a refined preparation obtained in our laboratory and a commercial one. Sodium tetrathionate was tested in the purification process to preserve the enzymatic activity of the peptidase. Purification was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and cation exchange chromatography, using commercial pure papain as standard for a rapid comparison. The best purification yields (3.4%) were obtained in presence of 30 mM sodium tetrathionate for the crude extract prepared in our laboratory. The described purification method proved to be robust and reliable to obtain pure papain on a preparative scale.
Partially purified preparations with proteolytic activity, obtained from South American native pl... more Partially purified preparations with proteolytic activity, obtained from South American native plants, were used as biocatalysts in condensation reactions of N-protected arginine alkyl ester derivatives with decylamine and dodecylamine in low-water content systems. The final ...
In this paper we study the effect of different water-immiscible organic solvents (benzene, toluen... more In this paper we study the effect of different water-immiscible organic solvents (benzene, toluene, 1-butanol, 1-octanol, dichloroethane, dichloromethane, diethyl ether, hexane, chlorobenzene, acetophenone, n-dodecane, trichloroethylene, ethyl acetate) on the stability (residual caseinolytic activity after 4h) of soluble phytoproteases, such as araujiain, funastrain and papain in aqueous-organic biphasic systems. Besides, the effect of organic solvents on enzymatic catalysis was quantitatively studied
The proteolytic extract obtained from the latex of Funastrum clausum (Jacq.) Schlechter (Asclepia... more The proteolytic extract obtained from the latex of Funastrum clausum (Jacq.) Schlechter (Asclepiadaceae), a South American climbing plant, was assayed as a novel catalyst for peptide synthesis and compared with commercial papain under the same conditions. After ...
Http Dx Doi Org 10 1080 10826068 2011 544230, Mar 26, 2011
Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption/ionization... more Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry homogeneity by salt precipitation from two different crude extract sources: a refined preparation obtained in our laboratory and a commercial one. Sodium tetrathionate was tested in the purification process to preserve the enzymatic activity of the peptidase. Purification was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and cation exchange chromatography, using commercial pure papain as standard for a rapid comparison. The best purification yields (3.4%) were obtained in presence of 30 mM sodium tetrathionate for the crude extract prepared in our laboratory. The described purification method proved to be robust and reliable to obtain pure papain on a preparative scale.
Two novel arginine-based cationic surfactants were synthesized using as biocatalyst papain, an en... more Two novel arginine-based cationic surfactants were synthesized using as biocatalyst papain, an endopeptidase from Carica papaya latex, adsorbed onto polyamide. The classical substrate N (α)-benzoyl-arginine ethyl ester hydrochloride for the determination of cysteine and serine proteases activity was used as the arginine donor, whereas decyl- and dodecylamine were used as nucleophiles for the condensation reaction. Yields higher than 90 and 80 % were achieved for the synthesis of N (α)-benzoyl-arginine decyl amide (Bz-Arg-NHC10) and N (α)-benzoyl-arginine dodecyl amide (Bz-Arg-NHC12), respectively. The purification process was developed in order to make it more sustainable, by using water and ethanol as the main separation solvents in a single cationic exchange chromatographic separation step. Bz-Arg-NHC10 and Bz-Arg-NHC12 proved antimicrobial activity against both Gram-positive and Gram-negative bacteria, revealing their potential use as effective disinfectants as they reduced 99 % ...
Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by ... more Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by a simple purification procedure involving ultracentrifugation and ion exchange chromatography. Molecular weights of araujiain h II and araujiain h III were 23,718 and 23546 (mass spectrometry), respectively. The isoelectric point of araujiain h II was 8.9, whereas araujiain h III had a pI higher than 9.3. Maximum proteolytic activity on caseine was reached at pH 8.0-9.0 for both endopeptidases, which were irreversibly inhibited by iodoacetate and E-64, suggesting they belong to the cysteine protease family. Esterolytic activity was determined on N-alpha-CBZ-amino acid-p-nitrophenyl esters, and the highest kcat/Km values for the both enzymes were obtained with the glutamine derivative. The N-terminal sequences of araujiain h II and araujiain h III showed a high degree of homology with other plant cysteine endopeptidases.
ABSTRACT Lipase activity found in the insoluble fraction of Araujia sericifera Brot. (Apocynaceae... more ABSTRACT Lipase activity found in the insoluble fraction of Araujia sericifera Brot. (Apocynaceae) (ASL) latex, a native South American milkweed, was characterized by use of different test reactions and under different reaction conditions. In this context, hydrolytic lipase activity towards both natural and synthetic substrates and towards fatty acid esterifications was assayed at different temperatures, pH values, and biocatalyst loads. In the case of natural substrates (cottonseed oil), highest lipase activity was found at pH 8.5 and 60 °C. In the hydrolysis of synthetic substrates (p-nitrophenyl esters) the lipase showed preference for the lowest molecular-weight p-nitrophenyl ester assayed (butyrate). Results of the direct esterification of fatty acids of different chain length in organic media showed that esterification levels of up to 45% could be obtained in one hour of reaction. Activity results were compared with the activity shown by the commercial immobilized lipase Novozym 435 under defined reaction conditions. The high activity exhibited by ASL in the hydrolysis of natural substrates and particularly in the direct esterification of different fatty acids in organic medium, together with its high storage stability, suggests that this plant lipase is a promising biocatalyst for various biotechnological applications.
ABSTRACT This paper presents a rational strategy to purify the lipase B of Candida antarctica of ... more ABSTRACT This paper presents a rational strategy to purify the lipase B of Candida antarctica of the commercial extract Lipozyme® through size exclusion coupled with anionic exchange chromatography using a non conventional, easy to remove buffer system such as ammonia-ammonium. In this context, each step of the purification was followed through the determination of the protein content, esterase activity measurements, SDS-PAGE, agarose electrophoresis, UVspectroscopy and isoelectric focusing. The purification of the commercial extract afforded a sample that retains 47% of the proteins (being CALB the major enzymatic component of the purified sample) with a hydrolytic activity higher than the starting crude extract.
Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption/ionization... more Papain from latex of Carica papaya was purified up to matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry homogeneity by salt precipitation from two different crude extract sources: a refined preparation obtained in our laboratory and a commercial one. Sodium tetrathionate was tested in the purification process to preserve the enzymatic activity of the peptidase. Purification was checked by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and cation exchange chromatography, using commercial pure papain as standard for a rapid comparison. The best purification yields (3.4%) were obtained in presence of 30 mM sodium tetrathionate for the crude extract prepared in our laboratory. The described purification method proved to be robust and reliable to obtain pure papain on a preparative scale.
Partially purified preparations with proteolytic activity, obtained from South American native pl... more Partially purified preparations with proteolytic activity, obtained from South American native plants, were used as biocatalysts in condensation reactions of N-protected arginine alkyl ester derivatives with decylamine and dodecylamine in low-water content systems. The final ...
In this paper we study the effect of different water-immiscible organic solvents (benzene, toluen... more In this paper we study the effect of different water-immiscible organic solvents (benzene, toluene, 1-butanol, 1-octanol, dichloroethane, dichloromethane, diethyl ether, hexane, chlorobenzene, acetophenone, n-dodecane, trichloroethylene, ethyl acetate) on the stability (residual caseinolytic activity after 4h) of soluble phytoproteases, such as araujiain, funastrain and papain in aqueous-organic biphasic systems. Besides, the effect of organic solvents on enzymatic catalysis was quantitatively studied
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