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Eleni Gomes - One of the best experts on this subject based on the ideXlab platform.

  • Characterization, Thermal Stability, and Partial Sequence of a Novel Exo-Polygalacturonase from the Thermophilic Fungus Rhizomucor pusillus A13.36 Obtained by Submerged Cultivation
    2020
    Co-Authors: Vinícius Lucas, Carla Desagiacomo, Lourdes Teixeira, Moraes Polizeli, André Ricardo De Lima Damasio, Aline Margarete, Furuyama Lima, Eleni Gomes
    Abstract:

    This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45 ∘ C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5-47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61 ∘ C. It presents thermal stability between 30 and 60 ∘ C, has 70% activation in the presence of Ca 2+ , and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. ( ) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and Δ ( ) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot

  • biochemical characterization thermal stability and partial sequence of a novel exo polygalacturonase from the thermophilic fungus Rhizomucor pusillus a13 36 obtained by submerged cultivation
    BioMed Research International, 2016
    Co-Authors: Lucas Vinicius Trindade, Eleni Gomes, Maria De Lourdes Teixeira De Moraes Polizeli, Carla Desagiacomo, Andre Damasio, Aline Margarete Furuyama Lima, Gustavo Orlando Bonillarodriguez
    Abstract:

    This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5-47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. Ea(d) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and ΔG(d) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot.

  • Partial purification, immobilization and preliminary biochemical characterization of lipases from
    2013
    Co-Authors: Rhizomucor Pusillus, Eleni Gomes, Roberto Da Silva, Ana Lúcia Ferrarezi, Daniele H. Pivetta, Gustavo Orl, O Bonilla-rodriguez, José Manuel Guisan, Benevides Costa Pessela
    Abstract:

    Copyright © 2013 Ana Lúcia Ferrarezi et al. This is an open access article distributed under the Creative Commons Attribution Li-cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lipases have important applications in bio-technological processes, motivating us to pro-duce, purify, immobilize and perform a bio-chemical characterization of the lipase from Rhizomucor pusillus. The fungus was cultivated by solid state fermentation producing lipolytic activity of about 0.5 U/mL(4U/g). A partial purifi-cation by gel filtration chromatography in Sephacryl S-100 allowed obtaining a yield of about 85 % and a purification factor of 5.7. Our results revealed that the purified enzyme is very stable with some significant differences in it

  • Protease Production by Different Thermophilic Fungi
    Applied Biochemistry and Biotechnology, 2007
    Co-Authors: Mariana M. Macchione, Eleni Gomes, Carolina W. Merheb, Roberto Da Silva
    Abstract:

    A comparative study was carried out to evaluate protease production in solid-state fermentation (SSF) and submerged fermentation (SmF) by nine different thermophilic fungi — Thermoascus aurantiacus Miehe, Thermomyces lanuginosus, T. lanuginosus TO.03, Aspergillus flavus 1.2, Aspergillus sp. 13.33, Aspergillus sp. 13.34, Aspergillus sp. 13.35, Rhizomucor pusillus 13.36 and Rhizomucor sp. 13.37 — using substrates containing proteins to induce enzyme secretion. Soybean extract (soybean milk), soybean flour, milk powder, rice, and wheat bran were tested. The most satisfactory results were obtained when using wheat bran in SSF. The fungi that stood out in SSF were T. lanuginosus, T. lanuginosus TO.03, Aspergillus sp. 13.34, Aspergillus sp. 13.35, and Rhizomucor sp. 13.37, and those in SmF were T. aurantiacus, T. lanuginosus TO.03, and 13.37. In both fermentation systems, A. flavus 1.2 and R. pusillus 13.36 presented the lowest levels of proteolytic activity.

  • production of saccharogenic and dextrinogenic amylases by Rhizomucor pusillus a 13 36
    Journal of Microbiology, 2005
    Co-Authors: Tony M. Silva, Maurício Boscolo, Derlene Attiliangelis, Ana Flavia Azevedo Carvalho, Roberto Santana Da Silva, Eleni Gomes
    Abstract:

    Laboratorio de Bioquimica e Microbiologia IBILCE-Instituto de Instituto de Biociencias, Letras e Ciencias Exatas UNESP-Universidade Estadual Paulista, Sao Jose do Rio Preto-SP

Trindade, Lucas Vinicius - One of the best experts on this subject based on the ideXlab platform.

  • Biochemical characterization, thermal stability, and partial sequence of a novel exo-polygalacturonase from the thermophilic fungus Rhizomucor pusillus a13.36 obtained by submerged cultivation
    'Hindawi Limited', 2017
    Co-Authors: Trindade, Lucas Vinicius, Gomes Eleni, Desagiacomo Carla, Polizeli, Maria De Lourdes Teixeira De Moraes, Damasio, André Ricardo De Lima, Lima, Aline Margarete Furuyama, Bonilla-rodriguez, Gustavo Orlando
    Abstract:

    Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45 degrees C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5-47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61 degrees C. It presents thermal stability between 30 and 60 degrees C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26% E-a(d) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and Delta G((d)) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot.This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45 degrees C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, b2016110CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCNQP - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)SEM INFORMAÇÃOSEM INFORMAÇÃO11/23991-710/12624-0This work was funded by CAPES, CNPq, and FAPESP (11/23991-7 and 10/12624-0).The authors thank the National Laboratory of Science and Technology of Bioethanol (CTBE), in Campinas, SP (Brazil), for mass spectrometry analysis and also Ms. Fatima Zapata fro

  • Purificação e estudos bioquímicos de exo-poligalacturonase (pectinase) produzida pelo fungo termofílico Rhizomucor pusillus, em cultivo submerso e aplicação na extração de sucos de frutas e hidrólise enzimática do bagaço de cana-de-açúcar
    Universidade Estadual Paulista (UNESP), 2015
    Co-Authors: Trindade, Lucas Vinicius
    Abstract:

    Pectinases are a group of enzymes capable of degrading pectic polysaccharides (pec-tin). These enzymes are used in the industry for production of fruit juices, wines, tex-tiles, paper, tea, oils, etc. This study aimed to produce, purify and characterize an exo-poligacturonase (exo-PG - EC 3.2.1.67) from the thermophilic fungus Rhizomucor pu-sillus. The enzyme was produced by submerged cultivation and purified by chromatog-raphy and salting out. This has proved to be the most appropriate using 95 % ammoni-um sulfate, and with this technique it was possible to obtain the enzyme almost free of contaminants and with a yield of approximately 74 %. A subsequent step of hydropho-bic interaction chromatography is able to remove the remaining contaminants. The pure enzyme has molecular mass around 43.5-47 kDa, optimum pH of 4.0, optimum tem-perature of 61 °C, is stable at a pH range between 3.5 and 6.5, presents thermal stability from 30 to 60 °C and is activated in the presence Ca2+. The isoelectric point of the exo-PG is 6.2 and thermodynamic studies have shown that it is a thermophilic enzyme, highlighting its half-life t1/2 of 2,310 minutes at 50 °C. The profile of hydrolysis was ana-lyzed by capillary electrophoresis and studies indicate that the enzyme in this study has a pattern of sequential hydrolysis (exo). The identification of amino acids by mass spec-trometry MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with the corresponding enzyme from Aspergillus fumigatus. The application tests of the enzymes of the crude extract showed increased extra juice extraction and a higher yield in the hydrolysis process, but this was not observed with the pure enzymeAs pectinases são um grupo de enzimas capazes de degradar polissacarídeos pécticos (pectinas). Estas enzimas são utilizadas na indústria de produção de sucos de frutas, vinhos, têxtil, papel, chá, óleos e etc. O presente trabalho teve por objetivo produzir, purificar e caracterizar a pectinase exo-poligacturonase (exo-PG - EC 3.2.1.67) do fun-go termofílico Rhizomucor pusillus. A enzima foi produzida por cultivo submerso e puri-ficada por salting-out e cromatografia. Este método se revelou como o mais adequado empregando 95 % de sulfato de amônio, sendo que com esta técnica foi possível obter a enzima quase livre de contaminantes e com um rendimento de aproximadamente 74 %. Um passo subsequente de cromatografia de interação hidrofóbica é capaz de re-mover os contaminantes remanescentes. A enzima pura possui massa molecular ao redor de 43,5-47 kDa, pH ótimo de 4,0, temperatura ótima de 61 °C, é estável a uma faixa de pH que varia de 3,5 a 6,5, possui estabilidade térmica de 30 até 60 °C e é ati-vada na presença de Ca+2. O ponto isoelétrico da exo-PG é 6,2 e estudos termodinâ-micos mostraram se tratar de uma enzima termofílica, destacando seu tempo de meia-vida t1/2 de 2.310 min., a 50 °C. O perfil da hidrólise foi analisado por eletroforese capi-lar e os estudos indicam que a enzima em estudo apresenta um padrão de hidrólise sequencial (exo). A identificação dos aminoácidos por espectrometria de massa do tipo MALDI-TOF e a comparação com bancos de dados revelou a maior identidade dos fragmentos sequenciados da exo-PG de R. pusillus com a enzima correspondente de Aspergillus fumigatus. Os testes de aplicação das enzimas do extrato bruto mostraram um aumento da extração de suco e um auxílio no processo de hidrólise, sendo que este não foi observado com a enzima puraCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Sueharu Horinouchi - One of the best experts on this subject based on the ideXlab platform.

  • replacements of amino acid residues at subsites and their effects on the catalytic properties of Rhizomucor pusillus pepsin an aspartic proteinase from Rhizomucor pusillus
    Journal of Biochemistry, 2001
    Co-Authors: Junichi Aikawa, Youngnam Park, Masakazu Sugiyama, Makoto Nishiyama, Sueharu Horinouchi, Teruhiko Beppu
    Abstract:

    : Site-directed mutagenesis was carried out to investigate the functional roles of amino acid residues of Rhizomucor pusillus pepsin (RMPP) in substrate-binding and catalysis. Mutations of two amino acid residues, E13 in the S3 subsite and N219 in the S3/S4 subsites, caused marked changes in kinetic parameters for two substrate peptides with different sequences. Further site-directed mutagenesis at E13 suggested that E13 plays a critical role in forming the correct hydrogen bond network around the active center. In the crystal structure of Rhizomucor miehei pepsin (RMMP), which is an aspartic proteinase produced by Rhizomucor miehei and shows 81% amino acid identity to RMPP, the Oepsilon atom of N219 forms a hydrogen bond with the N-H of isovaline in pepstatin A, a statine-type inhibitor, at the P3 position, suggesting that the loss of the hydrogen bond causes an unfavorable arrangement of the P3 residue. Among the mutants constructed, the E13A mutant showed a 5-fold increase in the ratio of clotting versus proteolytic activity without significant loss of clotting activity. This mutant may present a promising candidate for a useful milk coagulant.

  • Characterization of an alg2 mutant of the zygomycete fungus Rhizomucor pusillus
    Glycobiology, 1999
    Co-Authors: Kyoko Takeuchi, Haruka Yamazaki, Norihiko Shiraishi, Yasuo Ohnishi, Yoshihisa Nishikawa, Sueharu Horinouchi
    Abstract:

    The zygomycete fungus Rhizomucor pusillus secretes an aspartic proteinase (MPP) that contains asparagine ( N )-linked oligosaccharides at two sites. Mutant strain 1116 defective in N -glycosylation secretes MPP with truncated oligo-saccharide chains. Lipid-linked oligosaccharides in mutant 1116 were labeled with [6-(3)H]glucosamine and [2-(3)H]mannose, prepared by cycles of solvent extraction, and analyzed by gel filtration chromatography on a Bio-Gel P-4 column after mild acid-hydrolysis. Mutant 1116 accumulated an intermediate, Man(1)GlcNAc(2)-dolichol pyrophosphate (PP-Dol), whereas wild-type strain F27 synthesized the fully assembled oligosaccharide precursor Glc(3)Man(9)GlcNAc(2)-PP-Dol. Consistent with this, alg2 encoding a mannosyltransferase in the lipid-linked oligosaccharide biosynthetic pathway in mutant 1116 had a 5 bp insertion that generated a stop codon in the middle of the coding sequence. Transformation of mutant 1116 with the intact alg2 gene on a pUC19-derived plasmid generated transformants that contained multicopies of alg2 at the alg2 locus. Glycosylation of the total proteins in the transformants was recovered to the same level as in strain F27, as determined with peroxidase-concanavalin A. These transformants produced MPP mainly with the same N -linked oligosaccharides as that produced by strain F27, but still with truncated oligosaccharides in small amounts. All of these data show that Alg2 is an alpha-1,3 or alpha-1,6 mannosyltransferase that elongates Man(1)GlcNAc(2)-PP-Dol to Man(2)GlcNAc(2)-PP-Dol. The slower growth of mutant 1116 was significantly recovered on introduction of alg2. The viability of the alg2 mutants of the zygomycete R.pusillus makes a contrast with the lethal effect of ALG2 mutations in the yeast Saccharomyces cerevisiae.

  • genetic transformation of a Rhizomucor pusillus mutant defective in asparagine linked glycosylation production of a milk clotting enzyme in a less glycosylated form
    Applied Microbiology and Biotechnology, 1999
    Co-Authors: Haruka Yamazaki, Kyoko Takeuchi, Norihiko Shiraishi, Yasuo Ohnishi, N Mori, Y Sakata, Hitoshi Suzuki, Sueharu Horinouchi
    Abstract:

    Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms. For development of a genetic transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet (UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5′-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R. pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi. The amino acid sequence of R. pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi. The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R. pusillus 1116U17 by polyethylene glycol-assisted transformation. Transformation under optimized conditions yielded 5 Ura+ transformants with 1 μg pRPPyr4 DNA and 1 × 107 viable protoplasts. Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4 sequence were integrated into the genome by homologous recombination at the pyr4 locus. For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R. pusillus 1116U17. Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man0∼1GlcNAc2 at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain. The transformation system for R. pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains.

  • Characterization of an alg2 mutant of the zygomycete fungus Rhizomucor pusillus
    1999
    Co-Authors: Kyoko Takeuchi, Haruka Yamazaki, Norihiko Shiraishi, Yasuo Ohnishi, Yoshihisa Nishikawa, Sueharu Horinouchi
    Abstract:

    The zygomycete fungus Rhizomucor pusillus secretes an aspartic proteinase (MPP) that contains asparagine (N)-linked oligosaccharides at two sites. Mutant strain 1116 defective in N-glycosylation secretes MPP with truncated oligosaccharide chains. Lipid-linked oligosaccharides in mutant 1116 were labeled with [6- 3 H]glucosamine and [2- 3 H]mannose, prepared by cycles of solvent extraction, and analyzed by gel filtration chromatography on a Bio-Gel P-4 column after mild acid-hydrolysis. Mutant 1116 accumulated an intermediate, Man 1GlcNAc 2-dolichol pyrophosphate (PP-Dol), whereas wild-type strain F27 synthesized the fully assembled oligosaccharide precursor Glc 3Man 9GlcNAc 2-PP-Dol. Consistent with this, alg2 encoding a mannosyltransferase in the lipid-linked oligosaccharide biosynthetic pathway in mutant 1116 had a 5 bp insertion that generated a stop codon in the middle of the coding sequence. Transformation of mutant 1116 with the intact alg2 gene on a pUC19-derived plasmid generated transformants that contained multicopies of alg2 at the alg2 locus. Glycosylation of the total proteins in the transformants was recovered to the same level as in strain F27, as determined with peroxidase-concanavalin A. These transformants produced MPP mainly with the same N-linked oligosaccharides as that produced by strain F27, but still with truncated oligosaccharides in small amounts. All of these data show that Alg2 is an α-1,3 or α-1,6 mannosyltransferase that elongates Man 1 GlcNAc 2-PP-Dol to Man 2 GlcNAc 2-PP-Dol. The slower growth of mutant 1116 was significantly recovered on introduction of alg2. The viability of the alg2 mutants of the zygomycete R.pusillus makes a contrast with the lethal effect of ALG2 mutations in the yeast Saccharomyces cerevisiae. Key words: alg2/asparagine-linked glycosylation/ mannosyltransferase/Rhizomucor pusillus/zygomycete fungu

  • involvement of a residue at position 75 in the catalytic mechanism of a fungal aspartic proteinase Rhizomucor pusilus pepsin replacement of tyrosine 75 on the flap by asparagine enhances catalytic efficiency
    Protein Engineering, 1996
    Co-Authors: Youngnam Park, Junichi Aikawa, Makoto Nishiyama, Sueharu Horinouchi, Teruhiko Beppu
    Abstract:

    Residue 75 on the flap, a beta hairpin loop that partially covers the active site cleft, is tyrosine in most members of the aspartic proteinase family. Site-directed mutagenesis was carried out to investigate the functional role of this residue in Rhizomucor pusillus pepsin, an aspartic proteinase with high milk-clotting activity produced by the fungus Rhizomucor pusillus. A set of mutated enzymes with replacement of the amino acid at position 75 by 17 other amino acid residues except for His and Gly was constructed and their enzymatic properties were examined. Strong activity, higher than that of the wild-type enzyme, was found in the mutant with asparagine (Tyr75Asn), while weak but distinct activity was observed in Tyr75Phe. All the other mutants showed markedly decreased or negligible activity, less than 1/1000 of that of the wild-type enzyme. Kinetic analysis of Tyr75Asn using a chromogenic synthetic oligopeptide as a substrate revealed a marked increase in kcat with slight change in K(m), resulting in a 5.6-fold increase in kcat/K(m). When differential absorption spectra upon addition of pepstatin, a specific inhibitor for aspartic proteinase, were compared between the wild-type and mutant enzymes, the wild-type enzyme and Tyr75Asn, showing strong activity, had spectra with absorption maxima at 280, 287 and 293 nm, whereas the others, showing decreased or negligible activity, had spectra with only two maxima at 282 and 288 nm. This suggests a different mode of the inhibitor binding in the latter mutants. These observations suggest a crucial role of the residue at position 75 in enhancing the catalytic efficiency through affecting the mode of substrate-binding in the aspartic proteinases.

Trindade, Lucas Vinícius [unesp] - One of the best experts on this subject based on the ideXlab platform.

  • Purificação e estudos bioquímicos de exo-poligalacturonase (pectinase) produzida pelo fungo termofílico Rhizomucor pusillus, em cultivo submerso e aplicação na extração de sucos de frutas e hidrólise enzimática do bagaço de cana-de-açúcar
    Universidade Estadual Paulista (UNESP), 2015
    Co-Authors: Trindade, Lucas Vinícius [unesp]
    Abstract:

    Pectinases are a group of enzymes capable of degrading pectic polysaccharides (pec-tin). These enzymes are used in the industry for production of fruit juices, wines, tex-tiles, paper, tea, oils, etc. This study aimed to produce, purify and characterize an exo-poligacturonase (exo-PG - EC 3.2.1.67) from the thermophilic fungus Rhizomucor pu-sillus. The enzyme was produced by submerged cultivation and purified by chromatog-raphy and salting out. This has proved to be the most appropriate using 95 % ammoni-um sulfate, and with this technique it was possible to obtain the enzyme almost free of contaminants and with a yield of approximately 74 %. A subsequent step of hydropho-bic interaction chromatography is able to remove the remaining contaminants. The pure enzyme has molecular mass around 43.5-47 kDa, optimum pH of 4.0, optimum tem-perature of 61 °C, is stable at a pH range between 3.5 and 6.5, presents thermal stability from 30 to 60 °C and is activated in the presence Ca2+. The isoelectric point of the exo-PG is 6.2 and thermodynamic studies have shown that it is a thermophilic enzyme, highlighting its half-life t1/2 of 2,310 minutes at 50 °C. The profile of hydrolysis was ana-lyzed by capillary electrophoresis and studies indicate that the enzyme in this study has a pattern of sequential hydrolysis (exo). The identification of amino acids by mass spec-trometry MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with the corresponding enzyme from Aspergillus fumigatus. The application tests of the enzymes of the crude extract showed increased extra juice extraction and a higher yield in the hydrolysis process, but this was not observed with the pure enzymeAs pectinases são um grupo de enzimas capazes de degradar polissacarídeos pécticos (pectinas). Estas enzimas são utilizadas na indústria de produção de sucos de frutas, vinhos, têxtil, papel, chá, óleos e etc. O presente trabalho teve por objetivo produzir, purificar e caracterizar a pectinase exo-poligacturonase (exo-PG - EC 3.2.1.67) do fun-go termofílico Rhizomucor pusillus. A enzima foi produzida por cultivo submerso e puri-ficada por salting-out e cromatografia. Este método se revelou como o mais adequado empregando 95 % de sulfato de amônio, sendo que com esta técnica foi possível obter a enzima quase livre de contaminantes e com um rendimento de aproximadamente 74 %. Um passo subsequente de cromatografia de interação hidrofóbica é capaz de re-mover os contaminantes remanescentes. A enzima pura possui massa molecular ao redor de 43,5-47 kDa, pH ótimo de 4,0, temperatura ótima de 61 °C, é estável a uma faixa de pH que varia de 3,5 a 6,5, possui estabilidade térmica de 30 até 60 °C e é ati-vada na presença de Ca+2. O ponto isoelétrico da exo-PG é 6,2 e estudos termodinâ-micos mostraram se tratar de uma enzima termofílica, destacando seu tempo de meia-vida t1/2 de 2.310 min., a 50 °C. O perfil da hidrólise foi analisado por eletroforese capi-lar e os estudos indicam que a enzima em estudo apresenta um padrão de hidrólise sequencial (exo). A identificação dos aminoácidos por espectrometria de massa do tipo MALDI-TOF e a comparação com bancos de dados revelou a maior identidade dos fragmentos sequenciados da exo-PG de R. pusillus com a enzima correspondente de Aspergillus fumigatus. Os testes de aplicação das enzimas do extrato bruto mostraram um aumento da extração de suco e um auxílio no processo de hidrólise, sendo que este não foi observado com a enzima pur

Castañeda Ruiz Rafael - One of the best experts on this subject based on the ideXlab platform.

  • Evaluación de la producción experimental de enzimas coagulantes de leche utilizando cepas de Rhizomucor spp
    Universidad Nacional de Colombia - Sede Bogotá - Instituto de Biotecnología, 2015
    Co-Authors: Morillo, Osmar Thomas, Guerrero Cardena, Balmore Ruizdael, Torres Vielma Yzoleth, García Lugo, Pablo José, Castañeda Ruiz Rafael
    Abstract:

    Título en ingles: Evaluation of experimental production of milk-clotting enzymes using Rhizomucor spp strainsTítulo corto: Producción de enzimas coagulantes de lecheResumen: La producción experimental de enzimas coagulante de leche se llevó a cabo en un medio de cultivo de laboratorio durante 190 h de incubación, utilizando tres cepas certificadas de Rhizomucor pusillus,  R. miehei y dos especies nativas de Rhizomucor spp. BIOMI-12 y 13. La evaluación se realizó midiendo la concentración de glucosa y proteína durante la incubación, estimación de la productividad, actividad específica, índice fuerza de cuajo/actividad proteolítica en los extractos enzimáticos crudos, determinación de los pesos moleculares y actividad proteolítica en los extractos enzimáticos parcialmente purificado. Todas las cepas mostraron un consumo de glucosa similar, el mismo comportamiento se observó en el contenido de proteína, excepto la cepa BIOM-13. Los incrementos en el contenido de proteínas después del descenso, coincidieron con la máxima actividad coagulante registrada por cada cepa, siendo el extracto crudo de la cepa BIOMI-13 la de mayor actividad coagulante (148,15 FC), productividad (3,09 FC/h), índice fuerza de cuajo/actividad proteolítica (142,60 FC/U) y actividad específica (1.062,00 FC/mg). Los extractos enzimáticos parcialmente purificados de las cepas R miehei 37, Rhizomucor spp BIOM-12 y 13, presentaron proteínas con pesos moleculares en aproximadamente 22,6 y 46,52 KDa, mientras el extracto R pusillus 39 presentó una banda adicional de 39,6 KDa. En el zimograma se observó para todas las cepas actividad proteolítica en las bandas comprendidas entre 40-50 KDa y 20-22 KDa, no así para el R pusillus 36, donde fue escasa. Finalmente se determinó que la cepa BIOMI-13, tiene la mayor capacidad para producir enzimas coagulantes de la leche.Palabras clave: renina microbiana, fuerza de cuajo, actividad proteolítica, productividad.Key words: microbial rennet, rennet strength, proteolitic activity, productivity.Recibido: mayo 14 de 2014  Aprobado: abril 21 de 2015Experimental production of milk clotting enzymes was conducted on a laboratory culture medium for 190 h incubation, using three certified strains of Rhizomucor pusillus, miehei and two native Rhizomucor spp. BIOMI-12 and 13. The evaluation was performed by measuring the concentration of glucose and protein during incubation, estimate productivity, specific activity, rennet strength/proteolytic activity index in the crude enzyme extracts, determining the molecular weights and proteolytic activity in the partially purified enzyme extracts. All strains showed consumption rates of glucose, the same behavior observed protein content, except strain BIOMI-13. The increase in protein content after descent coincided with the recorded maximum coagulant activity each strains, being the crude extract of strain BIOMI-13 higher coagulant activity (148,15 FC), productivity (3.09 HR / h), rennet strength/proteolytic activity index (142,60 FC/U) and specific activity (1,062 FC/mg). The partially purified enzyme extracts from strains R miehei 37, Rhizomucor spp BIOMI-12 and 13, presented proteins with molecular weights in approximately 22,6 kDa and 46.52, while the extract R pusillus 39 present an additional band of 39,6 KDa. In the zymogram was observed for all strains, proteolytic activity in the bands between 40-50 KDa and 20-22 KDa, but not for the R pusillus 36, where activity was very dim. Finally it was determined that the strain BIOMI-13, has the greatest capacity to produce milk clotting enzymes.Key words: microbial rennet, rennet strength, proteolitic activity, productivity