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Partial purification and characterization of polygalacturonases of Geotrichum candidum.
Jäger, Jakub ; Breierová, Emília (oponent) ; Stratilová, Eva (vedoucí práce)
This work discusses the possibilities of using microbial degradation of grape pomace, main waste material from wine production, to preparate industrially important enzymes. The issue is focused on the production of pectolytic enzymes, particularly polygalacturonase, by Geotrichum candidum CCY 16-1-29 via solid state fermentation on grape pomace. The theoretical part of the bachelor thesis focuses on studying plant cells and saccharides from which the plant cell wall is made of, mainly pectin. Cell wall sacharides were used as a carbon source for solid state fermentation (SSF) and pectin as an inductor of pectolytic enzymes. This bachelor thesis also deals with the enzymatic degradation of cell wall polysacharides. The greatest attention is paid to degrade pectin and pectolytic enzyme function. Production of pectolytic enzymes is mentioned subsequently. The last chapter from the theoretical part is dedicated to technical use of pectolytic enzymes. In the experimental part of this work I deal with the partial purification and characterization of majority polygalacturonase produced on the seventh day of cultivation, when another increase of extracellular polygalacturonase activity occurred. The yield of cultivation was 43,5 mg of protein extract /100 g of grape pomace. The extract contained protein, and its activity was lyophilisate. Its specific activity was protein. The enzyme was produced in at least four forms differing in pH optimum (4,0; 4,4; 4,8; 5,2). The pH optimum for majority polygalacturonase was 4,8. Action pattern of this enzyme determined as the dependence of polymeric substrate viscosity decrease on its degradation showed that the enzyme is a typical polygalacturonase with random action pattern (EC 3.2.1.15).Value of Km reached indicating a high affinity for this substrate. The amino acid sequence "SNNVVSNVNILSSQVVNSDNGVR" obtained by mass spectrometry after SDS-PAGE and tryptic digestion, was identified as a stretch of primary structure of polygalacturonase of Ap2PG1 G. candidum based on the comparison with proteins from the Uniprot database. It shows the highest similarity with other polygalacturonases of G. candidum S31PG1, S31PG2 and G. klebahnii PSE3. On the basis of this similarity to enzymes produced by phytopathogenic strains of G. candidum and the fact that this enzyme was not produced only in the early stages of cultivation, it can be assumed, that the strain of G. candidum CCY 16-1-29 acted also as a phytopathogenic strain.
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Biotyping of Cryptococcus laurentii group using mass spectrometry
Jäger, Jakub ; Breierová, Emília (oponent) ; Stratilová, Eva (vedoucí práce)
Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human and animal infection have been already reported. This species is reported to be heterogenous. The taxonomy of yeast Cryptococcus laurentii was always highly ambiguous. The application of molecular biology and bioinformatic methods led to dividing of searched strains to two distinct phylogenetic groups, some varieties were recognized as species and the locution „Cryptococcus laurentii group“ was introduced. The taxonomy of this group is likely not definitive and with advancing knowledge will change. Our aim was the identification of individual species within this group based on matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI – TOF MS), which has recently been described as a rapid, reliable, cost-effective and powerful tool for analyzing of microorganisms, even on variety level. Generally, the yeasts of genus Cryptococcus form highly resistant polysaccharide capsules and produce large amount of extracellular polysaccharides, therefore belong to so called „difficult“ cases for biotyping. The experimental protocol has been optimized for MS analysis of this genus on the selected strains of Cr. neoformans, Cr. laurentii and Cr. magnus from the Culture Collection of Yeasts (CCY).Thirty-three strains, originally classified as Cryptococcus laurentii has been identified by chosen method. These strains were distributed into six different groups according their spectra similarities. It was selected at least one strain of each group, which was classified based on the sequence analysis of the D1/D2 domains of the LSU rRNA gene. This strain (with known sequence) became representative for its group. Type strain of Cryptococcus laurentii (CCY 17-3-2) belongs to the group I. Its MS spectrum of ribosomal proteins differ from mass spectra of all other biotyped species, even with strains identified as Cryptococcus laurentii was the similarity of the spectra low, which could be caused by identification of two different varieties. The group II is represented by Cryptococcus laurentii CCY 17-3-17. Except this strain, thirteen more strains belong to the group II. The group III represents Cryptococcus flavescens CCY 17-3-29. This group included 12 additional strains with almost identical mass spectra. Group IV included only one strain (CCY 17-3-13), which was identified as Cryptococcus carnescens based on gene sequence analysis. Similarly, one representative (CCY 17-3-5) has the group V. Strain CCY 17-3-5 was identified as Cryptococcus flavus. The last group VI of three members represents strain 17-3-35 identified as Bulleromyces albus. While Cr. laurentii and Cr. flavescens belong to phylogenetic group I and Cr. carnescens to the phylogenetic group II, four strains giving two types of different MS spectra and identified as Cr. flavus (1 strain) and Bulleromyces albus (3 strains) were excluded from „Cr. laurentii group.“
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Biotyping of Cryptococcus laurentii group using mass spectrometry
Jäger, Jakub ; Breierová, Emília (oponent) ; Stratilová, Eva (vedoucí práce)
Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human and animal infection have been already reported. This species is reported to be heterogenous. The taxonomy of yeast Cryptococcus laurentii was always highly ambiguous. The application of molecular biology and bioinformatic methods led to dividing of searched strains to two distinct phylogenetic groups, some varieties were recognized as species and the locution „Cryptococcus laurentii group“ was introduced. The taxonomy of this group is likely not definitive and with advancing knowledge will change. Our aim was the identification of individual species within this group based on matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI – TOF MS), which has recently been described as a rapid, reliable, cost-effective and powerful tool for analyzing of microorganisms, even on variety level. Generally, the yeasts of genus Cryptococcus form highly resistant polysaccharide capsules and produce large amount of extracellular polysaccharides, therefore belong to so called „difficult“ cases for biotyping. The experimental protocol has been optimized for MS analysis of this genus on the selected strains of Cr. neoformans, Cr. laurentii and Cr. magnus from the Culture Collection of Yeasts (CCY).Thirty-three strains, originally classified as Cryptococcus laurentii has been identified by chosen method. These strains were distributed into six different groups according their spectra similarities. It was selected at least one strain of each group, which was classified based on the sequence analysis of the D1/D2 domains of the LSU rRNA gene. This strain (with known sequence) became representative for its group. Type strain of Cryptococcus laurentii (CCY 17-3-2) belongs to the group I. Its MS spectrum of ribosomal proteins differ from mass spectra of all other biotyped species, even with strains identified as Cryptococcus laurentii was the similarity of the spectra low, which could be caused by identification of two different varieties. The group II is represented by Cryptococcus laurentii CCY 17-3-17. Except this strain, thirteen more strains belong to the group II. The group III represents Cryptococcus flavescens CCY 17-3-29. This group included 12 additional strains with almost identical mass spectra. Group IV included only one strain (CCY 17-3-13), which was identified as Cryptococcus carnescens based on gene sequence analysis. Similarly, one representative (CCY 17-3-5) has the group V. Strain CCY 17-3-5 was identified as Cryptococcus flavus. The last group VI of three members represents strain 17-3-35 identified as Bulleromyces albus. While Cr. laurentii and Cr. flavescens belong to phylogenetic group I and Cr. carnescens to the phylogenetic group II, four strains giving two types of different MS spectra and identified as Cr. flavus (1 strain) and Bulleromyces albus (3 strains) were excluded from „Cr. laurentii group.“
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Partial purification and characterization of polygalacturonases of Geotrichum candidum.
Jäger, Jakub ; Breierová, Emília (oponent) ; Stratilová, Eva (vedoucí práce)
This work discusses the possibilities of using microbial degradation of grape pomace, main waste material from wine production, to preparate industrially important enzymes. The issue is focused on the production of pectolytic enzymes, particularly polygalacturonase, by Geotrichum candidum CCY 16-1-29 via solid state fermentation on grape pomace. The theoretical part of the bachelor thesis focuses on studying plant cells and saccharides from which the plant cell wall is made of, mainly pectin. Cell wall sacharides were used as a carbon source for solid state fermentation (SSF) and pectin as an inductor of pectolytic enzymes. This bachelor thesis also deals with the enzymatic degradation of cell wall polysacharides. The greatest attention is paid to degrade pectin and pectolytic enzyme function. Production of pectolytic enzymes is mentioned subsequently. The last chapter from the theoretical part is dedicated to technical use of pectolytic enzymes. In the experimental part of this work I deal with the partial purification and characterization of majority polygalacturonase produced on the seventh day of cultivation, when another increase of extracellular polygalacturonase activity occurred. The yield of cultivation was 43,5 mg of protein extract /100 g of grape pomace. The extract contained protein, and its activity was lyophilisate. Its specific activity was protein. The enzyme was produced in at least four forms differing in pH optimum (4,0; 4,4; 4,8; 5,2). The pH optimum for majority polygalacturonase was 4,8. Action pattern of this enzyme determined as the dependence of polymeric substrate viscosity decrease on its degradation showed that the enzyme is a typical polygalacturonase with random action pattern (EC 3.2.1.15).Value of Km reached indicating a high affinity for this substrate. The amino acid sequence "SNNVVSNVNILSSQVVNSDNGVR" obtained by mass spectrometry after SDS-PAGE and tryptic digestion, was identified as a stretch of primary structure of polygalacturonase of Ap2PG1 G. candidum based on the comparison with proteins from the Uniprot database. It shows the highest similarity with other polygalacturonases of G. candidum S31PG1, S31PG2 and G. klebahnii PSE3. On the basis of this similarity to enzymes produced by phytopathogenic strains of G. candidum and the fact that this enzyme was not produced only in the early stages of cultivation, it can be assumed, that the strain of G. candidum CCY 16-1-29 acted also as a phytopathogenic strain.
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