Parciální purifikace a charakterizace polygalakturonázy z Geotrichum candidum.
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Authors
Jäger, Jakub
ORCID
Advisor
Stratilová, Eva
Referee
Breierová, Emília
Mark
A
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Vysoké učení technické v Brně. Fakulta chemická
Abstract
Táto práca sa zaoberá možnosťami využitia mikrobiálnej degradácie hroznových výliskov ako hlavného odpadového materiálu z výroby vína na produkciu priemyselne významných enzýmov. Problematika je orientovaná na produkciu pektolytických enzýmov, najmä polygalakturonáz. Ako produkčný kmeň bol použitý kvasinkovitý mikroorganizmus Geotrichum candidum CCY 16-1-29 a ako kultivácia s najväčším výťažkom bola využitá „solid state“ fermentácia (SSF). Teoretická časť práce bola zameraná na stavbu rastlinnej bunky a sacharidové zloženie bunkovej steny, hlavne pektín. Sacharidy bunkových stien slúžili všeobecne ako zdroj uhlíka pri SSF kultivácií a pektín ako induktor pektolytických enzýmov mikroorganizmu. Práca sa ďalej zaoberá enzymatickou degradáciou polysacharidov bunkovej steny, pričom je najväčšia pozornosť opäť venovaná degradácii pektínu a funkcii pektolytických enzýmov. Posledná kapitola teoretickej časti sa zaoberá technologickým využitím týchto enzýmov. Experimentálna časť práce bola zameraná na čiastočnú purifikáciu a charakterizáciu majoritnej polygalakturonázy produkovanej v siedmy deň kultivácie, kedy dochádza k opätovnému nárastu aktivity extracelulárnych polygalakturonáz. Výťažok z kultivácie bol 43,5 mg proteínového extraktu/100 g hroznových výliskov. Extrakt obsahoval 20,68 % bielkovín, pričom jeho aktivita bola lyofilizátu a špecifická aktivita bielkoviny. Enzým bol produkovaný minimálne v štyroch formách líšiacich sa pH optimom (4,0; 4,4; 4,8; 5,2). Majoritná polygalakturonáza mala pH optimum 4,8. Spôsob účinku tohto enzýmu na polymérny substrát stanovený koreláciou poklesu viskozity roztoku substrátu a stupňa jeho degradácie ukázal, že enzým je typická polygalakturonáza s náhodným spôsobom účinku (EC 3.2.1.15). Michaelis-Mentenovej konštanta na polymérny substrát dosahovala hodnotu čo naznačuje vysokú afinitu k tomuto substrátu. Aminokyselinová sekvencia „SNNVVSNVNILSSQVVNSDNGVR“, ktorá bola získaná hmotnostnou spektrometriou po SDS-PAGE a tryptickom štiepení, bola na základe porovnania s proteínmi z Uniprot databázy identifikovaná ako úsek primárnej štruktúry polygalakturonázy Ap2PG1 G. candidum, ktorá má najvyššiu podobnosť s ďalšími polygalakturonázami G. candidum S31PG1, S31PG2 a G. klebahnii PSE3. Na základe tejto podobnosti s enzýmom produkovaným fytopatogénnym kmeňom G. candidum a toho, že tento enzým nebol produkovaný len v prvých fázach kultivácie, sa dá predpokladať, že použitý kmeň G. candidum CCY 16-1-29, bol tiež fytopatogénnym kmeňom.
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.
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.
Description
Citation
JÄGER, J. Parciální purifikace a charakterizace polygalakturonázy z Geotrichum candidum. [online]. Brno: Vysoké učení technické v Brně. Fakulta chemická. 2012.
Document type
Document version
Date of access to the full text
Language of document
sk
Study field
Biotechnologie
Comittee
prof. Ing. Peter Šimko, DrSc. (předseda)
doc. Ing. Jiřina Omelková, CSc. (místopředseda)
prof. RNDr. Ivana Márová, CSc. (člen)
prof. RNDr. Milan Potáček, CSc. (člen)
doc. Ing. Bohuslav Rittich, CSc. (člen)
doc. RNDr. Alena Španová, CSc. (člen)
Date of acceptance
2012-06-19
Defence
1. Student seznámil členy komise s náplní a cílem bakalářské práce.
2. Byly přečteny posudky na bakalářskou práci.
3. Student akceptoval všechny připomínky oponenta a na všechny otázky odpověděl v plné šíři.
Diskuse:
doc. Márová: Jedná se o jednosubstrátovou reakci ?
Result of defence
práce byla úspěšně obhájena
Document licence
Standardní licenční smlouva - přístup k plnému textu bez omezení