Partial purification and characterization of polygalacturonases of Geotrichum candidum.

Abstract

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