Investigation into the mechanism of action and biological role of Saccharomyces cerevisiae mannoproteins which reduce visible haziness in white wine

Abstract

Heat induced protein haze is a common problem in white wine. Grape derived pathogenesis related proteins slowly denature and aggregate during wine storage and this gives rise to light dispersing haze. Protein haze formation is currently prevented by removing proteins using bentonite, an aluminium silicate clay, but this method has drawbacks. A potential alternative or complementary method is the use of haze protective factors ( HPF ), specific mannoproteins from Saccharomyces cerevisiae that visually reduce protein haze. Hpf1p was originally isolated from Muscat Gordo Blanco wine and Hpf2p from a synthetic grape juice ferment. Based on partial amino acid sequences, putative structural genes, HPF1 and HPF2, for these proteins were identified. HPF1 has a homologue, HPF1 ', ( 71 % similarity ) in S. cerevisiae. Sequence analysis suggests that Hpf1p, Hpf1 ' p and Hpf2p are localised to the cell wall or plasma membrane. This study aimed to determine the biological function of the HPF genes in S. cerevisiae. HPF overexpression and deletion strains were constructed and analysed for cell wall related phenotypes. Under a number of conditions, including cold temperature and ethanol stress, the hpf1 Δ hpf1 ' Δ strain was more tolerant than the wild type strain. However, mating efficiency of the hpf1 Δ hpf1 ' Δ strain was significantly less than the wild type strain and this was found to be correlated with the persistence of a septum between the mating partners. The decreased mating efficiency was also mating type specific, only occurring in MAT α cells. This study also aimed to establish conclusively that the HPF genes do indeed encode proteins with haze protective properties. Haze protective activity of the material from ferment supernatants was assessed. Material from the HPF deletion strains exhibited significantly less haze protective activity than the wild type. Moreover, material derived from HPF1 and HPF1 ' overexpressors was more active than material from the wild type. A 6xHis - tagged Hpf2p was expressed and purified using immobilised metal affinity chromatography. This Hpf2p had significant haze protective activity. Modification of N - glycans of 6xHis - Hpf2p by Endoglycosidase H decreased its haze protective activity.visually reduce protein haze. Hpf1p was originally isolated from Muscat Gordo Blanco wine and Hpf2p from a synthetic grape juice ferment. Based on partial amino acid sequences, putative structural genes, HPF1 and HPF2, for these proteins were identified. HPF1 has a homologue, HPF1 ', ( 71 % similarity ) in S. cerevisiae. Sequence analysis suggests that Hpf1p, Hpf1 ' p and Hpf2p are localised to the cell wall or plasma membrane. This study aimed to determine the biological function of the HPF genes in S. cerevisiae. HPF overexpression and deletion strains were constructed and analysed for cell wall related phenotypes. Under a number of conditions, including cold temperature and ethanol stress, the hpf1 Δ hpf1 ' Δ strain was more tolerant than the wild type strain. However, mating efficiency of the hpf1 Δ hpf1 ' Δ strain was significantly less than the wild type strain and this was found to be correlated with the persistence of a septum between the mating partners. The decreased mating efficiency was also mating type specific, only occurring in MAT α cells. This study also aimed to establish conclusively that the HPF genes do indeed encode proteins with haze protective properties. Haze protective activity of the material from ferment supernatants was assessed. Material from the HPF deletion strains exhibited significantly less haze protective activity than the wild type. Moreover, material derived from HPF1 and HPF1 ' overexpressors was more active than material from the wild type. A 6xHis - tagged Hpf2p was expressed and purified using immobilised metal affinity chromatography. This Hpf2p had significant haze protective activity. Modification of N - glycans of 6xHis - Hpf2p by Endoglycosidase H decreased its haze protective activity.