Recombinant Thaumatin-Like Protein (rTLP) and Chitinase (rCHI) from Vitis vinifera as Models for Wine Haze Formation

Abstract

Cross-linking net aggregates of thermolabile thaumatin-like proteins (TLPs) and chitinases (CHIs) are the primary source of haze in white wines. Although bentonite fining is still routinely used in winemaking, alternative methods to selectively remove haze proteins without affecting wine organoleptic properties are needed. The availability of pure TLPs and CHIs would facilitate the research for the identification of such technological advances. Therefore, we proposed the usage of recombinant TLP (rTLP) and CHI (rCHI), expressed by Komagataella phaffii, as haze-protein models, since they showed similar characteristics (aggregation potential, melting point, functionality, glycosylation levels and bentonite adsorption) to the native-haze proteins from Vitis vinifera. Hence, rTLP and rCHI can be applied to study haze formation mechanisms on a molecular level and to explore alternative fining methods by screening proteolytic enzymes and ideal adsorptive resins.

Peptidomics as a Tool to Assess the Cleavage of Wine Haze Proteins by Peptidases from Drosophila suzukii Larvae

“…We expressed rTLP and rCHI, each bearing a His 6 -tag, in the yeast Komagataella phaffii (formerly Pichia pastoris ). Plasmid vectors (described in Albuquerque et al [ 27 ]) containing the genes encoding a thermolabile TLP (4JRU isoform) and a class IV CHI were introduced into K. phaffii cells by electroporation. The yeast cells were cultured for 4 days in buffered methanol-complex medium (BMMY) with a daily feed of 1% methanol.…”

“…The yeast cells were cultured for 4 days in buffered methanol-complex medium (BMMY) with a daily feed of 1% methanol. The proteins were recovered from the culture medium and purified by chromatography, as previously described [ 27 ]. Briefly, the culture supernatant was loaded onto a 5-mL HiTrap IMAC FF column (Cytiva, Freiburg, Germany) and eluted with 0.2 M phosphate buffer (pH 6.0) containing 250 mM imidazole (Carl Roth, Karlsruhe, Germany) directly into a HiLoad 16/600 Superdex 75 column (Cytiva) mounted on an NGC fast protein liquid chromatography (FPLC) system (Bio-Rad Laboratories, Munich, Germany).…”

“…Therefore, we extracted peptidases from D. suzukii larvae and characterized the purified enzymes to evaluate their ability to cleave haze-forming proteins at pH 3.5. Recombinant TLP and CHI (rTLP and rCHI) were expressed, purified [ 27 ], and digested with the larval peptidases at pH 3.5. The cleaved peptides were detected by LC-MS/MS analysis, and the resulting peptidomics data were analyzed by de novo sequencing.…”

Mass Spectrometry-Based Proteomic Profiling of a Silvaner White Wine

“…In brief, the wine proteome is expected to be highly diverse. Among all grape proteins, a major research focus is on thermolabile proteins, such as thaumatin-like proteins (TLPs) and chitinases (CHIs), which are assumed to be responsible for major economic losses through their key role as wine haze promoters [ 24 , 25 ].…”