Electroextraction of Insoluble Proteins from the Organic Matrix of the Nacreous Layer of the Japanese Pearl Oyster, Pinctada fucata.

The nacreous layer of shells and pearls is composed of aragonite crystals arranged in an organic matrix. The organic matrix contains chitin and several proteins that regulate the formation of the nacreous layer. Owing to their strong interactions in the organic matrix, the current method for extraction of insoluble proteins from the pre-powdered nacreous layer involves heating to high temperatures in the presence of a detergent (e.g., sodium dodecyl sulfate, SDS) and reductant (e.g., dithiothreitol, DTT), which is likely to induce protein degradation. Therefore, we have developed an electroextraction method to isolate proteins from the organic matrix of a nacreous organic sheet, that was obtained following the decalcification of shells in their original shape. Our electroextraction method employs milder conditions without heating or detergent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the electro-extracted proteins (EEPs) under non-reduced and reduced conditions revealed that this method yielded a greater number of different proteins compared with the conventional extraction method and the isolated EEPs retained their disulfide bonds. Our method is able to easily extract insoluble proteins from the nacreous layer under mild conditions and will undoubtedly aid future analyses into the functions of the nacreous layer proteins.

Overexpression of PkINO1 improves ethanol resistance of Pichia kudriavzevii N77-4 isolated from the Korean traditional fermentation starter nuruk.

The yeast Pichia kudriavzevii N77-4 was isolated from the Korean traditional fermentation starter nuruk. In this study, fermentation performance and stress resistance ability of N77-4 was analyzed. N77-4 displayed superior thermotolerance (up to 44°C) in addition to enhanced acetic acid resistance compared to Saccharomyces cerevisiae. Moreover, N77-4 produced 7.4 g/L of ethanol with an overall production yield of 0.37 g/g glucose in 20 g/L glucose medium. However, in 250 g/L glucose medium the growth of N77-4 slowed down when the concentration of ethanol reached 14 g/L or more and ethanol production yield also decreased to 0.30 g/g glucose. An ethanol sensitivity test indicated that N77-4 was sensitive to the presence of 1% ethanol, which was not the case for S. cerevisiae. Furthermore, N77-4 displayed a severe growth defect in the presence of 6% ethanol. Because inositol biosynthesis is critical for ethanol resistance, expression levels of the PkINO1 encoding a key enzyme for inositol biosynthesis was analyzed under ethanol stress conditions. We found that ethanol stress clearly repressed PkINO1 expression in a dose-dependent manner and overexpression of PkINO1 improved the growth of N77-4 by 19% in the presence of 6% ethanol. Furthermore, inositol supplementation also enhanced the growth by 13% under 6% ethanol condition. These findings indicate that preventing downregulation in PkINO1 expression caused by ethanol stress improves ethanol resistance and enhances the utility of P. kudriavzevii N77-4 in brewing and fermentation biotechnology.