Study On Disruption Of Saccharomyces Cerevisiae PEP4 Gene

The stability of beer foams is one of the most difficult problem in the beer quality control nowdays. How to improve the stability of beer foam has become focus in the beer industry.Decrease in beer foam stability due to proteinase A activity has been reported by several research groups. Proteinase A from brewers'yeast may digest wort proteins responsible for the beer foam stability, leaving peptides with reduced or no foaming properties and, hence, leading to a final product with poor foam stability.Proteinase A is a vacuole aspartic protease encoded by the PEP4 gene (1218bp encoding a protein of 405 aminoacids).In this paper,the new plasmid for disruption of the Saccharomyces cerevisiae PEP4 gene was constructed,and it containing the marker module flanked by 40-41 bp of DNA homologous to the genomic target locus and KanMX molecules is used to screen out transformants. This new plasmid was transformed into the Saccharomyces cerevisiae cell and the mutants lacking proteinase A were isolated after the mutant strains were verified by resistance, PCR product, single site restrication enzyme single and APE overlay test for CPY activity.The mutant strain inheritance and ferment stability is well and the time of foam stability is 1.5min longer than the initial strain. And the same time, we test every guideline of liquid made by brewing respectively. After be domesticated, the mutant strain was carried out experiments of fermentation. In the meantime, we respectively test the activity of Proteinase A about the liquid by brewing of the initial strain and the mutant strain through advanced experimental method of Proteinase A in the world. At last, we found that the activity of Proteinase A in the liquid by brewing of the mutant strain decreased 33.2%～49.8% in the end through comparing to the liquid by brewing of the initial strain.