Anchored Expression Of Saccharifying Amylase In Saccharomyces Cerevisiae

Saccharifying amylase are widely used in the food and brewing industy, which are ableto directly hydrolyze starch into maltose or glucose. Since absence of saccharifying amylasegene in Saccharomyces cerevisia cells, they can not utilize starch as substrate. In recent years,with the development of molecular biology technology, construction of recombinant S.cerevisiae which could directly digest starch was attracted extensively, among which yeastsurface display technology was paid much attention. To employ the yeast cell efficientlyhydrolyzing starch material, two saccharifying amylase were anchored on the cell surface of S.cerevisiae SA. The surface display technology of these enzymes fulfilled the digestion ofstarch by recombinant yeast cell.In my study, fungal α-amylase gene from Rhizopus oryzae and β-amylase gene frombarley were anchored on the cell surface of S. cerevisiae by using the α-agglutinin system.They expressed on cell surface of S. cerevisiae and the recombinant strains of SAA and SBAwere obtained, the activities of the surface displayed amylase were found to be128U/g drycell and131U/g dry cell respectively.Secondly, the property of the surface displayed amylases anchored on the cell surface ofthe S. cerevisiae strains were characterized. The optimal reaction temperature of fungalα-amylase and β-amylase was55℃and50℃, respectively. The optimal reaction pH wasbetween5.0-5.5, and both of them showed high stability below60℃. With the temperaturekept at30℃for1h, the cell-surface displayed β-amylase maintained its stability betweenpH3.0-10.0and showed90%enzymatic activity. The surface displayed fungal α-amylasemaintained excellent stability between pH4.0-7.5. It was also observed that the concentrationof5.0mmol/L Fe3+had strong inhibition on these two enzymes.Lastly, further experiments were carried out to explore the preliminary applications ofthe recombinant S. cerevisiae. The recombinant strain SAA and SBA were able to make use ofstarch as the carbon source to grow and fermentation. As a new-type of regulated enzyme, thiswhole-cell catalyst was used in the production of maltose syrup. A more than45%maltosesyrup was abtained from20%maltodextrin by using surface displayed β-amylase per gram ofsubstrate for five times repeat after incubation at pH5.0and40℃for24h. The recombinantstrains SAA expressing of cell-wall anchored fungal α-amylase and SBA expressing ofcell-wall anchored β-amylase obtained were studied through process of brewing with mediumcontaining starch. It was observed that the recombinant S. cerevisiae had good fermentingproperties. They are able to reduce residual sugar effectively in fermentation broth, andincrease the yield of alcohol, which holds significant economic benefits for improving theutilization ratio of raw materials.