Increased Synthesis and Secretion of Recombinant Barley a-Amylase by Saccharomyces cerevisiae in Glycerol-enriched Medium

摘要

High-throughput screening of barley α-amylase mutants expressed in Saccharomyces cerevisiae was hampered by the interference of the glucose used in YEPD. An alternative carbon source was investigated, so that enzyme activity could be monitored without affecting the expression and secretion of the recombinant enzyme. The !!!-amylase cDNA gene was isolated from a barley cDNA library and cloned into pYEX yeast expression vector. Transformants were cultured in YEPD or YEPG, and enzyme activity and cell density were monitored at various time intervals. Proteins in yeast extract and culture medium were analyzed by SDS-PAGE. Western blot of intra- and extracellular proteins was sequentially probed with anti-amylase antibody and anti-rabbit HRP conjugate, followed by chemiluminescent detection. The enzyme activity of recombinant barley a-amylase secreted by the yeast clone DY150[pYEX-Amyl] showed a significant increase when the culture medium included glycerol as the carbon source. The enhancement reached a 4.5 fold increase at 120 hr, and the effect was strain nonspecific. Intra- and extracellular proteins increased significantly with time in both the yeast clone and the control grown in YEPG. Proteins in YEPD and YEPG cultures showed very different band patterns, indicating that the secretory pathway was altered. Western blotting indicates that the recombinant amylase accumulated inside yeast cells, at a relatively low level, compared with that in the culture medium. Using YEPG medium consistently increased synthesis and secretory efficiency of the recombinant enzyme. More than 90% of the synthesized recombinant enzyme was secreted, resulting in very little intracellular accumulation. This investigation demonstrates that the use of glycerol as a carbon source for S. cerevisiae allows direct monitoring of α-amylase activity in yeast clones. It also enhances synthesis and secretion of the recombinant enzyme while suppressing cell growth.