Abstract
In alcohol manufacturing, glucoamylase (GA, 1, 4-α-D-glucan-glucohydrolase, EC3.2.1.3) is widely used for starch saccharification. Many scientists are interested in producing this enzyme by microorganisms to approach enormous quantity and high quality. Due to the enzyme is an extracellular one and can be secreted into the medium, we tried to analyze its signal peptide by mutation screening. The gene encoding glucoamylase from Rhizopus oryzae was cloned into a yeast-compatible vector, and the secretion of GA was investigated.This study was focused on the screening method. Based on the nature of glucoamylase, we have developed two methods to assay the mutants of GA. It was observed that a colony of yeast with glucoamylase on a starch plate would produce clear zone once GA was secreted. Bigger clear zones corresponded to better GA secretion in a colony. The other method was the in vitro activity assay. Using fructose instead of glucose as the carbon source for yeast growth, we could test the glucoamylase activity by measuring the concentration of glucose in the medium.According to the report by Perlman, D., and Halvorson, H. O. in 1983 (21), there are three domain, the N-domain, the H-domain, and the C-domain, in a secretory signal peptide. We introduced rational mutations in the area to see the features of these domains. One clone of our interest was identified with no secretion in expression of yeast. Besides the two negatively-charged residues introduced (Gln to Asp? and a Tyr to Glu?), the change of the cleavage location recognized by signal peptidase may be the reason for the down secretion.