BISC110: Series 4 Lab 11 Enzymology: Difference between revisions

Yeast make invertase (as well as other enzymes) only when there is an insufficient amount of glucose in the environment. In order to determine whether or not the genes for invertase production are being expressed, you will perform an invertase assay in lab today. Using this assay you will determine the amount of invertase produced by normal, "wild type" yeast grown in YEP+2% glucose (normal glucose medium) and in YEP+0.05% glucose (minimal glucose medium).  

A strain of yeast that is unable to make invertase due to a genetic mutation is also available for investigation.  The inability to produce invertase should adversely affect the growth of the mutant cells in a medium that contains sucrose as the sole sugar.  The mutants should also grow poorly in a medium containing only minimal glucose because, after the small amount of glucose available is exhausted, growth will cease. However, the mutants should grow almost as well as the wild type cells on YEP+2% glucose.  

Based on genetic regulation of invertase gene expression by glucose repression, it is expected that the wild type and mutant strains of yeast will make very little invertase when grown in media containing 2% glucose.  Only the wild type yeast cells are expected to produce invertase when grown in media containing 0.05% glucose.  The mutant yeast strain cannot make invertase when it is growing in minimal glucose medium due to a defective invertase gene.  

Our invertase assay uses a yellow reagent called DNS (43.9 mM 3,5-dinitrosalicylic acid and 250mM NaOH) that changes to an amber color in the presence of reducing sugars, such as glucose and fructose.  Reducing sugars can act as mild reducing agents due to an aldehyde group.  Because sucrose is not a reducing sugar, the activity of the invertase enzyme can be determined by the intensity of the amber color in the reaction tubes after time is allowed for sucrose hydrolysis and subsequent addition of DNS reagent.  Invertase catalyzes the conversion of sucrose into glucose and fructose, which results in the DNS reagent turning amber.  The intensity of the amber color can be measured in a spectrophotometer as absorbance, which is proportional to the amount of glucose and fructose produced during hydrolysis.