Studies Of Microencapsulated α-galactosidase In Saccharomyces Cerevisiae Spores

In response to nitrogen starvation in the presence of a non-fermentive carbon source, diploid cells of the Saccharomyces cerevisiae undergo meiosis package the haploid nuclei produced in meiosis into spores. The spore wall has a muti-layer structure which consists of mannan, β-glucan, chitosan and dityrosine from the innermost layer to outmost layer. Saccharomyces cerevisiae spore wall has pore size. As mutants defective in spore wall defected mutants could make spore wall loose and increase the substrate permeability, the enzymatic activity of the target protein on the spores could be improved. Therefore, we knocked out the spore wall related genes to adjust its pore size. As a result, the activity of encapsulated enzyme was increased. In order to screen mutants with the highest activity, the multicopy plasmid pRS426-TEFpr-MEL1 expressing α-galactosidase(Mel1p) enzyme was transformed into the S. cerevisiae wild type and mutants, and the activity assay of the encapsulated enzyme showed that osw2? mutant has highest catalytic activity. Moreover, stability test suggested that the encapsulated enzyme of osw2? mutant was stable. Therefore, the osw2? mutant was the best candidate for enzyme encapsulation.It was reported that OSW2 gene participated in the assembly of spore wall, but the detailed function of OSW2 gene is unclear. To study the pore sizes of AN120 wild type and osw2? mutant spore wall, three different oligosaccharides(melibiose, raffinose and stachyose) were used as substrates to detect the catalytic activity of encapsulated α-galactosidase(Mel1p) on yeast spores. The results indicated that the pore size of AN120 wild-type spore wall was larger than trisaccharide but smaller than tetrasaccharide; however, the pore size of osw2? mutant yeast spore wall was larger than tetrasaccharide but smaller than pentasaccharide probably.One of advantages for spores as carriers for enzyme enzyme encapsulation is that the unique structures of the spore wall will provide protection for the encapsulated enzymes. To study the protections of spores, AN120 wild type and osw2? mutant spores containing encapsulated enzymes were treated by 2% and 10% SDS using free enzyme as a control. The results suggested that enzyme encapsulated on wild type and osw2? mutant spores could completely resist 2% SDS treatment and partially resist 10% SDS treatment, while the free enzymes completely lost its activity.Urea is a low molecular weight compound and can make protein denatured. To test whether encapsulated enzyme on spores can resist urea treatment, wild-type and osw2Δ mutant spores containing encapsulated enzyme were treated by 8 mol·L-1 urea. Wild type spore can resist the treatment by 8 mol·L-1 urea, whereas osw2? mutant spore was sensitive to urea. Furthermore, western blot analysis showed that the amounts of enzyme in the wild type spores were stable after urea treatment, but the amounts of enzyme on osw2? mutant spores decreased significantly. These results demonstrated the enzyme encapsulated on osw2? mutant spores enzyme could not resist the treatment of urea compared with wild type spores. The possible reason was that the spore wall of osw2? mutant is weaker than wild type spore wall.