Journal: Microorganisms

Article Title: Differential Processing of a Bacillus subtilis GH5 Endoglucanase During Yeast Surface Display in Saccharomyces cerevisiae

doi: 10.3390/microorganisms14051061

Figure Lengend Snippet: Zymographic analysis of recombinant endoglucanase activity in different cellular fractions of Saccharomyces cerevisiae JARC06. Lane 1 shows the prestained protein molecular weight marker. Lane 2 shows the zymogram of the intracellular (cytoplasmic) enzyme extract. Lane 3 shows the zymogram of the cell wall-associated enzyme fraction released after DTT treatment. Lane 4 shows the zymogram of the enzyme fraction secreted into the culture medium. Lanes 5–7 show SDS–PAGE gel runs parallel with the zymogram and stained with Coomassie Brilliant Blue G-250 to visualize the protein-banding pattern. Lane 5 shows the cytoplasmic protein fraction, lane 6 shows the cell wall-associated proteins, and lane 7 shows the proteins present in the culture supernatant.

Article Snippet: Protein concentrations in the different enzyme extracts were determined using the Bradford method with the Coomassie PlusTM Protein Assay Kit (Thermo Scientific, Carlsbad, CA, USA) [ ], using bovine serum albumin (BSA) as the standard.

Techniques: Recombinant, Activity Assay, Molecular Weight, Marker, SDS Page, Staining

Journal: Microorganisms

Article Title: Differential Processing of a Bacillus subtilis GH5 Endoglucanase During Yeast Surface Display in Saccharomyces cerevisiae

doi: 10.3390/microorganisms14051061

Figure Lengend Snippet: Zymographic analysis of endoglucanase activity in cytoplasmic extracts of Saccharomyces cerevisiae. SDS–PAGE gels copolymerized with 0.5% ( w / v ) carboxymethyl cellulose (CMC) were used to evaluate enzymatic activity. Cytoplasmic extracts from the recombinant strain expressing egl S and the control strain ( S. cerevisiae EBY100 harboring the empty pYD1 vector) were analyzed under identical conditions. Lanes: (1) recombinant strain, (2) control strain, (3) molecular weight marker. ( A , B ) Same gel: ( A ) Coomassie Brilliant Blue staining and ( B ) Congo Red staining showing hydrolytic activity as clear zones. ( C , D ) Independent replicate: ( C ) Congo Red staining and ( D ) subsequent Coomassie staining. Hydrolytic activity is detected exclusively in the recombinant strain.

Article Snippet: Protein concentrations in the different enzyme extracts were determined using the Bradford method with the Coomassie PlusTM Protein Assay Kit (Thermo Scientific, Carlsbad, CA, USA) [ ], using bovine serum albumin (BSA) as the standard.

Techniques: Activity Assay, SDS Page, Recombinant, Expressing, Control, Plasmid Preparation, Molecular Weight, Marker, Staining