Editing Yeast Fermentation (section)

=== Analyzing the enzymatic activity of the recombinant Endo H ===

The hydrolytic activity of Endo H from ''P''. ''pastoris'' and commercial source (heterlogously expressed in ''E''. ''coli'') was compared with carboxypeptidase Y (CPY) and human erythropoietin (EPO) as substrates. About 10 U of Purified recombinant Endo H or commercial Endo H was added to 10 μg of CPY (Sigma, USA) and 10 μg of EPO (Cusabio, China), and incubated in proper buffer at 37°C for 1 h. Meanwhile, 10 U of PNGase F (NEB, USA) was used to deglycosylate EPO under the same condition. All samples were loaded for SDS-PAGE.

RNase B (10 μg) was denatured with 1× Glycoprotein Denaturing Buffer (0.5% SDS, 40 mM DTT) at 100°C for 10 minutes. After this treatment, 1 μl of reaction buffer (50 mM sodium citrate, pH 5.5) was added along with 1 μl of the dilutedrecombinant Endo H-P (5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%); dH<sub>2</sub>O were added to the final volume of 10 μl. The mixture was incubated at 37°C for 1 h. SDS-PAGE was used to detect the hydrolyzed products. One unit (U) of Endo H was defined as the amount of enzyme required to remove >95% of carbohydrates from 10 μg of denatured RNase B in 1 h at 37°C in a total reaction volume of 10 μl (10NEB units = 1 IUB milliunit). The optimum temperature was determined by incubating 10 μg denatured RNase B with 1 μl reaction buffer and in a final volume of 10 μl at the proper temperature for 1 h. The optimum pH was determined by incubating 10 μg denatured RNase B with 1 μl reaction buffer adjusted to a series of pH values from 5.0 to 7.5 in a final volume of 10 μl at 37°C for 1 h. The experiments were performed in triplicate.
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