反应详情

Culture supernatants were sampled at approximately 50 and 100 hours, treated with 2 mM PMSF final concentration, and frozen. To estimate NOVAMYL™ expression, supernatants were mixed with an equal volume of 2X Laemmli loading buffer, immediately boiled, and loaded on 14% or 8-16% acrylamide TRIS-glycine gels purchased from a commercial source (NOVEX, San Diego, Calif.). Known amounts of a NOVAMYL™ standard were also loaded on the same gel to estimate the amount of NOVAMYL™ produced. In some cases, NOVAMYL™ titer was determined using maltotriose as substrate. Specifically, a sample of the enzyme is incubated with maltotriose at pH 5.0 and 37° C. for 30 minutes. The reaction is then stopped by adjusting the pH to approximately 11. The amount of glucose produced from the breakdown of maltotriose to glucose and maltose is then measured with glucose dehydrogenase and NADH at 340 nm under standard conditions. Known amounts of a NOVAMYL™ standard (Novo Nordisk A/S, Bagsvaerd, Denmark) are also run.

1. aliquotCulture supernatants were sampled at approximately 50 and 100 hours
2. concentrationconcentration
3. temperaturefrozen
4. additionwere mixed with an equal volume of 2X Laemmli loading buffer
5. customproduced