No abstract

A protocol combining recombination PCR and long-distance PCR is demonstrated to be highly accurate and rapid for site-directed mutagenesis of large (> 10 kb) plasmids. Application of this protocol to the generation of mutant rabies virus glycoproteins expressed by the baculovirus/insect cell system illustrates the usefulness of this approach in facilitating structure-function relationships in this important eukaryotic expression system.

Corn oil, peanut oil, sunflower oil, soybean oil, canola oil, olive oil and butter oil were tested as replacements for nonaqueous solvents in the lipase mediated biosynthesis of ethyl butyrate, butyl butyrate, isoamyl butyrate and isoamyl acetate. The most effective oils tested were butter oil, soybean oil, peanut oil and olive oil (yield = 50–60% after 16 h incubation at 25°C) while corn oil was least effective (yield = 38%). Higher yields were obtained, for all ester syntheses, at a reaction temperature of 50°C. Drying agents were shown to have no effect or to inhibit the synthesis. Of the four enzymes tested, Candida cylindracea lipase was the most effective enzyme for ethyl butyrate synthesis.