R Mørkeberg | PubMed Health

PubMed1995

Induction and repression of alpha-amylase production in batch and continuous cultures of Aspergillus oryzae.

The intra- and extracellular concentrations of alpha-amylase in Aspergillus oryzae have been measured during batch culture of a wild-type strain and two recombinant strains. The mean intracellular level for the two recombinant strains was about four to five times the level of the wild-type strain. The recombinant strains also had a higher alpha-amylase productivity, whereas the residence time of the intracellular alpha-amylase pool was approximately the same for the three strains. At high glucose concentrations there was a low constitutive synthesis of alpha-amylase, whereas at low glucose concentrations derepression resulted in an increased production rate. Shifts from a glucose- to a maltose-limited chemostat showed that maltose induces both the production and secretion of alpha-amylase. Finally, from immunoblots, both a glycosylated and an unglycosylated alpha-amylase have been detected.

Amidohydrolases↗

PubMed1994

Efficient sampling of protein sequence space for multiple mutants.

We describe here a method capable of generating a very large population of multiple mutants, the size of which is primarily limited by volume constraints. This method, referred to as recombination-enhanced mutagenesis, combines the power of in vitro mutagenesis with the high frequencies of in vivo recombination that can be achieved using single-stranded transduction systems. The recombination frequency between two mutations separated by as little as 19 amino acids is 0.02; this frequency approaches a value of 0.1 for mutations separated by more than 38 amino acids. Up to 10(8) independent recombinants were generated in 1 ml of an E. coli culture, and this number scales linearly (or better) with increasing volume. To prove the method's effectiveness, we applied it to the problem of reverting multiple mutants of mouse dihydrofolate reductase, which could not be reverted using mutagenesis alone. Thus, given an appropriate screen or selection scheme, recombination-enhanced mutagenesis is well-suited for addressing a range of combinatorially complex problems, such as antigen recognition, enzyme catalysis, protein folding, and transport/transduction across biomembranes.

Animals↗