The United States of America As Represented by the Secretary of

United States of America, As Represented by the Secretary of Agriculture

Herbert A Wyckoff

Gregory L Cote

Peter Biely

Rodney J Bothast

Bruce S Dien

Robert B Hespell

Recombinant bacteria are disclosed which are transformed with heterologous DNA coding for alcohol dehydrogenase (adh) and pyruvate decarboxylase (pdc), and which are effective for use in the production of ethanol, but which do not require the presence of antibiotics in the culture medium to maintain genetic stability and high ethanol productivity. These recombinant bacteria are produced using mutant host strains which are substantially deficient in the ability to fermentatively reduce pyruvate. When grown in an anaerobic environment, the recombinant pyruvate mutants transformed with the adh and pdc genes are genetically stable, maintaining the inserted genes and ethanol productivity even in the absence of antibiotics.

Stabilization of pet operon plasmids and ethanol production in bacterial strains lacking lactate dehydrogenase and pyruvate formate lyase activities

A new enzyme, alternanase, which is effective for the endo-hydrolytic cleavage of alternan, producing a thinned composition of low molecular weight fractions which exhibit reduced viscosity and increased solubility relative to native alternan, is described. The enzyme is produced and secreted extracellularly by a plurality of novel bacteria isolated from soil. One of the fractions present in the thinned alternan resulting from hydrolysis with alternanase is a the cyclic tetrasaccharide, cyclo{-6)-.alpha.-D-Glcp-(1,3)-.alpha.-D-Glcp-(1,6)-.alpha.-D-Glcp-(1,3)-. alpha.-D-Glcp-(1-}. A novel method for isolating strains of microorganisms which produce endo-.alpha.-D-glucanases such as alternanase effective for the endo-hydrolytic cleavage or thinning of alternan is also described. Cultures of the subject strains are contacted with a test substrate of alternan coupled to a detectable indicator. Detection of released indicator provides an indication of endo-.alpha.-D-glucanase activity.

Bacteria and enzymes for production of alternant fragments

Bacteria and enzymes for production of alternan fragments

Growing community of inventors

Roscoe, IL, United States of America

Herbert A Wyckoff