Thermogen Inc.

Veronika Vonstein

Malcolm J Casadaban

David C Demirjian

Nikos C Pagratis

J Mark Weber

Michael Fonstein

John Aikens

Larry Allen

David DeMirjian

We have developed a new gene transfer system for extreme thermophiles of the genus Thermus, including ., using a chromosomal gene, and a thermostable derivative of the kanamycin-resistance gene (kan ). A plasmid mediated gene-replacement process is used to insert it into the chromosome resulting in the production of Leu Km transformants. This system not only allows stable, single-copy gene insertion into the chromosome of an extreme thermophile, but can be used in the thermo-genetic process described here to generate thermo-stabilized enzymes and proteins for industrial processes. This host-vector environment makes it possible to generate further thermo-stabilizing mutations in the kan gene beyond those levels previously reported.

Recombinant DNA and methods for producing thermostable enzymes

The instant invention encompasses isolated stable esterase enzymes characterized by the ability to remain stable at certain temperatures, substrate specificities, and activity profile.

Stable biocatalysts for ester hydrolysis

The instant invention encompasses isolated stable esterase enzymes characterized by the ability to remain stable at certain temperatures, substrate specificities, and activity profile; the expression vectors which can express, nucleic acids which encode for, and corresponding protein amino acid sequence of such proteins.

We have developed a new gene transfer system for extreme thermophiles of the genus Thermus, including Thermus flavus., using a chromosomal gene, and a thermostable derivative of the kanamycin-resistance gene (kan.sup.tr2). A plasmid mediated gene-replacement process is used to insert it into the chromosome resulting in the production of Leu.sup.- Km.sup.r transformants. This system not only allows stable, single-copy gene insertion into the chromosome of an extreme thermophile, but can be used in the thermo-genetic process described here to generate thermo-stabilized enzymes and proteins for industrial processes. This host-vector environment makes it possible to generate further thermo-stabilizing mutations in the kan gene beyond those levels previously reported.

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Chicago, IL, United States of America

Veronika Vonstein