Subtilin variant of enhanced stability and activity
AssigneeUniversity of Maryland
InventorJ. Norman Hansen
Life of this patent
Estimating the term…
Abstract
A subtilin mutant substituting isoleucine for Glu.sub.4 of the native sequence (SEQ ID NO: 7) exhibits a 57-fold improvement in stability, resisting modification of the dehydroalanine residue at position 5. This stable mutant exhibits 3-4 times the specific activity, in suppression of bacterial spore outgrowth, of the native bacteriocin. A method for site-specific mutagenesis, as well as the resulting mutant gene, plasmid and transformant is similarly set forth.
Claims11
An isolated gene encoding a polypeptide which, when expressed and processed in Bacillus subtilis, consists of the amino acid sequence of native subtilin, but in which the 4-position is an isoleucine residue.
An isolated gene encoding a polypeptide which, when expressed in Bacillus subtilis, is an analog of native subtilin in which the 4-position is an isoleucine residue and which has at least 10-fold superior stability and 3-fold superior activity than native subtilin.
An isolated gene encoding a polypeptide which consists of the Amino Acid sequence of SEQ ID NO:7:
- Met Ser Lys Phe Asp Asp Phe Asp Leu Asp Val Val Lys Val Ser Lys Gln Asp Ser Lys Ile Thr Pro Gln Trp Lys Ser Ile Ser Leu Cys Thr Pro Gly Cys Val Thr Gly Ala Leu Gln Thr Cys Phe Leu Gln Thr Leu Thr Cys Asn Cys Lys Ile Ser Lys.
A method of transforming Bacillus subtilis with a mutant subtilin gene, comprising:
- (A) replacing the native subtilin gene consisting essentially of a selectable marker gene such that the transformed host is unable to produce subtilin, and
- (B) subsequently replacing said selective marker gene with said mutant subtilin gene.
Dependencies are inferred from the claim text.