Abstract: The invention relates to the novel use of mutants of subtilisin in organic syntheses reactions in non-native environments. Especially the invention relates to methods for the use of mutant subtilisins in organic solvents for the catalysis of reactions involving ester formation and cleavage, including acylations and deacylations, and amidations and deamidations. The methods provide novel strategies which are useful in the synthesis of deoxynucleosides, dideoxynucleosides, peptides, sugars and the like.

Abstract: Cloned DNA is mutated by creating a single-stranded target region in a cloned DNA segment, and introducing a mutation into the single-stranded target region by treating the target region with a chemical or biological mutagenizing agent capable of introducing mutations into single-stranded DNA. The mutated target region then is rendered double-stranded and a microorganism is transformed with the mutated double-stranded DNA present in an expression vector. The transformed microorganism is cultivated under conditions wherein the mutated DNA is expressed to form an expression product, and the expression product is screened to identify a desired mutation in the DNA segment. Mutant subtilisins of enhanced thermal stability are also disclosed.