Amidases, nucleic acids encoding them and methods for making and using them

Abstract

The present invention provides amidases, polynucleotides encoding the amidases, methods of making and using these polynucleotides and polypeptides. In one aspect, the invention provides enzymes having secondary amidase activity, e.g., having activity in the hydrolysis of amides, including enzymes having peptidase, protease and / or hydantoinase activity. In alternative aspects, the enzymes of the invention can be used to used to increase flavor in food (e.g., enzyme ripened cheese), promote bacterial and fungal killing, modify and de-protect fine chemical intermediates, synthesize peptide bonds, carry out chiral resolutions, hydrolyze Cephalosporin C. The enzymes of the invention can be used to generate 7-aminocephalosporanic acid (7-ACA) and semi-synthetic cephalosporin antibiotics, including caphalothin, cephaloridine and cefuroxime. The enzymes of the invention can be used as antimicrobial agents, e.g., as cell wall hydrolytic agents. The invention also provides a fluorescent amidase substrate comprising 7-(epsilon-D-2-aminoadipoyladipoylamido)-4-methylcoumarin.

Description

Enzymes with secondary amidase activity and methods of use thereof technical field The present invention relates generally to molecular and cellular biology and biochemistry. The present invention provides amidase, polynucleotides encoding the amidase, applications of these polynucleotides and polypeptides, and, on the one hand, the present invention provides secondary amidase (secondaryamidase) activity such as having amidase Enzymes with hydrolytic activity include enzymes with peptidase, protease and / or hydantoinase activity. In other aspects, the enzymes of the invention can be used in food processing, e.g., to enhance food flavor (e.g., enzymatically ripened cheese), to facilitate the killing of bacteria and fungi, to modify and deprotect fine chemical intermediates, to synthesize peptide bonds, Perform chiral resolution and hydrolyze cephalosporin C. The enzymes of the invention can be used to produce 7-aminocephalosporanic acid (7-ACA) and semi-synthetic cephalospori...

AI Technical Summary

Problems solved by technology

The search for secondary amidases is limited by a lack of substrate / assay combinations suitable for high-throughput screening

Previous research efforts have used substrates or assays that suffer from low throughput, lack of sensitivity, and / or lack of specificity

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