MOLECULAR CLONING AND EXPRESSION OF GENES ENCODING PROTEOLYTIC ENZYMES

• Main Authors: VAN DER LAAN; JOHANNES C, MULLENERS; LEO J. S. M, VAN EEKELEN; CHRISTIAAN A. G
• Format: Patent
• Language: English
• Published: 08.06.1993
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS THEREOF; CULTURE MEDIA; ENZYMOLOGY; FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIREDCHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERSFROM A RACEMIC MIXTURE; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PROCESSES USING MICROORGANISMS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

A novel expression cassette comprises, in the direction of transcription, a transcriptional regulatory region and a translational initiation region functional in a Bacillus host cell, a DNA sequence encoding a high alkaline proteolytic enzyme and translational and transcriptional termination regions functional in the host cell, where expression of the DNA sequence is under regulatory control of the initiation and termination regions, and at least one of a marker gene and a temp.-sensitive origin of replication of a bacterial plasmid. Also claimed is a method for transforming alkalophilic Bacillus cells which comprises: (a) pretreating the Bacillus cells with lysozyme in an alkaline medium at 20-37 deg.C to form protoplasts, (b) sepg. the protoplasts from the lysozyme, (c) combining the protoplasts with a DNA construct in the presence of a fusogen at 20-37 deg.C, (d) diluting the fusogen and regenerating the Bacillus cells and (e) growing the regenerated Bacillus cells under selective conditions. Also claimed is a DNA sequence encoding a serine protease originating from Bacillus novo species PB92. Also claimed is an isolated DNA or RNA oligonucleotide comprising at least 15 sequential nucleotides capable of hybridising with a sequence that encodes an amino acid sequence of a high alkaline serine protease obtainable from Bacillus novo species PB92.