Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis.

Applied and Environmental Microbiology
C KlessenH Malke

Abstract

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased ...Continue Reading

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Jan 1, 1996·Journal of Basic Microbiology·J GumpertC Hoischen
Mar 21, 2001·Biotechnology & Genetic Engineering Reviews·L M BabéB F Schmidt
Nov 24, 2016·Nature Communications·Patrick StuderMartin J Loessner
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Oct 11, 2003·Biotechnology Advances·A K MohantyV K Batish

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