Summary
Col E1 hybrid plasmids carrying the phoA + structural gene of alkaline phosphatase, a periplasmic enzyme of Escherichia coli K-12, were identified from the Clarke and Carbon genomic bank. Wild-type (lky +) phoA + plasmid-bearing strains synthesized 14 times more intracellular enzyme than the haploid lky + strain. Phosphate-induced repression was maintained in transformed strains.
PhoA + plasmids carrying the phoB and phoR regulatory genes were introduced into a periplasmic-leaky (lky) recipient strain able to release alkaline phosphatase into the extracellular medium. Transformed lky mutants excreted up to 90% of total enzyme activity which corresponded to 3.5 times the amount of intracellular alkaline phosphatase made by the haploid lky + strain. The protein composition analysis of periplasmic and extracellular fractions showed that: (i) wild-type phoA + hybrid plasmidbearing clones did not excrete alkaline phosphatase but had a modified periplasmic content; (ii) alkaline phosphatase was the major excreted protein by transformed lky mutants. The use of periplasmic-leaky phoA + hybrid plasmid-bearing mutants for an easier production and purification of alkaline phosphatase is discussed.
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Lazzaroni, JC., Portalier, R. Production of extracellular alkaline phosphatase by Escherichia coli K-12 periplasmic-leaky mutants carrying phoA + Plasmids. European J. Appl. Microbiol. Biotechnol. 16, 146–150 (1982). https://doi.org/10.1007/BF00500744
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DOI: https://doi.org/10.1007/BF00500744