Production of extracellular alkaline phosphatase by Escherichia coli K-12 periplasmic-leaky mutants carrying phoA + Plasmids

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.