Summary
Penicillin acylase genes from Escherichia coli 194, of an overproducing mutant (194-3) of this strain, and of a similar overproducing mutant of Bacillus megaterium UN1 were cloned in E. coli DH1 on the plasmid vector pACYC184. The sizes of chromosomal DNA fragments essential for penicillin acylase production were found by Tn1000 mutagenesis and in vitro deletions to be between 2.2 and 2.5 kb in the case of both E. coli genes and between 2.3 and 2.7 kb in the case of the mutant Bacillus gene. Restriction mapping indicated substantial sequence differences between the E. coli and B. megaterium penicillin acylase genes. Enzyme production in E. coli recombinants from both overproducing mutants was found to be constitutive and higher than in the original strains. The Bacillus penicillin acylase was produced intracellularly in E. coli recombinants, which is in contrast to the normal extracellular production of this enzyme in B. megaterium. Recombinant plasmids containing penicillin acylase genes from either source were found to be unstable in the absence of selection pressure for retention of the vector.
Similar content being viewed by others
References
Balasingham K, Warburton D, Dunnill P, Lilly MD (1972) The isolation and kinetics of penicillin amidase from Escherichia coli. Biochim Biophys Acta 276:250–256
Bruns W, Hoppe J, Tsai H, Bruning HJ, Maywald F, Collins J, Mayer H (1985) Structure of the Penicillin acylase gene from Escherichia coli: A periplasmic enzyme that undergoes multiple proteolytic processing. J Molec Appl Genet 3:36–44
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Chiang C, Bennette RE (1967) Purification and properties of penicillin amidase from Bacillus megaterium. J Bacteriol 93:302–308
Close TJ, Rodriguez RL (1982) Construction and characterization of the chloramphenicol resistance gene cartridge: a new approach to the transcriptional mapping of extrachromosomal elements. Gene 20:305–316
Guyer MS (1978) The γ-Δ sequence of F is an insertion sequence. J Mol Biol 126:347–365
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Humphreys GO, Willshaw GA, Anderson ES (1975) A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta 383:457–463
Kutzbach C, Rauenbusch E (1974) Preparation and general properties of crystalline acylase from Escherichia coli ATCC 11 105. Z Physiol Chem 355:45–53
McCullough JE (1983) Gene cloning in bacilli related to enhanced penicillin acylase production. Biotechnology 1:879–882
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, USA, pp 458–459
Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3:208–218
Mayer H, Collins J, Wagner F (1979) Cloning of the penicillin G acylase gene of Escherichia coli ATCC 11 105 on multicopy plasmids. In Timmis KN, Puhler A (eds), Plasmids of medical, environmental and commercial importance. Elsevier/North Holland Biomedical Press, Amsterdam, pp 459–470
Mayer H, Collins J, Wagner F (1980) Cloning of the penicillin G acylase gene of Escherichia coli ATCC 11 105 on multicopy plasmids. In Weetall HH, Royer GP (eds) Enzyme engineering, vol. 5. Plenum Publishing Co., New York, pp 61–69
Meevootisom V, Somsuk P (1983) Penicillin acylase of Escherichia coli. J Sci Soc Thailand 9:143–152
Meevootisom V, Somsuk P, Prachaktam R, Flegel T (1983) Simple screening methods for isolation of penicillin acylase producing bacteria. Appl Environ Microbiol 46:1227–1229
Oliver G, Valle F, Rosetti F, Gomez-Pedrozo M, Santamaria P, Gosset G, Bolivar F (1985) A common precursor for the two subunits of the penicillin acylase from Escherichia coli ATCC 11 105. Gene 40:9–14
Szentirmai A (1964) Production of penicillin acylase. Appl Microbiol 12:185–187
Theeragool G (1985) Studies on penicillin acylase of Bacillus megaterium strain UN1 and some applications for future industrial use. M. Sc Thesis Mahidol University, Bangkok, Thailand
Vandamme EJ, Voets JP (1974) Microbial penicillin acylases. Adv Appl Microbiol 17:311–369
Vojtisek V, Slezak J (1975) Penicillin amidohydrolase in Escherichia coli: II Synthesis of the enzyme, kinetics and specificity of its induction and the effect of O2. Folia Microbiol 20:289–297
Wood WB (1966) Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. J Mol Biol 16:118–133
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Meevootisom, V., Saunders, J.R. Cloning and expression of penicillin acylase genes from overproducing strains of Escherichia coli and Bacillus megaterium . Appl Microbiol Biotechnol 25, 372–378 (1987). https://doi.org/10.1007/BF00252550
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00252550