Cloning of 1-aminocyclopropane-1-carboxylate deaminase gene from soil microorganism
1995
Theerachai Thanananta
From collected soil samples, 55 isolates of bacteria including 8 species were capable of growing on 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to alpha-ketobutyrate and ammonia. Among these bacterial isolates Pseudomonas fluorescens strain J2 had the highest growth rate. The chromosomal DNA of Pseudomonas fluorescens strain J2 was purified, cut with Sau3AI, inserted into BamHI site of pGEM-3Zf(+) plasmid and cloned in Escherichia coli strain XL-1-Blue. Six clones with high growth rate on the medium with ACC and ampicillin were selected for further investigation. ACC deaminase assay, restriction endonuclease digestion patterns, and hybridization result indicated that all of plasmid clones contained an overlapping region of ACC deaminase gene from the J2 genome. However, ACC deaminase activity of Escherichia coli containing smaller plasmid clones were less active than those with larger plasmid clones and all of the transformed Escherichia coli had lower ACC deaminase activity than that of Pseudomonas fluorescens strain J2. This might have been due to the incompletion of ACC deaminase gene or some factors from host cell or plasmid vector used.
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