Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis
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Abstract:
The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli. The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E. coli and Bacillus subtilis. The expression in B. subtilis carrying the recombinant plasmid is approximately two times higher than in the original B. sphaericus strain. A comparison of the purified enzyme from B. sphaericus and the expressed gene product in E. coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000.
Insights
The Bacillus sphaericus gene for penicillin V amidase was cloned into E. coli. This enzyme
Area of Science:
- Biotechnology
- Molecular Biology
- Enzymology
Background:
- Penicillin V amidase is crucial for producing 6-aminopenicillanic acid.
- Understanding its genetic basis facilitates biotechnological applications.
Purpose of the Study:
- To isolate and characterize the Bacillus sphaericus gene encoding penicillin V amidase.
- To enable heterologous expression and analysis of the enzyme.
Main Methods:
- Molecular cloning of the penicillin V amidase gene from B. sphaericus into Escherichia coli.
- Gene expression analysis in E. coli and Bacillus subtilis.
- Enzyme characterization through SDS-PAGE.
Main Results:
- The penicillin V amidase gene was successfully isolated on a 2.2-kilobase DNA fragment.
- The gene expressed functional penicillin V amidase in both E. coli and B. subtilis.
- Expression levels in B. subtilis were approximately double those in the native B. sphaericus strain.
- The native enzyme comprises four identical subunits, each with a molecular weight of 35,000.
Conclusions:
- The B. sphaericus penicillin V amidase gene can be effectively cloned and expressed in heterologous hosts.
- Enhanced expression is achievable in B. subtilis, offering potential for improved industrial production.
- The enzyme's quaternary structure was elucidated.