Related Experiment Videos
Penicillinase-releasing protease of Bacillus licheniformis: purification and general properties
Abstract:
The membrane penicillinase of Bacillus licheniformis 749/C is a phospholipoprotein which differs from the exoenzyme in that it has an additional sequence of 24 amino acid residues and a phosphatidylserine at the NH2 terminus. In exponential-phase cultures, the conversion of membrane penicillinase to exoenzyme occurs at neutral and alkaline pH. An enzyme that will cleave the membrane penicillinase to yield the exoenzyme is present (in small amounts) in exponential-phase cells and is released during their conversion to protoplasts. The enzyme is found in the filtrate of a stationary-phase culture of the uninduced penicillinase-inducible strain 749 and has been purified to apparent homogeneity from this source. The protease has an approximate molecular weight of 21,500 and requires Ca2+ ions for stabilization. It has a pH optimum of 7.0 to 9.5 for hydrolysis of casein and for the cleavage of membrane penicillinase. Both activities are inhibited by diisopropylfluorophosphate; hence, the enzyme is a serine protease. This enzyme may be entirely responsible for the formation of exopenicillinase by this organism, since the other neutral and alkaline proteases of strain 749 have little, if any, activity in releasing exopenicillinase. The enzyme has been termed penicillinase-releasing protease.
Insights
A novel penicillinase-releasing protease was identified in Bacillus licheniformis. This serine protease cleaves membrane penicillinase to form the exoenzyme, crucial for exopenicillinase production.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Bacillus licheniformis 749/C produces membrane-bound and extracellular penicillinase.
- The conversion of membrane penicillinase to exoenzyme is pH-dependent and involves enzymatic cleavage.
Purpose of the Study:
- To identify and characterize the enzyme responsible for converting membrane penicillinase to exoenzyme.
- To elucidate the properties and role of this enzyme in exopenicillinase formation.
Main Methods:
- Purification of the enzyme from stationary-phase culture filtrate.
- Characterization of protease activity, including pH optimum, molecular weight, and cofactor requirements.
- Inhibition studies using diisopropylfluorophosphate to determine enzyme class.
Main Results:
- A serine protease, termed penicillinase-releasing protease, was purified.
- The enzyme has a molecular weight of ~21,500, requires Ca2+ for stability, and has a pH optimum of 7.0-9.5.
- This protease specifically cleaves membrane penicillinase to yield exoenzyme and is responsible for exopenicillinase formation.
Conclusions:
- Penicillinase-releasing protease is the key enzyme for generating exopenicillinase from its membrane-bound precursor in Bacillus licheniformis.
- The enzyme's characteristics suggest a specific role in bacterial cell wall metabolism and enzyme secretion.