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NH2-terminal processing of Bacillus subtilis alpha-amylase
1Department of Molecular Biology, National Institute of Agrobiological Resources, Ibaraki, Japan.
The Journal of Biological Chemistry
|August 15, 1988
Summary
Bacillus subtilis alpha-amylase undergoes proteolytic maturation after secretion. This process involves at least two enzymes, with specific steps influenced by EDTA, calcium, and PMSF, revealing key insights into enzyme processing.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Bacillus subtilis alpha-amylase is secreted as a precursor and processed to its mature form.
- Processing involves the removal of an N-terminal sequence, but the exact mechanism and enzymes are not fully elucidated.
Purpose of the Study:
- To investigate the post-secretory processing mechanism of Bacillus subtilis alpha-amylase.
- To identify the factors and enzymes involved in the proteolytic maturation of alpha-amylase.
Main Methods:
- Cloning and expression of B. subtilis N7 alpha-amylase in B. subtilis.
- Analysis of extracellular alpha-amylase forms using native gel electrophoresis.
- Investigating the effects of EDTA, Ca2+, and phenylmethylsulfonyl fluoride (PMSF) on processing.
Main Results:
- Different forms of alpha-amylase (N34, N40, N42) were identified, with N34 being major in log phase and N40/N42 in stationary phase.
- Conversion of larger to smaller forms occurred in culture supernatants and partially purified samples.
- The N34 to N40 conversion was inhibited by EDTA and PMSF, while N40 to N42 was facilitated by Ca2+.
Conclusions:
- Proteolytic maturation of alpha-amylase occurs post-secretion.
- At least two distinct enzymatic processes are involved in the maturation.
- The signal peptidase cleavage site is likely between positions 30 and 35, with further processing occurring subsequently.