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Leuconostoc mesenteroides B-1355 Mutants Producing Alternansucrases Exhibiting Decreases in Apparent Molecular Mass
Abstract:
Mutants of Leuconostoc mesenteroides B-1355 exhibiting decreases in the apparent molecular mass of alternansucrase on sodium dodecyl sulfate (SDS)-polyacrylamide gels stained for enzyme activity were isolated after mutagenizing strain R15 with N-methyl-N(prm1)-nitro-N-nitrosoguanidine. Strain R15 was a UV mutant of strain B-1355 which was enriched for production of alternansucrase. All strains produced principal and minor alternansucrase bands on SDS gels when cultures were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of the principal and minor activity bands on our SDS gels did not result from dextran-enzyme complexes, because mutants constitutive for synthesis of glucosyltransferases (GTFs) on sugars other than sucrose produced activity bands after growth in glucose medium that were the same as those produced after growth in sucrose medium. Dextransucrase, which had been inactivated by heating at 45(deg)C, was reactivated when subjected to SDS-PAGE, showing that our SDS-PAGE conditions were reversibly denaturing. Thermal denaturation at 45(deg)C did not involve a dispersal of GTFs into subunits. Densitometry measurements showed a roughly linear relationship between enzyme activity and band intensity over a loading range of 0.2 to 0.8 mU per sample well. We concluded that SDS-PAGE followed by activity staining was a reliable method for estimating numbers and ratios of GTFs produced by Leuconostoc sp. in media containing sucrose.
Insights
Researchers developed a reliable method using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with activity staining to quantify glucosyltransferases (GTFs) in Leuconostoc species. This technique accurately estimates the numbers and ratios of these crucial enzymes.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Leuconostoc mesenteroides produces alternansucrase, a key glucosyltransferase (GTF).
- Understanding GTF production is crucial for industrial applications.
- Previous methods for analyzing GTF activity and quantity were limited.
Purpose of the Study:
- To develop and validate a reliable method for quantifying GTFs in Leuconostoc.
- To assess the accuracy of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with activity staining for GTF analysis.
- To investigate the properties of alternansucrase and other GTFs during SDS-PAGE.
Main Methods:
- Isolation of Leuconostoc mesenteroides mutants with altered alternansucrase apparent molecular mass.
- Application of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by activity staining.
- Investigation of dextran-enzyme complexes and thermal denaturation effects on GTFs.
Main Results:
- SDS-PAGE with activity staining reliably detected and differentiated principal and minor GTF activity bands.
- The method confirmed that observed bands were not artifacts of dextran-enzyme complexes.
- Reversible denaturation and linear correlation between enzyme activity and band intensity were demonstrated.
Conclusions:
- SDS-PAGE followed by activity staining is a dependable technique for estimating GTF numbers and ratios in Leuconostoc.
- This method provides a robust tool for studying GTF expression and enzyme engineering.
- The findings support the use of this technique for analyzing microbial enzyme production.
