Identification and Characterization of Components of the Proteolytic Enzyme System of Lactobacillus Helveticus Which Effect Bioactive Peptide Accumulation, Utah State University part

Principal Investigator: Dr. Bart Weimer

Project Summary:

Strains of lactobacilli (60) and brevibacteria (15) were screened for degradation of the alpha-casein 1-23. In Lactobacillus helveticus at least 5 patterns were found. Specific peptides are unique to strains. Each peptide profile is being investigated to determine unique peptides in each strain. Whole caseins are being screened for production of peptides with HPLC/MS. Knockout mutants provided by Jim Steele were screened for peptide production from alpha-casein 1-23, and found that Lb. helveticus CNRZ32 produces at least 2 proteases and that the peptide pattern is a product of those enzymes. Pure peptides with bioactivity have been synthesized and were as standards for further analysis of proteolytic patterns. The gene sequence is partially determined, which will be combined with the hydrolysis patterns to form a classification system. Accumulation of peptides is being determined as the hydrolysis progresses.

Hydrolysis of the alpha-casein 1-23 fragment by lactobacilli was complete within 15 minutes of incubation at 37�C. Identity of the resulting peptides were determined for CNRZ32 and protease deficient mutants by HPLC/MS and co--elution of synthetic peptide standards. Multiple peaks were observed with 11 dominate peptides being produced in the wild type. The most noteworthy is the 1-9 fragment that was produced at high levels in all strains tested. It appears that the substrate is hydrolyzed once in various locations that result in two fragments, opposed to hydrolysis of each fragment in multiple locations. For example, one fragment pair was 1-9 and 10-23. In some cases, one part of the pair was not observed. However, since every peak was not sequenced, it is possible that it was present and not identified. Alternatively, these peptides may have been metabolized by cultures during incubation, but this is unlikely considering the short incubation times used in the assay. These data are ready to be used to define a protease classification system.

The proteolytic system of L. helveticus is significantly different to that of lactococci. This strain contains at least 2 proteases. The first cloned and sequenced has a different substrate binding site and different substrate specificity to that of the lactococcal proteinase. The new proteinase changed the peptide pattern. In the deletion mutant, 3 bitter peptides were not produced, despite the large amount of 1-9 produced by all strains tested (>65 cultures).

Additional comparisons are in progress to group all the species into distinct classifications based on the peptides produced. This will be completed in the next few months.

Publications:

Pederson, J. A., G. J. Mileski, B. C. Weimer, J. L. Steele. 1999. Genetic Characterization of a Cell Envelope-Associated Proteinase from Lactobacillus helveticus CNRZ32. J. Bact. 181:4592-4597.

Theses:

Paul Joseph - Ph.D. candidate

Published Abstract:

Pederson, J. A., G. J. Mileski, B. C. Weimer, J. L. Steele. 1999. Genetic Characterization of a Cell Envelope-Associated Proteinase from Lactobacillus helveticus CNRZ32. 6th Lactic acid bacteria meetings (Veldhoven, The Netherlands). Sept. '99.

Presentations:

Weimer, B. C., P. Joseph, J. Petersen, and J. Steele. 2000. The proteinases of lactobacilli. International Dairy Federation Biennial Cheese Flavor Conference, Banff, Alberta.