Determination of the biological origin of enzyme preparations using SDS-PAGE and peptide mass fingerprinting

Abstract

Enzymes are mainly extracted from the culture broth of microorganisms. Various commercially available enzyme preparations (EPs) are derived from different microorganisms, and the source of the EP should be the same as that mentioned in the manufacture’s information. The development of analytical methods that can determine the origin of the final products is important for ensuring that the EPs are nontoxic, especially when used as food additives. In this study, various EPs were subjected to SDS-PAGE, and the main protein bands were excised. After in-gel digestion, the generated peptides were analysed using MALDI-TOF MS, and protein identification was performed by searching the set of peptide masses against protein databases. In total, 36 EPs including amylase, β-galactosidase, cellulase, hemicellulase and protease were analysed, and the information about the enzyme sources was obtained for 30 EPs. Among these, the biological sources determined for 25 EPs were consistent with the manufacturer’s information; for the remaining five, enzymes produced by closely-related species were shown as matching proteins due to high sequence similarity. Six enzymes derived from four microorganisms could not be identified because their protein sequences were not registered in the database. As these databases are expanded, this approach of using SDS-PAGE and peptide mass fingerprinting (PMF) can determine the biological origin of enzymes rapidly and contribute to ensuring the safety of EPs.

Keywords:

• Enzyme preparation
• biological origin
• MALDI-TOF MS
• peptide mass fingerprinting

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by a research grant from the Ministry of Health, Labour and Welfare of Japan [Grant No. 20KA1008].