Rapid classification of enzymes in cleaning products by hydrolysis, mass spectrometry and linear discriminant analysis

Abstract

A method for the rapid classification of proteases, lipases, amylases and cellulases used as enhancers in cleaning products, based on precipitation with acetone, hydrolysis with HCl, dilution of the hydrolysates with ethanol, and direct infusion into the electrospray ion source of an ion-trap mass spectrometer, has been developed. The abundances of the ([M+H]+ ions of the amino acids, from the hydrolysates of both the enzyme industrial concentrates and the detergent bases spiked with them, were used to construct linear discriminant analysis models, capable of distinguishing between the enzyme classes. For this purpose, the variables were normalized as follows: (A) the ion abundance of each amino acid was divided by the sum of the ion abundances of all the amino acids in the corresponding mass spectrum; (B) the ratios of pairs of ion abundances were obtained by dividing the ion abundance of each amino acid by each one of the ion abundances of the other 17 amino acids in the corresponding mass spectrum. Using normalization procedure B, excellent class-resolution between proteases, lipases, amylases and cellulases was achieved. In all cases, enzymes in industrial concentrates and manufactured cleaning products were correctly classified with >98% assignment probability.

Identification of industrial detergent enzymes by SDS-PAGE and MALDI-TOF mass spectrometry

“…However, despite the widespread application of enzymes in the detergent industry, analytical methods for enzymes in cleaning products have rarely been reported. Two methods have been developed based on the complete hydrolysis of a protein to its constituent amino acids followed by direct infusion into the electrospray ion source of an ion-trap mass spectrometer, 31 and derivatization with o-phthaldialdehyde, and then separation by HPLC. 32 In both of them, the obtained amino acid profiles were used to predict the type of enzyme by linear discriminant analysis, although further information is lost during complete hydrolysis.…”