Proteomic methods applied to the analysis of immobilized biocatalysts.
Methods adapted from proteomics can directly characterize proteins present in immobilized biocatalysts. Complete hydrolysis followed by HPLC analysis of Tyr and Phe estimates total protein bound, and is preferable to conventional difference methods, as tested with subtilisin Carlsberg on silica. This new method shows that various treatments give quantitative desorption of proteins immobilized by adsorption. Intact desorbed proteins may be analyzed by electrospray mass spectrometry. The Candida antarctica lipase B from Novozyme 435 was shown to be heavily glycosylated, while the lipase from Lipozyme RM IM was a mixture of four N-terminally truncated forms. Peptides from selective cleavage were analyzed by tandem mass spectrometry, leading to automatic identification of proteins present. A second major protein present in Lipozyme RM IM was thus found to be alpha-amylase from Aspergillus oryzae. These methods should be valuable complements to activity measurements in understanding immobilized enzyme activity and stability.
Petry
Inga
I
Westchem, Department of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, G1 1XL Glasgow, UK. inga.petry@strath.ac.uk
Ganesan
Ashok
A
Pitt
Andrew
A
Moore
Barry D
BD
Halling
Peter J
PJ
D000327:Adsorption; D001331:Automation; D019149:Bioreactors; D002384:Catalysis; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized / Q000032:analysis* / Q000737:chemistry; D005656:Fungal Proteins; D006031:Glycosylation; D008049:Lipase / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry; D040901:Proteomics / Q000379:methods*
D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica