Cross-linked aggregates of the hydroxynitrile lyase from Manihot esculenta:: Highly active and robust biocatalysts

The precipitation and cross-linking into CLEAs of the hydroxynitrile lyase (E.C. 4.1.2.10) from Manihot esculenta was investigated and an optimized procedure, which involved precipitation with (NH4)(2)SO4, was developed. It was found that a fast (photometric) assay severely underestimated the activity recovery in the CLEA due to rate-limiting diffusion, whereas in a synthetic assay the activity recovery was up to 93% of the starting activity. The CLEA was applied in the hydrocyanation of various aldehydes and ketones in microaqueous medium, conditions that rendered the non-enzymatic background reaction insignificant, even with unreactive substrates.