High-activity biocatalysts in organic media: solid-state buffers as the immobilisation matrix for protein-coated microcrystals.
Recently, we reported a new high-activity biocatalyst for use in organic media termed protein-coated microcrystals (PCMC) (Kreiner et al. [2001] Chem Commun 12:1096-1097). These novel particles consist of water-soluble micron-sized crystalline particles coated with the given biocatalyst(s) and are prepared in a one-step rapid dehydration process. In this study we extended the choice of immobilisation matrix from a simple inorganic salt, K(2)SO(4), to other compounds, both inorganic and zwitterionic, that act as solid-state buffers for biocatalysis in organic media. The catalytic activity of serine proteases subtilisin Carlsberg (SC) and alpha-chymotrypsin (CT) were significantly increased when coated onto the surface of solid-state buffers, as measured in acetonitrile/1wt% H(2)O. SC-PCMC with both organic and inorganic buffer carriers (Na-AMPSO, Na(2)CO(3), and NaHCO(3)) showed a 3-fold greater activity than that observed when using the unbuffered system (PCMC-SC/K(2)SO(4)). In comparison with freeze-dried preparations, this represents an approximately 3,000-fold increase in catalytic activity. Importantly, there is no improvement in catalytic activity upon external addition of any of the solid-state buffers to the reaction mixture. When acting in a solid-state buffer capacity, good buffering capacity was observed with SC-PCMC (3 wt% protein loading) prepared from a 1:1 mixture of AMPSO and AMPSO-Na. Alternatively, increasing the amount of solid-state buffer in the system allows improvement of the buffering. This can be achieved either by decreasing the protein loading of the SC/Na-AMPSO-PCMC or by addition of further external solid-state buffer to the reaction mixture. The catalytic activity of lipase-PCMC prepared from solid-state buffers was found less responsive to immobilisation.
Kreiner
Michaela
M
Department of Chemistry, University of Glasgow, Joseph Black Building, Glasgow G12 8QQ, Scotland, UK. mickr@chem.gla.ac.uk
Parker
Marie Claire
MC
D000818:Animals; D002021:Buffers; D002384:Catalysis; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D008049:Lipase / Q000737:chemistry*; D012492:Salts; D012997:Solvents; D020860:Subtilisin / Q000737:chemistry*; D013816:Thermodynamics
D002021:Buffers; D004800:Enzymes, Immobilized; D012492:Salts; D012997:Solvents; D008049:Lipase; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D020860:Subtilisin