High-activity biocatalysts in organic media: solid-state buffers as the immobilisation matrix for protein-coated microcrystals

• Published in: Biotechnology and bioengineering 2004-07, Vol.87 (1), p.24-33
• Main Authors: Kreiner, Michaela, Parker, Marie Claire
• Format: Article
• Language: English
• Subjects: Animals; biocatalysis; Biological and medical sciences; Biotechnology; Buffers; Catalysis; Catalysts; Cattle; Chymotrypsin - chemistry; Crystals; Enzymes, Immobilized - chemistry; Fundamental and applied biological sciences. Psychology; lipase; Lipase - chemistry; organic solvent; protease; protein-coated microcrystals (PCMC); Proteins; Salts; solid-state buffer; Solvents; Subtilisin - chemistry; Thermodynamics
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.20101

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, K2SO4, 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 α‐chymotrypsin (CT) were significantly increased when coated onto the surface of solid‐state buffers, as measured in acetonitrile/1wt% H2O. SC‐PCMC with both organic and inorganic buffer carriers (Na‐AMPSO, Na2CO3, and NaHCO3) showed a 3‐fold greater activity than that observed when using the unbuffered system (PCMC‐SC/K2SO4). In comparison with freeze‐dried preparations, this represents an ∼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. © 2004 Wiley Periodicals, Inc.