Particles covalent coupling methods

Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

Enzymes have been bound to insoluble cellulose derivatives by various methods such as (a) to insoluble carboxymethyl cellulose using the azide derivative, (b) to insoluble cellulose using the diimide reaction, etc. More recently H. Weetall described the covalent coupling of 1-amino-acid oxidase to porous silica glass particles. 

The following protocol for passive adsorption is based on methods reported for use with hydrophobic polymeric particles, such as polystyrene latex beads or copolymers of the same. Other polymer particle types also may be used in this process, provided they have the necessary hydrophobic character to promote adsorption. For particular proteins, conditions may need to be optimized to take into consideration maximal protein stability and activity after adsorption. Some proteins may undergo extensive denaturation after immobilization onto hydrophobic surfaces therefore, covalent methods of coupling onto more hydrophilic particle surfaces may be a better choice for maintaining native protein structure and long-term stability. 

Chitosan is of importance because of its primary amino groups that are susceptible for coupling reactions. Furthermore, porous spherical chitosan particles are commercially available (Chitopearl, Fuji Spinning) allowing noncovalent or covalent attachment of enzymes [55]. This support matrix can be easily prepared [56] and activation methods have been summarized [57]. Treatment with polyethyleneimine or with hexamethylenediamine and glut-ardialdehyde can improve the mechanical characteristics [53,58] of the biocatalyst, which is poor otherwise. However, this is often accompanied by some activity loss or increase of diffusional limitations. 

---