Design of Smart Biocatalysts

Immobilization of Enzymes on Smart Polymers Ipsita Roy and Munishwar N. Gupta

Summary

Smart polymers are water-soluble polymers that can be precipitated by an appropriate stimulus such as change of pH, ionic strength, temperature, or addition of a chemical species. Such polymers occur naturally (e.g., alginate, chitosan) but can also be synthesized chemically (e.g., methyl methacrylate polymers available commercially as Eudragit™). Linking of an enzyme to these polymers by noncovalent or covalent methods gives a biocatalyst that can be used as a homogeneous catalyst but can be recovered for possible re-use (after the reaction) by applying appropriate stimulus. The illustrative protocol shows that xylanase could be adsorbed on Eudragit L-100 and this reversibly soluble-insoluble bio-catalyst could be used for hydrolysis of xylan. Interestingly, the adsorption removed cellu-lase impurity. This is useful for paper pulp bleaching because xylanase should be free of cellulase activity. The soluble Eudragit-xylanase conjugate could be studied by circular dichroism spectroscopy to examine conformational changes in enzymes on immobilization on Eudragit L-100.

Key Words: Smart polymers; reversibly soluble-insoluble polymers; stimuli-sensitive polymers; xylanase; paper pulp bleaching; CD spectroscopy.

1. Introduction

Smart polymers undergo dramatic changes in their solubility in response to an appropriate stimulus. As these changes are reversible, these stimuli-sensitive-stimuli-responsive polymers have also been called reversibly soluble polymers (1-4). Immobilization of enzymes on such polymers normally does not alter the smart behavior of the polymers. Therefore, such bioconjugates are called "smart biocatalysts" (4-6). Smart biocatalysts combine the virtues of both homogeneous (catalysis is carried out by the soluble form) and heterogeneous (catalyst can be easily recovered) catalysts. Table 1 lists some of the smart bioconjugates described in the literature.

It is obvious from Table 1 that smart polymers can be both synthetic as well as naturally occurring. Eudragit™ is an example of an enteric polymer that has been extensively used by many workers. In view of its original intended application, this synthetic polymer is available easily in large amounts and is fairly economical. Alginate, chitosan, and K-carrageenan represent naturally occurring polysaccharides. All of these are fairly economical. The nontoxic nature of Eudragit, alginate, and K-carrageenan, allow the use of smart biocatalysts based on these polymers in food processing industries.

Table 1 also illustrates the fact that in many cases noncovalent immobilization has been used for preparing such bioconjugates. In fact, in some cases, attempts to use covalent coupling methods did not succeed because noncovalent interactions dominated the adsorption of the enzyme to the matrix (4,7). In principle, all cova-lent coupling methods described for immobilization on solid supports (8) can be used for conjugating proteins to these polymers.

The smart biocatalysts listed in Table 1 have been used in the bioconversion of starch, cellulose, chitin/chitosan, hydrolysis of xylan, and for obtaining protein hydrolysates. It has been observed that even with soluble matrices, the intrinsic rate of the immobilized preparation is different from that of the free enzyme. Enhanced Michaelis constant (Km) values for the bioconjugate have been often reported (9,10). In these cases, the steric hindrance from matrix is not totally abolished, even if the latter is soluble. A possible solution to this is to use the "end-group conjugation" method (11,12). In this method, smart oligomers (instead of polymer) of A/-isopropylacrylamide have been linked (to the enzyme) via its only one reactive end group. It was found that even with a ratio of oligomer to enzyme of 12:1, immobilized trypsin showed accessibility (toward soybean trypsin inhibitor) comparable to the free trypsin (12). Unfortunately, this conjugate showed worse stability (than even free trypsin) during thermal recycling, presumably as a result of "microenvironmental stress" (12). Smart bioconjugates, in which the polymer is linked to a specific site on the protein, have also been described (13,14). Such "site-specific conjugation" has been used for designing molecular switches (14).

As enzymes have also been used in organic solvents, it is natural that smart bioconjugates have also been evaluated in such media in terms of their re-usability and catalytic efficiency (15,16). A photosensitive bioconjugate of subtilisin gave a transesterification rate comparable to the free enzyme powder. The smart bioconjugate could be re-used four times without any significant loss of enzyme activity (15).

The smart bioconjugates, in their soluble forms, can be probed spectroscopi-cally or by circular dichroic measurements (17) (see Fig. 1). Thus, such systems also allow workers to gain further insight into the immobilization process. It should be added that one can also immobilize enzymes on smart hydrogels. Such hydrogels are insoluble but change their volume dramatically as a result of external stimuli (6). There is a growing interest in the design and applications of such smart biocatalysts as smart biocatalysts offer no general disadvantage over enzymes immobilized on solid supports.

Table 1

Smart polymer

Protein/Enzyme

Immobilization method Appropriate stimulus Reference co vo

MPM-06 Copolymer of

A-isopropylacrylamide and A-acryloxysuccinimide or glycidyl methacrylate A-isopropylacrylamide

Chitosan Eudragit S-100 Eudragit L-100 Alginate

Polymer of acrylamide 1-

(ß-methacryloxy)-ethyl-3,3]-dimethyl-6-nitrospiro (indoline-2,2[ [2^-1] benzopyran) Polymerized as1-casein

K-Carrageenan K-Carrageenan Hydroxypropyl methyl cellulose acetate succinate Poly[3-carbamoyl-1-(j»-vinyl-

benzyl)pyridinium chloride] Copolymer of A-4-phenyl-azophenyl acrylamide and A,A-dimethylacrylamide Elastin-like polypeptide (ELP) Poly(2-hydroxymethacrylate-co-dimethyl aminoethyl methacrylate)

Papain

IgG; alkaline phosphatase Trypsin

Laccase

Yeast lytic enzyme Xylanase Pectinase a-Chymotrypsin

Phosphoglyceromutase Pectinase

Alcohol dehydrogenase Chitinase

Trypsin

Endoglucanase 12A

Carbodiimide coupling pH

Adsorption

Coupling via dicyclohexyl carbodiimide and A-hydroxysuccinimide Carbodiimide coupling Covalent coupling Adsorption Adsorption Encapsulation

Conjugation using A-succinimidyl 3-(2-pyridylthio)propionate Adsorption Covalent coupling Covalent coupling

Graft polymerization

Site-specific conjugation

Thioredoxin-ELP fusion protein Adsorption Cholesterol oxidase Entrapment

Temperature and Ionic strength

Temperature pH pH pH

Ca2+

Ca2+

Redox couple of

Na2S2O4 and H2O2 UV/Vis illumination

Temperature Cholesterol

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