Interfacial and monophasic nonaqueous chemical processing using chemically modified enzymes.

摘要

Traditional aqueous enzymatic processing often encounters problems in dealing with hydrophobic substances because of their insolubility in aqueous solutions. In recent years, rapid development in non-aqueous biocatalysis greatly broadened the potential of enzymes for chemical processing especially for organic chemicals. However, the insolubility of native or immobilized enzymes in organic solvents usually leads to low catalytic efficiency. In this work, chemical solidification was applied to significantly improve enzymes' solubility and compatibility in organic media. Horseradish peroxidase (HRP) and proleather from bacillus sp. were chemically modified with decanoyl chloride. The modified enzymes are highly soluble and active in various organic solvents. The solubility of modified proleather in chloroform was 44 mg/ml, while much higher solubility was obtained with modified HRP. The modified HRP showed improved activity in organic solvents compared with HRP immobilized on glass powder. The organic-soluble proleather efficiently catalyzed the polymerization of ethyl lactate. The reaction rate was 4--22 times higher than that of native proleather.; On the other hand, many valuable biotransformation processes involve both hydrophobic and hydrophilic substrates. Monophasic reactions are mostly inefficient, because of the solubility limitation of substrates. Organic-aqueous biphasic systems have been employed widely to overcome such solubility limitations. Mass transfer resistance across the organic-aqueous interface, however, still exists. An additional issue for traditional biphasic reactions is the inactivation of enzymes at the liquid interface. To overcome these problems, water-soluble enzymes including alpha-chymotrypsin, beta-galactosidase, and chloroperoxidase were modified with functionalized polystyrene to form surfactant-like conjugates. The polymer-enzyme conjugates showed stable assembly at an organic-aqueous interface as tested by protein dye agent and interfacial tension measurement. Catalytic efficiency of the polymer-enzyme conjugates in biphasic systems were investigated for reactions involving both hydrophobic and hydrophilic substrates. Experimental results showed that the transgalactosylation reaction catalyzed by polystyrene-(beta-galactosidase) was over 150 times faster than the native enzyme in a toluene-buffer system. The epoxidation of styrene catalyzed by polystyrene-chloroperoxidase conjugate in a styrene-buffer system showed 2.5-fold enhancement of productivity versus native CPO under the same batch reaction conditions, while much more significant improvement was realized with continuous feeding operations that are not feasible with native CPO. In both reactions, undesired side-reactions were effectively suppressed and improved enzyme stability was also observed.; This novel interfacial biocatalysis may provide new avenues for the study of membrane-binding proteins, generate alternative strategies for the encapsulation and delivery of protein therapeutic agents, and lead to the development of novel approaches to enzymatic bioprocessing of hydrophobic chemicals.