Reversibly soluble biocatalyst: optimization of trypsin coupling to Eudragit S-100 and biocatalyst activity in soluble and precipitated forms

Eudragit S-100, a copolymer of methacrylic acid and methyl methacrylate is soluble at pH above 5 and insoluble at pH below 4.5. pH-dependent solubilit y of the polymer is used for the development of reversibly soluble biocatal yst, which combines the advantages of both soluble and immobilized biocatal ysts. Activity of trypsin, covalently coupled to Eudragit S-100, was improv ed by protecting the active site of the enzyme with benzamidine and removin g the noncovalently bound proteins with Triton X-100 in 0.15 M Tris buffer (pH 7.6). Accurate choice of coupling conditions combined with proper washi ng protocol produced highly active enzyme-polymer conjugate with no noncova lently bound protein. Two conjugates with 100-fold difference in the conten t of trypsin coupled to Eudragit S-100 were studied when the preparations w ere in soluble and precipitated forms. The K-m values of the soluble enzyme to the lower molecular weight substrate was less than that of the free enz yme, whereas that to the higher molecular weight substrate was closer to th at of the free enzyme. Activities of the soluble and precipitated immobiliz ed trypsin with higher molecular weight substrate were completely inhibited by soy bean trypsin inhibitor. whereas complete inhibition with soy bean t rypsin inhibitor was never achieved with lower molecular weight substrate, indicating reduced access of high-molecular weight substrate/inhibitor to s ome of the catalytically active enzyme molecules in trypsin-Eudragit conjug ate. (C) 2000 Elsevier Science Inc. All rights reserved.