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Oriented and selective enzyme immobilization on functionalized silica carrier using the cationic binding module Z basic2 : Design of a heterogeneous D ‐amino acid oxidase catalyst on porous glass
Author(s) -
Bolivar Juan M.,
Nidetzky Bernd
Publication year - 2012
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.24423
Subject(s) - chemistry , immobilized enzyme , glucose oxidase , enzyme , catalysis , d amino acid oxidase , oxidase test , chromatography , combinatorial chemistry , organic chemistry
D ‐Amino acid oxidase from Trigonopsis variabilis ( Tv DAO) is applied in industry for the synthesis of pharmaceutical intermediates. Because free Tv DAO is extremely sensitive to exposure to gas–liquid interfaces, biocatalytic processing is usually performed with enzyme immobilizates that offer enhanced stability under bubble aeration. We herein present an “Immobilization by Design” approach that exploits engineered charge complementarity between enzyme and carrier to optimize key features of the immobilization of Tv DAO. A fusion protein between Tv DAO and the positively charged module Z basic2 was generated, and a corresponding oppositely charged carrier was obtained by derivatization of mesoporous glass with 3‐(trihydroxysilyl)‐1‐propane‐sulfonic acid. Using 250 mM NaCl for charge screening at pH 7.0, the Z basic2 fusion of Tv DAO was immobilized directly from E. coli cell extract with almost absolute selectivity and full retention of catalytic effectiveness of the isolated enzyme in solution. Attachment of the homodimeric enzyme to the carrier was quasi‐permanent in low‐salt buffer but fully reversible upon elution with 5 M NaCl. Immobilized Tv DAO was not sensitive to bubble aeration and received substantial (≥tenfold) stabilization of the activity at 45°C as compared to free enzyme, suggesting immobilization via multisubunit oriented interaction of enzyme with the insoluble carrier. The Z basic2 enzyme immobilizate was demonstrated to serve as re‐usable heterogeneous catalyst for D ‐amino acid oxidation. Z basic2 ‐mediated binding on a sulfonic acid group‐containing glass carrier constitutes a generally useful strategy of enzyme immobilization that supports transition from case‐specific empirical development to rational design. Biotechnol. Bioeng. 2012; 109:1490–1498. © 2012 Wiley Periodicals, Inc.