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

D-amino acid oxidase from Trigonopsis variabilis (TvDAO) is applied in industry for the synthesis of pharmaceutical intermediates. Because free TvDAO 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 TvDAO. A fusion protein between TvDAO 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 TvDAO 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 TvDAO 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.     

Production of biocommodities and bioelectricity by cell‐free synthetic enzymatic pathway biotransformations: Challenges and opportunities

Cell‐free synthetic (enzymatic) pathway biotransformation (SyPaB) is the assembly of a number of purified enzymes (usually more than 10) and coenzymes for the production of desired products through complicated biochemical reaction networks that a single enzyme cannot do. Cell‐free SyPaB, as compared to microbial fermentation, has several distinctive advantages, such as high product yield, great engineering flexibility, high product titer, and fast reaction rate. Biocommodities (e.g., ethanol, hydrogen, and butanol) are low‐value products where costs of feedstock carbohydrates often account for ～30–70% of the prices of the products. Therefore, yield of biocommodities is the most important cost factor, and the lowest yields of profitable biofuels are estimated to be ca. 70% of the theoretical yields of sugar‐to‐biofuels based on sugar prices of ca. US$ 0.18 per kg. The opinion that SyPaB is too costly for producing low‐value biocommodities are mainly attributed to the lack of stable standardized building blocks (e.g., enzymes or their complexes), costly labile coenzymes, and replenishment of enzymes and coenzymes. In this perspective, I propose design principles for SyPaB, present several SyPaB examples for generating hydrogen, alcohols, and electricity, and analyze the advantages and limitations of SyPaB. The economical analyses clearly suggest that developments in stable enzymes or their complexes as standardized parts, efficient coenzyme recycling, and use of low‐cost and more stable biomimetic coenzyme analogs, would result in much lower production costs than do microbial fermentations because the stabilized enzymes have more than 3 orders of magnitude higher weight‐based total turn‐over numbers than microbial biocatalysts, although extra costs for enzyme purification and stabilization are spent. Biotechnol. Bioeng. 2010. 105: 663–677. © 2009 Wiley Periodicals, Inc.