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Design and characterization of a prototype enzyme microreactor: Quantification of immobilized transketolase kinetics
Author(s) -
Matosevic S.,
Lye G. J.,
Baganz F.
Publication year - 2009
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.319
Subject(s) - microreactor , transketolase , chemistry , immobilized enzyme , bioreactor , bioconversion , chromatography , kinetics , glycolaldehyde , biocatalysis , enzyme catalysis , enzyme kinetics , combinatorial chemistry , enzyme , catalysis , organic chemistry , active site , reaction mechanism , physics , quantum mechanics , fermentation
In this work, we describe the design of an immobilized enzyme microreactor (IEMR) for use in transketolase (TK) bioconversion process characterization. The prototype microreactor is based on a 200‐μm ID fused silica capillary for quantitative kinetic analysis. The concept is based on the reversible immobilization of His 6 ‐tagged enzymes via Ni‐NTA linkage to surface derivatized silica. For the initial microreactor design, the mode of operation is a stop‐flow analysis which promotes higher degrees of conversion. Kinetics for the immobilized TK‐catalysed synthesis of L ‐erythrulose from substrates glycolaldehyde (GA) and hydroxypyruvate (HPA) were evaluated based on a Michaelis–Menten model. Results show that the TK kinetic parameters in the IEMR ( V max(app) = 0.1 ± 0.02 mmol min –1 , K m(app) = 26 ± 4 mM) are comparable with those measured in free solution. Furthermore, the k cat for the microreactor of 4.1 × 10 5 s −1 was close to the value for the bioconversion in free solution. This is attributed to the controlled orientation and monolayer surface coverage of the His 6 ‐immobilized TK. Furthermore, we show quantitative elution of the immobilized TK and the regeneration and reuse of the derivatized capillary over five cycles. The ability to quantify kinetic parameters of engineered enzymes at this scale has benefits for the rapid and parallel evaluation of evolved enzyme libraries for synthetic biology applications and for the generation of kinetic models to aid bioconversion process design and bioreactor selection as a more efficient alternative to previously established microwell‐based systems for TK bioprocess characterization. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010