Characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube
The concept of de novo metabolic engineering through novel synthetic pathways offers new directions for multi-step enzymatic synthesis of complex molecules. This has been complemented by recent progress in performing enzymatic reactions using immobilized enzyme microreactors (IEMR). This work is c...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier B.V.
2013
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| Subjects: | |
| Online Access: | http://irep.iium.edu.my/47769/ http://irep.iium.edu.my/47769/ http://irep.iium.edu.my/47769/ http://irep.iium.edu.my/47769/1/Characterization_and_multi-step_tra_nsketolase-%CF%89-transaminase_bioconversions_in_an_immobilized_enzyme_microreactor_%28IEMR%29_with_packed_tube..pdf |
| Summary: | The concept of de novo metabolic engineering through novel synthetic pathways offers new directions for
multi-step enzymatic synthesis of complex molecules. This has been complemented by recent progress
in performing enzymatic reactions using immobilized enzyme microreactors (IEMR). This work is concerned
with the construction of de novo designed enzyme pathways in a microreactor synthesizing chiral
molecules. An interesting compound, commonly used as the building block in several pharmaceutical
syntheses, is a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT). This chiral amino alcohol can
be synthesized from simple achiral substrates using two enzymes, transketolase (TK) and transaminase
(TAm). Here we describe the development of an IEMR using His6-tagged TK and TAm immobilized onto
Ni-NTA agarose beads and packed into tubes to enable multi-step enzyme reactions. The kinetic parameters
of both enzymes were first determined using single IEMRs evaluated by a kinetic model developed for
packed bed reactors. The Km(app) for both enzymes appeared to be flow rate dependent, while the turnover
number kcat was reduced 3 fold compared to solution-phase TK and TAm reactions. For the multi-step
enzyme reaction, single IEMRs were cascaded in series, whereby the first enzyme, TK, catalyzed a model
reaction of lithium-hydroxypyruvate (HPA) and glycolaldehyde (GA) to l-erythrulose (ERY), and the second
unit of the IEMR with immobilized TAm converted ERY into ABT using (S)--methylbenzylamine
(MBA) as amine donor. With initial 60 mM (HPA and GA each) and 6 mM (MBA) substrate concentration
mixture, the coupled reaction reached approximately 83% conversion in 20 min at the lowest flow
rate. The ability to synthesize a chiral pharmaceutical intermediate, ABT in relatively short time proves
this IEMR system as a powerful tool for construction and evaluation of de novo pathways as well as for
determination of enzyme kinetics. |
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