Characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube

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...

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Main Authors: Abdul Halim, Amanatuzzakiah, Szita, Nicolas, Baganz, Frank
Format: Article
Language:English
Published: Elsevier B.V. 2013
Subjects:
TP155 Chemical engineering
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
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spelling iium-477692016-02-23T07:27:58Z http://irep.iium.edu.my/47769/ Characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube Abdul Halim, Amanatuzzakiah Szita, Nicolas Baganz, Frank TP155 Chemical engineering 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. Elsevier B.V. 2013-09-17 Article PeerReviewed application/pdf en 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 Abdul Halim, Amanatuzzakiah and Szita, Nicolas and Baganz, Frank (2013) Characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube. Journal of Biotechnology, 168 (4). pp. 567-575. ISSN 0168-1656 http://www.sciencedirect.com/science/article/pii/S0168165613003921 10.1016/j.jbiotec.2013.09.001
repository_type Digital Repository
institution_category Local University
institution International Islamic University Malaysia
building IIUM Repository
collection Online Access
language English
topic TP155 Chemical engineering
spellingShingle TP155 Chemical engineering
Abdul Halim, Amanatuzzakiah
Szita, Nicolas
Baganz, Frank
Characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube
description 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.
format Article
author Abdul Halim, Amanatuzzakiah
Szita, Nicolas
Baganz, Frank
author_facet Abdul Halim, Amanatuzzakiah
Szita, Nicolas
Baganz, Frank
author_sort Abdul Halim, Amanatuzzakiah
title Characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube
title_short Characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube
title_full Characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube
title_fullStr Characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube
title_full_unstemmed Characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube
title_sort characterization and multi-step transketolase-ω- transaminase bioconversions in an immobilized enzyme microreactor (iemr) with packed tube
publisher Elsevier B.V.
publishDate 2013
url 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
first_indexed 2023-09-18T21:07:55Z
last_indexed 2023-09-18T21:07:55Z
_version_ 1777411058302976000

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