Semiquantitative Process Screening for the Biocatalytic Synthesis of <scp>d</scp>-Xylulose-5-phosphate

Shaeri, Jobin; Wohlgemuth, Roland; Woodley, John M.

doi:10.1021/op050254a

Cited by 43 publications

(22 citation statements)

References 19 publications

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“…TK mediates the transfer of a two-carbon ketol moiety from a ketose to an aldose. The reaction makes the TK very attractive as a biocatalyst in industrial synthesis (Shaeri et al, 2006). The enzyme has previously been cloned by Ingram et al, (2007) and variants of the wild type enzyme engineered with a His 6 -tag (Hibbert et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Design and characterization of a prototype enzyme microreactor: Quantification of immobilized transketolase kinetics

Matosevic¹,

Lye²,

Baganz³

2009

Biotechnology Progress

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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-microm 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 x 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.

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Section: Introductionmentioning

confidence: 99%

Design and characterization of a prototype enzyme microreactor: Quantification of immobilized transketolase kinetics

Matosevic¹,

Lye²,

Baganz³

2009

Biotechnology Progress

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“…The use of process modeling via the use of windows of operation is a further tool in the quest to develop biocatalytic processes as rapidly as possible (Lye et al, 2002) in order to give the best possible basis for industrial implementation. Subsequently we have used the characterization data gathered for this model system to identify a series of process flow-sheets (Shaeri et al, 2006) which are the next step for establishing a simplified synthesis of this metabolite in a robust and scalable multi-step process.…”

Section: Discussionmentioning

confidence: 99%

Characterization of enzymatic D‐xylulose 5‐phosphate synthesis

Shaeri

Wright²,

Rathbone³

et al. 2008

Biotech & Bioengineering

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In this article we report on the characterization of the enzymatic synthesis of D-xylulose 5-phosphate using triosephosphate isomerase and transketolase. Two potential starting substrates are possible with this scheme. The data presented here allow a comparison of both routes for the synthesis, based on experimental information on reaction kinetics. Operational guidelines are proposed which should assist in the scale-up of such syntheses.

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“…With this approach, 5 out of 20 process options have been selected in the bioprocess for the synthesis of d-xylulose 5-phosphate. [129] Many of the bioprocess engineering aspects relevant to onepot reactions have been reported in the present paper; however, their use should be considered from case to case. More comprehensive references on process synthesis in general exist in the literature.…”

Section: Enzyme-enzyme and Chemo-enzymatic Reactionsmentioning

confidence: 96%

Multi‐Enzymatic Cascade Reactions: Overview and Perspectives

Brucher²,

2011

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Multi-enzymatic cascade reactions, i.e., the combination of several enzymatic transformations in concurrent one-pot processes, offer considerable advantages: the demand of time, costs and chemicals for product recovery may be reduced, reversible reactions can be driven to completion and the concentration of harmful or unstable compounds can be kept to a minimum. This review summarizes the developments in multi-enzymatic cascades employed for the asymmetric synthesis of chiral alcohols, amines and amino acids, as well as for C À C bond formation. In addition, a general classification of biocatalytic cascade systems is provided and bioprocess engineering aspects associated with the topic are discussed.

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Semiquantitative Process Screening for the Biocatalytic Synthesis of d-Xylulose-5-phosphate

Cited by 43 publications

References 19 publications

Design and characterization of a prototype enzyme microreactor: Quantification of immobilized transketolase kinetics

Design and characterization of a prototype enzyme microreactor: Quantification of immobilized transketolase kinetics

Characterization of enzymatic D‐xylulose 5‐phosphate synthesis

Multi‐Enzymatic Cascade Reactions: Overview and Perspectives

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