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

Biotechnology Progress
S MatosevicF Baganz

Abstract

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 ...Continue Reading

References

Aug 1, 1991·Applied Biochemistry and Biotechnology·B SzajániL Töke
Jan 1, 1988·Methods in Enzymology·B Mattiasson
Sep 1, 1966·The Biochemical Journal·M D LillyE M Crook
Nov 1, 1994·Trends in Biotechnology·D S Clark
Apr 26, 2001·Biotechnology and Bioengineering·Y D KimD S Clark
Jun 16, 2001·Biotechnology and Bioengineering·P WangB H Davison
Jan 29, 2002·Analytical Chemistry·Hanbin MaoPaul S Cremer
Nov 7, 2002·Journal of the American Chemical Society·Gi Hun Seong, Richard M Crooks
Mar 26, 2003·Biotechnology and Bioengineering·Hana StiborovaWilfred Chen
Apr 3, 2003·Chemical Communications : Chem Comm·Masaya MiyazakiHideaki Maeda
May 13, 2003·Biotechnology and Bioengineering·Moo-Yeal LeeJonathan S Dordick
Sep 11, 2003·Analytical Chemistry·Gi Hun SeongRichard M Crooks
Jan 10, 2004·Applied Microbiology and Biotechnology·S J Lee, S Y Lee
Jan 22, 2004·Angewandte Chemie·Klaus JähnischManfred Baerns
Apr 2, 2004·Journal of Nanobiotechnology·Richard Barry, Dimitri Ivanov
Jul 14, 2004·Langmuir : the ACS Journal of Surfaces and Colloids·Nathaniel J Gleason, Jeffrey D Carbeck
Oct 2, 2004·Biotechnology and Bioengineering·Aravind SrinivasanJonathan S Dordick
Nov 27, 2004·Electrophoresis·Jana Krenková, Frantisek Foret
Jan 4, 2005·Proteomics·Taewoon ChaXiao-Yang Zhu
Apr 29, 2005·Biomolecular Engineering·Edward G HibbertPaul A Dalby
Oct 13, 2005·Chemical Communications : Chem Comm·Takeshi HondaHideaki Maeda
Mar 16, 2006·Analytical Chemistry·Guoyue ShiStephen G Weber
Aug 8, 2006·Biotechnology Progress·Bosung KuJonathan S Dordick
Dec 15, 2006·Analytical Chemistry·Matthew B KerbyAnubhav Tripathi
Sep 11, 2007·Journal of Biotechnology·Edward G HibbertPaul A Dalby

❮ Previous
Next ❯

Citations

Dec 14, 2011·Analytical Chemistry·Piro SiutiMitchel J Doktycz
Jul 21, 2011·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Yuya AsanomiHideaki Maeda

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.