Supramolecular-mediated thermostabilization of phenylalanine dehydrogenase modified with β-cyclodextrin derivatives

Villalonga, Reynaldo; Tachibana, Sanro; Cao, Roberto; Ramírez, Héctor L.; Asano, Yasuhisa

doi:10.1016/j.bej.2006.01.013

Cited by 22 publications

(23 citation statements)

References 21 publications

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“…Unfortunately, only a small fraction of thermophilic proteins in nature have been identified, and natural thermophilic proteins with desired activity and selectivity are not always available. Chemical modification and immobilization have been used for improving enzyme kinetic stability by reducing conformational flexibility (Abian et al, 2004;Srimathi et al, 2006;Villalonga et al, 2006). However, reduced conformational flexibility by modification and immobilization usually results in a significant loss of enzymatic activity (Fernández-Lafuente et al, 2001;Kranz et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Enzyme stabilization by domain insertion into a thermophilic protein

Kim

Pierre

Ostermeier

et al. 2009

Protein Engineering Design and Selection

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Insufficient kinetic stability of exoinulinase (EI) restricts its application in many areas including enzymatic transformation of inulin for production of ultra-high fructose syrup and oligofructan, as well as fermentation of inulin into bioethanol. The conventional method for enzyme stabilization involves mutagenesis and therefore risks alteration of an enzyme's desired properties, such as activity. Here, we report a novel method for stabilization of EI without any modification of its primary sequence. Our method employs domain insertion of an entire EI domain into a thermophilic scaffold protein. Insertion of EI into a loop of a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) resulted in improvement of kinetic stability (the duration over which an enzyme remains active) at 37 degrees C without any compromise in EI activity. Our analysis suggests that the improved kinetic stability at 37 degrees C might originate from a raised kinetic barrier for irreversible conversion of unfolded intermediates to completely inactivated species, rather than an increased energy difference between the folded and unfolded forms.

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Section: Introductionmentioning

confidence: 99%

Enzyme stabilization by domain insertion into a thermophilic protein

Kim

Pierre

Ostermeier

et al. 2009

Protein Engineering Design and Selection

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“…The results showed that L-PheDH from S. ureae has a much lower K m for the substrates compared to the enzymes from B. badius, Brevibacterium sp., and Rhodococcus sp. M4 [9,17,27], which were 0.215, 0.4, and 0.75 mM, respectively.…”

Section: Temperature-dependent Activity and Stability Variationsmentioning

confidence: 99%

“…In order to increase its stability and economical application, special attention is currently devoted on the modification and the immobilization of L-PheDH. For instance, L-PheDH immobilization on tresylated poly(vinyl alcohol) beads [16], modification with cyclodextrin derivatives [17], biosensor coated amino-activated cellulose membrane by cross-linking with glutaraldehyde (GDA) [18], L-adamantanyl modified L-PheDH immobilization on β-cyclodextrins coated Au electrode [19], and carbon paste electrode with 3,4-dihydroxybenzaldehyde as an electron mediator [20] can be given as examples to the limited research in this area.…”

Section: Introductionmentioning

confidence: 99%

Immobilization of Phenylalanine Dehydrogenase and Its Application in Flow-Injection Analysis System for Determination of Plasma Phenylalanine

Tarhan

Ayar-Kayali

2010

Appl Biochem Biotechnol

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Phenylalanine dehydrogenase (L-PheDH) from Sporosarcina ureae was immobilized on DEAE-cellulose, modified initially with 2-amino-4,6-dichloro-s-triazine followed by hexamethylenediamine and glutaraldehyde. The highest activity of immobilized PheDH was determined as 95.75 U/g support with 56% retained activity. The optimum pH value of immobilized L-PheDH was shifted from pH 10.4 to 11.0. The immobilized L-PheDH showed activity variations close to the maximum value in a wider temperature range of 45-55 °C, whereas it was 40 °C for the native enzyme. The pH and the thermal stability of the immobilized L-PheDH were also better than the native enzyme. At pH 10.4 and 25 °C, K (m) values of the native and the immobilized L-PheDH were determined as K(m Phe) = 0.118, 0.063 mM and K(m NAD)(+) = 0.234, 0.128 mM, respectively. Formed NADH at the exit of packed bed reactor column was detected by the flow-injection analysis system. The conversion efficiency of the reactor was found to be 100% in the range of 5-600 μM Phe at 9 mM NAD(+) with a total flow rate of 0.1 mL/min. The reactor was used for the analyses of 30 samples each for 3 h per day. The half-life period of the reactor was 15 days.

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“…In view of important applications of PheDH some attempts were made to improve its enzymatic activity [11][12][13][14]. For example, Villalonga et al [14] covalently attached two derivatives of cyclodextrins (CDs) to the PheDH molecule.…”

Section: Introductionmentioning

confidence: 99%

“…For example, Villalonga et al [14] covalently attached two derivatives of cyclodextrins (CDs) to the PheDH molecule. In this way, two new chemically modified PheDH enzymes were obtained.…”

Section: Introductionmentioning

confidence: 99%