Entrapment in E. coli improves the operational stability of recombinant β-glycosidase CelB from Pyrococcus furiosus and facilitates biocatalyst recovery

Kamrat, Thomas; Nidetzky, Bernd

doi:10.1016/j.jbiotec.2006.11.020

Cited by 8 publications

(3 citation statements)

References 34 publications

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“…These findings indicate that arrangement of enzymes within the multienzyme fusion protein can have significant impacts on the activity of the individual enzymes. It has been previously suggested that CelB is inhibited by magnesium, 30 which is essential for GLUK activity, but we found no loss in CelB activity in the presence of 10 mM magnesium in either citrate or HEPES buffer at pH 7.0 (Supplementary Figure S5). The HEPES data suggest that coordination of magnesium by citrate is not a factor.…”

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confidence: 51%

Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle

et al. 2013

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Developing methods for investigating coupled enzyme systems under conditions that mimic the cellular environment remains a significant challenge. Here we describe a biomimetic approach for constructing densely packed and confined multienzyme systems through the co-encapsulation of 2 and 3 enzymes within a virus-like particle (VLP) that perform a coupled cascade of reactions, creating a synthetic metabolon. Enzymes are efficiently encapsulated in vivo with known stoichiometries, and the kinetic parameters of the individual and coupled activities are characterized. From the results we develop and validate a mathematical model for predicting the expected kinetics for coupled reactions under co-localized conditions.

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confidence: 51%

Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle

et al. 2013

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“…7–9 Non-covalent and covalent attachment strategies have previously been employed for immobilizing hyperthermophilic enzymes. 10–14 However, based on electrostatic and hydrophobic multipoint interactions between the protein and immobilization surface, physical adsorption of hyperthermophilic enzymes may not be sufficiently strong at elevated temperatures to prevent displacement. Immobilization by covalent attachment often involves using toxic chemicals, such as carbodiimides and succinic anhydride, 15,16 to modify the support surface and bind the enzyme.…”

Section: Introductionmentioning

confidence: 99%

N‐terminal fusion of a hyperthermophilic chitin‐binding domain to xylose isomerase from Thermotoga neapolitana enhances kinetics and thermostability of both free and immobilized enzymes

Harris

Epting

Kelly

2010

Biotechnology Progress

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Immobilization of a thermostable D-xylose isomerase (EC 5.3.1.5) from Thermotoga neapolitana 5068 (TNXI) on chitin beads was accomplished via a N-terminal fusion with a chitin-binding domain (CBD) from a hyperthermophilic chitinase produced by Pyrococcus furiosus (PF1233) to create a fusion protein (CBD-TNXI). The turnover numbers for glucose to fructose conversion for both unbound and immobilized CBD-TNXI were greater than the wild-type enzyme: kcat (min−1) was approximately 1000, 3800, and 5800 at 80°C compared to 1140, 10350, and 7000 at 90°C, for the wild-type, unbound, and immobilized enzymes, respectively. These kcat values for the glucose to fructose isomerization measured are the highest reported to date for any XI at any temperature. Enzyme kinetic inactivation at 100°C, as determined from a bi-phasic inactivation model, showed that the CBD-TNXI bound to chitin had a half-life approximately three times longer than the soluble wild-type TNXI (19.9 hours vs. 6.8 hours, respectively). Surprisingly, the unbound soluble CBD-TNXI had a significantly longer half-life (56.5 hours) than the immobilized enzyme. Molecular modeling results suggest that the N-terminal fusion impacted subunit interactions, thereby contributing to the enhanced thermostability of both the unbound and immobilized CBD-TNXI. These interactions likely also played a role in modifying active site structure, thereby diminishing substrate-binding affinities and generating higher turnover rates in the unbound fusion protein.

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“…1992; Siso and Doval 1994; Carvalho Lins and Rocha Leao 2002). Recently, the wet cells of Kluyveromyces lactis and recombinant cells of Escherichia coli with plasmid‐encoded expression β‐glycosidase from Pyrococcus woesei were used for this purpose (Kamrat and Nidetzky 2007; Lee et al . 2004).…”

Section: Introductionmentioning

confidence: 99%

Β-Galactosidase ACTIVITY OF MEIOTHERMUS RUBER CELLS

Synowiecki

Sinkiewicz

Zakrzewska

et al. 2011

Journal of Food Biochemistry

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Freeze-dried cells of Meiothermus ruber catalyses cleavage of o-nitrophenyl-b-Dgalactopiranoside (oNPb-gal) and conversion of lactose into glucose and galactose. The permeabilization with 2% toluene,20% ethanol and 20% acetone increased enzymatic activity from 74.87 U/g of lyophilized cells up to 129.44, 114.38 and 90.19 U/g, respectively. Ethanol was an effective permeabilizing agent and its efficiency was dependent on the concentration, the incubation time and incubation temperature. The Km values for the untreated and permeabilized cells were 2.94 mM and 2.26 mM but Vmax values were 122 mmol/min and 193 mmol/min, respectively. The optimum pH for the b-galactosidase activity in the untreated and permeabilized cells were 6.5 and optimum of temperatures 65C.The stability of enzymatic activity in M.ruber cells incubated for 1 h at pH 6.5 was almost unchanged at temperatures below 65C. PRACTICAL APPLICATIONSThe current study shows that freeze-dried permeabilized cells of Meiothermus ruber can be used instead of isolated b-galactosidase. It leads to elimination of expensive purification procedures and assures longer half-life time of enzyme activity. Application of such biocatalyst for lactose hydrolysis in milk products for human consumption is limited until designation of the Generally Recognized As Safe status of Meiothermus ruber. It can be employed, however, for other purposes, e.g., for production of alkyl glycosides, a group of non-ionic surfactants with a variety of applications in detergents, cleaning agents, personal care products and pharmaceuticals.

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Entrapment in E. coli improves the operational stability of recombinant β-glycosidase CelB from Pyrococcus furiosus and facilitates biocatalyst recovery

Cited by 8 publications

References 34 publications

Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle

Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle

N‐terminal fusion of a hyperthermophilic chitin‐binding domain to xylose isomerase from Thermotoga neapolitana enhances kinetics and thermostability of both free and immobilized enzymes

Β-Galactosidase ACTIVITY OF MEIOTHERMUS RUBER CELLS

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