Use of Ionic Liquids to Increase the Yield and Enzyme Stability in the β-Galactosidase Catalysed Synthesis of <i>N</i>-Acetyllactosamine

Kaftzik, Nicole; Wasserscheid, Peter; Kragl, Udo

doi:10.1021/op0255231

Cited by 192 publications

(120 citation statements)

References 50 publications

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“…Following the stimulating work of Kaftzik et al (2002) on b-galactosidase-catalyzed synthesis of N-acetyl-lactosamine in ionic liquids, this is the first report in which the influence of a water-miscible ionic liquid cosolvent on the transglycosylation properties of a glycosidase is described in some detail. The results show that the transferase activity of CelB can be improved significantly by addition of the widely used ionic liquid [MMIm] [MeSO 4 ].…”

Section: Discussionmentioning

confidence: 97%

Influence of ionic liquid cosolvent on transgalactosylation reactions catalyzed by thermostable β-glycosylhydrolase CelB fromPyrococcus Furiosus

Lang

Kamrat

Nidetzky

2006

Biotechnol. Bioeng.

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The synthesis of glycosides by enzymatic transglycosylation is a kinetically controlled reaction performed in the context of a non-favorable thermodynamic equilibrium. An unreactive organic cosolvent which increases the selectivity of the enzyme for glycosyl transfer to the acceptor nucleophile compared with water (Ksel) could improve maximum product yield. Here we report on the effect of the ionic liquid 1,3-dimethylimidazoliummethylsulfate on hydrolase and transferase activities of the hyperthermostable beta-glycosidase CelB from the archaeon Pyrococcus furiosus. CelB retained full catalytic efficiency for lactose hydrolysis at 80 degrees C in a 50% (by vol.) solution of ionic liquid in sodium citrate buffer, pH 5.5. It was inactive but not irreversibly denatured at 70% ionic liquid. Using lactose (0.15 M) as galactosyl donor, values of Ksel for a representative series of eight acceptor alcohols were determined in kinetic assays at 80 degrees C and found to increase between 1.3-fold (D-xylose) and 3.1-fold (glycerol) in 45% ionic liquid. Enhancement of Ksel was dependent on ionic liquid concentration and higher than expected from the decrease in water activity caused by the cosolvent. Experimental molar ratios of D-glucose and D-galactose produced during enzymatic conversion of lactose (75-150 mM) in the presence of D-xylose (0.5 M) or glycerol (0.5 M) showed excellent agreement with predictions based on Ksel values and confirm a significant, yet moderate effect of 45% ionic liquid on increasing the yield of D-galactoside product, by < or = 10%.

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Section: Discussionmentioning

confidence: 97%

Influence of ionic liquid cosolvent on transgalactosylation reactions catalyzed by thermostable β-glycosylhydrolase CelB fromPyrococcus Furiosus

Lang

Kamrat

Nidetzky

2006

Biotechnol. Bioeng.

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“…It was shown that the enzyme activity of β-galactosidase could be vastly increased in an IL environment. 93 Preliminary studies of our lab have demonstrated that β-galactosidase was active when BGs were re-suspended in the IL [Bmim]PF 6 .…”

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confidence: 99%

The bacterial ghost platform system

Langemann¹,

et al. 2010

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The Bacterial Ghost (BG) platform technology is an innovative system for vaccine, drug or active substance delivery and for technical applications in white biotechnology. BGs are cell envelopes derived from Gram-negative bacteria. BGs are devoid of all cytoplasmic content but have a preserved cellular morphology including all cell surface structures. Using BGs as delivery vehicles for subunit or DNA-vaccines the particle structure and surface properties of BGs are targeting the carrier itself to primary antigenpresenting cells. Furthermore, BGs exhibit intrinsic adjuvant properties and trigger an enhanced humoral and cellular immune response to the target antigen. Multiple antigens of the native BG envelope and recombinant protein or DNA antigens can be combined in a single type of BG. Antigens can be presented on the inner or outer membrane of the BG as well as in the periplasm that is sealed during BG formation. Drugs or supplements can also be loaded to the internal lumen or periplasmic space of the carrier. BGs are produced by batch fermentation with subsequent product recovery and purification via tangential flow filtration. For safety reasons all residual bacterial DNA is inactivated during the BG production process by the use of staphylococcal nuclease A and/or the treatment with β-propiolactone. After purification BGs can be stored long-term at ambient room temperature as lyophilized product. The production cycle from the inoculation of the pre-culture to the purified BG concentrate ready for lyophilization does not take longer than a day and thus meets modern criteria of rapid vaccine production rather than keeping large stocks of vaccines. The broad spectrum of possible applications in combination with the comparably low production costs make the BG platform technology a safe and sophisticated product for the targeted delivery of vaccines and active agents as well as carrier of immobilized enzymes for applications in white biotechnology.

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“…The first reaction was carried out at 40 C in 100 mM phosphate buffer at pH 6.5, supplemented with MgCl 2 (10 mM), starting from lactose 2 (1 M) as a galactosyl donor and from GlcNAc 1 (250 mM) as the acceptor (Scheme 1). Various galactosylated derivatives of GlcNAc were found in the media after 5 h of reaction catalyzed by KlbGal (1,014 U mL À1 ) or BcbGal (142 U mL…”

Section: Resultsmentioning

confidence: 99%

“…Kinetics studies were performed at 40 C with a diluted solution of the crude preparation of KlbGal (1:500 dilution) or BcbGal (1:250 dilution) prepared in 100 mM phosphate buffer, pH 6.5 (10 mM MgCl 2 ), on a chromogenic substrate (o-nitrophenyl-b-D-galactopyranoside). Continuous changes in absorbance at 415 nm were monitored in spectrophotometric cuvettes of 1-cm path length using a UV-visible spectrophotometer (Lambda 650; Perkin Elmer), associated with a PTP1 (Peltier Temperature Programmer) system.…”

Section: Determination Of Enzyme Activity By Spectrophotometric Assaymentioning

confidence: 99%

A comparative study of the regioselectivity of the β‐galactosidases from Kluyveromyces lactis and Bacillus circulans in the enzymatic synthesis of N‐Acetyl‐lactosamine in aqueous media

Bridiau¹,

Maugard²

2011

Biotechnology Progress

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The enzymatic synthesis of N-acetyl-lactosamine (LacNAc) was studied in aqueous media with high substrate concentrations using the transgalactosylation of N-acetyl-D-glucosamine (GlcNAc), starting from lactose as a galactosyl donor. The efficiency and regioselectivity of the β-galactosidases from Kluyveromyces lactis (KlβGal) and Bacillus circulans (BcβGal) were compared. The reaction was optimized by varying the experimental conditions (pH, catalytic activity concentration, and mass concentration ratio of the substrates), which enhanced the synthesis yields with both enzymes and especially with BcβGal. BcβGal catalyzed the formation of the maximal LacNAc concentration obtained (101 mM or 39 g L(-1), corresponding to a yield of 11% on the basis of GlcNAc conversion), after 5 h at pH 6.5 and for a substrate mass concentration ratio of 1. This enzyme also gave an optimal synthesis yield of about 17.5%. No change in regioselectivity was observed when using KlβGal, whereas the regioselectivity of BcβGal proved to be subject to variations, the 1-4 and 1-6 linkages being favored under kinetic and thermodynamic control conditions, respectively. Finally, it was demonstrated that the N-acetyl-allolactosamine synthesized during the GlcNAc transgalactosylation catalyzed by BcβGal was a thermodynamic product and did not result from a chemical and/or enzymatic isomerization of LacNAc.

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Use of Ionic Liquids to Increase the Yield and Enzyme Stability in the β-Galactosidase Catalysed Synthesis of N-Acetyllactosamine

Cited by 192 publications

References 50 publications

Influence of ionic liquid cosolvent on transgalactosylation reactions catalyzed by thermostable β-glycosylhydrolase CelB fromPyrococcus Furiosus

Influence of ionic liquid cosolvent on transgalactosylation reactions catalyzed by thermostable β-glycosylhydrolase CelB fromPyrococcus Furiosus

The bacterial ghost platform system

A comparative study of the regioselectivity of the β‐galactosidases from Kluyveromyces lactis and Bacillus circulans in the enzymatic synthesis of N‐Acetyl‐lactosamine in aqueous media

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