ISOLATION AND SOME PROPERTIES OF THE THERMOSTABLE Β-Galactosidase OF PYROCOCCUS WOESEI EXPRESSED IN ESCHERICHIA COLI

Synowdecki, Jozef; Maciuńska, Jadwiga

doi:10.1111/j.1745-4514.2002.tb00049.x

Cited by 7 publications

(7 citation statements)

References 14 publications

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“…We first investigated the substrate spectrum for the hydrolysis of various nitrophenyl (NP)-glycosides. Kinetic data between 0.8 and 2.9 mM were already described [ 27 , 30 ]. We therefore tested substrate concentrations between 15 and 30 mM for optimal activity measurements ( Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…In this study, we focused on Pyrococcus woesei β- d -galactosidase (GenBank accession number AF043283.1) clustered in glycoside hydrolase 1 (GH-1) family with a retaining catalytic mechanism. The hyperthermophilic enzyme has a high sequence identity (99.8%) to the β-galactosidase of Pyrococcus furiosus [ 25 ] and has been recombinantly produced in E. coli expression strains and characterized for its hydrolytic activity [ 25 , 26 , 27 , 28 ]. Directed evolution of the synthetic gene (GenBank accession number EF090269) switched the β-galactosidase activity to β-glucuronidase activity by exchange of seven key amino acid residues [ 29 ].…”

Section: Introductionmentioning

confidence: 99%

“…However, this enzyme originating from shallow marine volcanic vents [ 31 ] facilitates universal biotechnological applications supported by the choice of donor substrates at very high temperatures. Optimal hydrolytic enzymatic activities were reported up to 100 °C [ 27 , 30 ].…”

Section: Introductionmentioning

confidence: 99%

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Microwave-Assisted Synthesis of Glycoconjugates by Transgalactosylation with Recombinant Thermostable β-Glycosidase from Pyrococcus

Henze¹,

Merker²,

Elling³

2016

IJMS

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The potential of the hyperthermophilic β-glycosidase from Pyrococcus woesei (DSM 3773) for the synthesis of glycosides under microwave irradiation (MWI) at low temperatures was investigated. Transgalactosylation reactions with β-N-acetyl-d-glucosamine as acceptor substrate (GlcNAc-linker-tBoc) under thermal heating (TH, 85 °C) and under MWI at 100 and 300 W resulted in the formation of (Galβ(1,4)GlcNAc-linker-tBoc) as the main product in all reactions. Most importantly, MWI at temperatures far below the temperature optimum of the hyperthermophilic glycosidase led to higher product yields with only minor amounts of side products β(1,6-linked disaccharide and trisaccharides). At high acceptor concentrations (50 mM), transgalactosylation reactions under MWI at 300 W gave similar product yields when compared to TH at 85 °C. In summary, we demonstrate that MWI is useful as a novel experimental set-up for the synthesis of defined galacto-oligosaccharides. In conclusion, glycosylation reactions under MWI at low temperatures have the potential as a general strategy for regioselective glycosylation reactions of hyperthermophilic glycosidases using heat-labile acceptor or donor substrates.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Microwave-Assisted Synthesis of Glycoconjugates by Transgalactosylation with Recombinant Thermostable β-Glycosidase from Pyrococcus

Henze¹,

Merker²,

Elling³

2016

IJMS

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“…Cells were lysed in homogenizer at 2000 psig in two passes. Ultrafiltration and gel column chromatography (Synowiecki 2002) were employed for the purification of lactase. The specific activity and fold purification of lactase was found to be 568.61 & 21.2 respectively ( Table 2).…”

Section: Downstream Processing and Characterization Of Lactasementioning

confidence: 99%

Lactase Production from Lactobacillus acidophilus

Akolkar

Sajgure

Lele

2005

World J Microbiol Biotechnol

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A lactobacillus strain isolated from fermented Ragi (Eleusine coracana Gaertn.) was characterized as Lactobacillus acidophilus. The isolate was found to be homofermentative, slime-forming and a lactase (b-galactosidase) producer. Production, recovery, characterization and performance of lactase were studied at laboratory scale from 100 ml to 5 l under stationary and stirred conditions. 1.5% lactose was found to be the best carbon source for lactase production. The lactose content could be reduced to 0.75% by supplementing with 1% ragi, thus making the media economically more attractive. A 6.5-fold increase (5400 U ml )1 ) was achieved on scale-up. Performance of the lactase obtained from this strain was found to be slightly better than the commercial lactase produced by Kluyveromyces lactis.

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“…coli − In our previous study, we characterized and utilized recombinant Pw Gly for the synthesis of defined galacto-oligosaccharides using microwave irradiation . We herein report on the synthesis of Gal-EMA on gram scale by recombinant Pw Gly and utilization of Gal-EMA in glycopolymer synthesis for lectin-binding studies (Scheme ).…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Synthesis of 2-(β-Galactosyl)-ethyl Methacrylate by β-Galactosidase fromPyrococcus woeseiand Application for Glycopolymer Synthesis and Lectin Studies

et al. 2020

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Glycosidases have long been used for the synthesis of glycosides by transglycosylation reactions. Especially glycosidases from hyperthermophilic bacteria are useful for reactions under extreme reaction conditions, e.g., in the presence of organic solvents. We herein report the facile enzymatic synthesis and purification of 2-(β-galactosyl)-ethyl methacrylate (Gal-EMA) with the recombinant hyperthermostable glycosidase from Pyrococcus woesei in high yields. Optimized reaction conditions resulted in gram-scale synthesis of the galactosylated monomer with 88% transglycosylation yield. The product Gal-EMA was characterized by high-performance liquid chromatography−electrospray ionization−mass spectrometry (HPLC-ESI-MS), nuclear magnetic resonance (NMR) spectroscopy, and infrared (IR) spectroscopy. Gal-EMA was utilized to synthesize sugar-functionalized acrylate polymers with defined amounts of incorporated galactose (0−100%). Analysis of the binding affinity of the lectin RCA 120 from Ricinus communis to the glycopolymers using an enzyme-linked lectin assay (ELLA) revealed K D values between 0.24 and 6.2 nM, depending on the amount of incorporated Gal-EMA. The potential of Gal-EMA for the synthesis of acrylate-functionalized glycan oligomers was demonstrated by sequential elongation of the terminal galactose by two glycosyltransferases, resulting in the terminal glycan N-acetyllactosamine (LacNAc) epitope. In conclusion, the enzymatic synthesis of Gal-EMA opens new routes to a series of novel monomeric building blocks for the synthesis of glycan-functionalized polyacrylates.

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ISOLATION AND SOME PROPERTIES OF THE THERMOSTABLE Β-Galactosidase OF PYROCOCCUS WOESEI EXPRESSED IN ESCHERICHIA COLI

Cited by 7 publications

References 14 publications

Microwave-Assisted Synthesis of Glycoconjugates by Transgalactosylation with Recombinant Thermostable β-Glycosidase from Pyrococcus

Microwave-Assisted Synthesis of Glycoconjugates by Transgalactosylation with Recombinant Thermostable β-Glycosidase from Pyrococcus

Lactase Production from Lactobacillus acidophilus

Enzymatic Synthesis of 2-(β-Galactosyl)-ethyl Methacrylate by β-Galactosidase fromPyrococcus woeseiand Application for Glycopolymer Synthesis and Lectin Studies

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