Enzymatic synthesis of sialyl-Lewisa-libraries with two non-natural monosaccharide units

Baisch, Gabi; Öhrlein, Reinhold; Streiff, Markus; Frank, Konstantin

doi:10.1016/s0960-894x(98)00092-4

Cited by 26 publications

(11 citation statements)

References 15 publications

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“…Vent DNA polymerase was purchased from New England Biolabs (Beverly, Mass.). Nickel-nitrilotriacetic acid (Ni-NTA) agarose, a PCR purification kit, a QIAEX II gel extraction kit, and a DNA miniprep spin kit were obtained from Qiagen (Santa Clarita, Calif. 2 , 0.5 U of Vent DNA polymerase, and 1ϫ buffer. After the mixture was heated at 94°C for 2 min, 30 cycles consisting of 30 s at 94°C, 60 s at 55°C, and 90 s at 72°C were performed, followed by a final 10 min of elongation at 72°C.…”

Section: Methodsmentioning

confidence: 99%

Donor Substrate Regeneration for Efficient Synthesis of Globotetraose and Isoglobotetraose

Shao

Zhang

Kowal

et al. 2002

Appl Environ Microbiol

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Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAc␤133Gal␣134Gal␤134Glc) and isoglobotetraose (GalNAc␤133Gal␣133Gal␤13 4Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant ␤-1,3-N-acetylgalactosaminyltransferase from Haemophilus influenzae strain Rd can transfer N-acetylgalactosamine to a wide range of acceptor substrates with a terminal galactose residue. The donor substrate UDP-GalNAc can be regenerated by a six-enzyme reaction cycle consisting of phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, phosphate acetyltransferase, pyruvate kinase, and inorganic pyrophosphatase from Escherichia coli, as well as UDP-N-acetylglucosamine C4 epimerase from Plesiomonas shigelloides. All these enzymes were overexpressed in E. coli with six-histidine tags and were purified by one-step nickel-nitrilotriacetic acid affinity chromatography. Multiple-enzyme synthesis of globotetraose or isoglobotetraose with the purified enzymes was achieved with relatively high yields.

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Section: Methodsmentioning

confidence: 99%

Donor Substrate Regeneration for Efficient Synthesis of Globotetraose and Isoglobotetraose

Shao

Zhang

Kowal

et al. 2002

Appl Environ Microbiol

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“…Generally speaking, multiple enzyme systems have been assigned to successive reactions [10][11][12] or assistant actions to the main reactions by removing byproducts or enzyme pre-treatment [13,14]. The former approach, also called combinatorial biocatalysis, can obviate some separation steps and simplify purification procedures.…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Interesterification of Palm Stearin and Coconut Oil by a Dual Lipase System

Ibrahim

Guo

2007

J Americ Oil Chem Soc

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Enzymatic interesterification of palm stearin with coconut oil was conducted by applying a dual lipase system in comparison with individual lipase-catalyzed reactions. The results indicated that a synergistic effect occurred for many lipase combinations, but largely depending on the lipase species mixed and their ratios. The combination of Lipozyme TL IM and RM IM was found to generate a positive synergistic action at all test mixing ratios. Only equivalent amount mixtures of Lipozyme TL IM with Novozym 435 or Lipozyme RM IM with Novozym 435 produced a significant synergistic effect as well as the enhanced degree of interesterification. The interesterification catalyzed by Lipozyme TL IM mixed with thermally inactivated immobilized lipase preparations indicated that the carrier property may play an important role in affecting the interaction of two mixed lipases and the subsequent reactions. A dual enzyme system, consisting of immobilized lipases and a non-immobilized one (Lipase AK), in most cases apparently endows the free lipase with a considerably enhanced activity. 70% Lipase AK mixed with 30% immobilized lipase (Lipozyme TL IM, RM IM and Novozym 435) can achieve an increase in activity greater than 100% over the theoretical value when the reaction proceeds for 2 h. The co-immobilization action of the carrier of the immobilized lipases towards the free lipase was proposed as being one of the reasons leading to the synergistic effect and this has been experimentally verified by a reaction catalyzed by a Lipase AK-inactivated preparation. No apparently synergistic effect of the combinations of Lipozyme TL IM and RM IM was observed when the dual enzyme systems applied to the continuous reaction performed in a packed bed reactor.In brief, this work demonstrated the possibility of increasing the reaction rate or enhancing the degree of conversion by employing a dual lipase system as a biocatalyst.

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“…So far, a large number of mammalian glycosyltransferases have been cloned and employed in oligosaccharide synthesis; these enzymes include bovine α1,3-galactosyltransferase [24]and human α 2,3-sialyltransferase [25] and α1,3-fucosyltransferase [26]. However, the utility of these enzymes in large-scale synthesis of glycoconjugates is partially limited by the high cost of eukaryotic cell culture and by the low level of protein expression.…”

Section: Discussionmentioning

confidence: 99%

Reassembled Biosynthetic Pathway for a Large-scale Synthesis of CMP-Neu5Ac

Song

Zhang

et al. 2003

Marine Drugs

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CMP-Neu5Ac is an important sugar nucleotide for biosynthesis of sialic acid and its conjugates. In this paper, a large-scale production system of CMP-Neu5Ac by a single strain is reported. The co-expression of Neu5Ac aldolase (EC4.1.3.3) and CMPNeu5Ac synthetase (EC 2.7.7.43) was achieved by constructing individual genes into one plasmid and having a single culture that has both NeuAc aldolase and CMP-Neu5Ac synthetase activities. Overall this system only employed N-acetylmannosamine, excess of pyruvate and CTP to produce CMP-Neu5Ac. This work has demonstrated that a largescale synthesis of sialic acid-derived oligosaccharides could be achieved economically and efficiently through a single, biosynthetic pathway engineered microorganism.

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Enzymatic synthesis of sialyl-Lewisa-libraries with two non-natural monosaccharide units

Cited by 26 publications

References 15 publications

Donor Substrate Regeneration for Efficient Synthesis of Globotetraose and Isoglobotetraose

Donor Substrate Regeneration for Efficient Synthesis of Globotetraose and Isoglobotetraose

Enzymatic Interesterification of Palm Stearin and Coconut Oil by a Dual Lipase System

Reassembled Biosynthetic Pathway for a Large-scale Synthesis of CMP-Neu5Ac

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