Mechanism‐Oriented Redesign of an Isomaltulose Synthase to an Isomelezitose Synthase by Site‐Directed Mutagenesis

Görl, Julian; Timm, Malte; Seibel, Jürgen

doi:10.1002/cbic.201100576

Cited by 16 publications

(20 citation statements)

References 33 publications

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“…This result is consistent with the conclusion that Phe256 and Phe280 in MutB were essential for product specificity [13,25]. Gorl et al observed a similar result that mutagenesis of Phe297 and Phe321 in SmuA abolished the isomaltulose synthesis activity [48]. …”

Section: Resultssupporting

confidence: 88%

The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

et al. 2013

PLoS ONE

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Sucrose isomerase NX-5 from Erwinia rhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonas mesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations.

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Section: Resultssupporting

confidence: 88%

The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

et al. 2013

PLoS ONE

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“…As previously mentioned, several microbial α‐glucosidases has been already used to produce isomelezitose, a rare trisaccharide found in honey, which shows potential nutraceutical and prebiotic properties (Görl et al , ). However, and as far as we know, the obtained yields when using native enzymes or cellular extracts were lower than those reported in this work.…”

Section: Resultsmentioning

confidence: 99%

“…In this context, Munir and Vogel () patented the synthesis of isomelezitose using immobilized cells of Protaminobacter and 50ºC with yields close to 11% of total sugars. Besides, Görl et al () transformed a sucrose isomerase into an isomelezitose synthase, obtaining a protein variant by mutation that yielded 70% of isomelezitose using 100 g l −1 sucrose, but this amount was calculated based on sucrose consumed (~ 32%) instead of the total sucrose added to the reaction mixture, which means to produce about 22.4 g l −1 of this trisaccharide. More recently, Côté et al () obtained an engineered glucansucrase from the bacteria Leuconostoc mesenteroides that produced isomelezitose very efficiently using ~ 800 g l −1 sucrose.…”

Section: Resultsmentioning

confidence: 99%

Efficient production of isomelezitose by a glucosyltransferase activity in Metschnikowia reukaufii cell extracts

García-González

Plou

Cervantes

et al. 2019

Microbial Biotechnology

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Summary Metschnikowia reukaufii is a widespread yeast able to grow in the plants’ floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l−1, which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload‐sucrose reactions. These products were purified and structurally characterized by MS‐ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α‐(1→1), α‐(1→3) and α‐(1→6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l−1, the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l−1 of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications.

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“…Although several uses for isomelezitose have been proposed, including prebiotics [7] and pharmaceutical excipients [Backström et al, 1999, Powder formulations containing melezitose as a diluents, US Patent 6,004,574. ], applications are limited due to the high cost and relative scarcity of this compound.…”

Section: Streptococcus Sobrinus 6715 Gtfi Variantsmentioning

confidence: 99%

“…], applications are limited due to the high cost and relative scarcity of this compound. Isomelezitose has been isolated in small amounts from several enzymatic reaction mixtures [1,4,5], but only one instance of a high-yielding synthesis has been reported [7]. In that example, the yield was reportedly over 70% from sucrose, but that number was calculated from the amount of sucrose consumed, not from the total sucrose added to the reaction.…”

Section: Streptococcus Sobrinus 6715 Gtfi Variantsmentioning

confidence: 99%

Production of isomelezitose from sucrose by engineered glucansucrases

Côté

Dunlap

Vermillion

et al. 2017

Amylase

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Certain lactic acid bacteria produce glycosyltransferases known as glucansucrases, which synthesize α-D-glucans via glucosyl transfer from sucrose. We recently reported on the formation of the unusual trisaccharide isomelezitose in low yields by a variety of glucansucrases. Isomelezitose is a rare non-reducing trisaccharide, with the structure α-d-glucopyranosyl- (1→6)-β-d-fructofuranosyl-(2↔1)-α-d-glucopyranoside. In this work, we describe the synthesis of isomelezitose in high yields by variants of glucansucrases engineered to contain a single point mutation at a key leucine residue involved in acceptor substrate binding. Some variants produce isomelezitose in yields up to 57%. This method is amenable to large-scale production of isomelezitose for food, industrial and biomedical applications.

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Mechanism‐Oriented Redesign of an Isomaltulose Synthase to an Isomelezitose Synthase by Site‐Directed Mutagenesis

Cited by 16 publications

References 33 publications

The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

Efficient production of isomelezitose by a glucosyltransferase activity in Metschnikowia reukaufii cell extracts

Production of isomelezitose from sucrose by engineered glucansucrases

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