Development of a nucleotide sugar purification method using a mixed mode column &amp; mass spectrometry detection

Eastwood, Heather; Xia, Fang; Lo, Mei-Chu; Zhou, Jing; Jordan, John B.; McCarter, John D.; Barnhart, Wesley W.; Gahm, Kyung-Hyun

doi:10.1016/j.jpba.2015.08.001

Cited by 7 publications

(2 citation statements)

References 23 publications

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“…The exploitation of Mf GM35E for production on a preparative scale would allow the synthesis of about 15 g/L GDP- l -Gal and 5 g/L GDP- l -Gul starting from 100 g/L GDP-Man. Subsequently, the recovery of the nucleotide sugars might be realized by an anion exchange chromatography protocol [58,59]. Interestingly, the availability of these NDP-sugars forms a gateway to glycorandomization, a process in which the carbohydrate moiety of antibiotics is altered in order to tailor their pharmacological properties and/or biological activity [60,61,62].…”

Section: Discussionmentioning

confidence: 99%

Characterization of the First Bacterial and Thermostable GDP-Mannose 3,5-Epimerase

Gevaert

Overtveldt

Beerens

et al. 2019

IJMS

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GDP-mannose 3,5-epimerase (GM35E) catalyzes the conversion of GDP-mannose towards GDP-l-galactose and GDP-l-gulose. Although this reaction represents one of the few enzymatic routes towards the production of l-sugars and derivatives, it has not yet been exploited for that purpose. One of the reasons is that so far only GM35Es from plants have been characterized, yielding biocatalysts that are relatively unstable and difficult to express heterologously. Through the mining of sequence databases, we succeeded in identifying a promising bacterial homologue. The gene from the thermophilic organism Methylacidiphilum fumariolicum was codon optimized for expression in Escherichia coli, resulting in the production of 40 mg/L of recombinant protein. The enzyme was found to act as a self-sufficient GM35E, performing three chemical reactions in the same active site. Furthermore, the biocatalyst was highly stable at temperatures up to 55 °C, making it well suited for the synthesis of new carbohydrate products with application in the pharma industry.

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Section: Discussionmentioning

confidence: 99%

Characterization of the First Bacterial and Thermostable GDP-Mannose 3,5-Epimerase

Gevaert

Overtveldt

Beerens

et al. 2019

IJMS

View full text Add to dashboard Cite

show abstract

“…Since the late 1970s, several HPLC methods have been developed to purify and analyze sugar nucleotides [10] , [11] , [12] , [13] . In addition, Mass Spectrometry [11] , [24] , [25] , capillary electrophoresis [26] , [27] , and NMR methods [11] , [28] , [29] have been put forth to study sugar nucleotide levels. However, these instruments are expensive and require extensive expertise to operate.…”

Section: Methods Detailsmentioning

confidence: 99%