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Noda Institute for Scientific Research GRANT2014 Young Investigator Research GrantOutline of Research Result

Functional analysis of cellobiose 2-epimerase and relatedenzymes and their applications for the efficient synthesis ofuseful carbohydratesWataru SaburiLaboratory of Biochemistry, Research Group of Molecular Bioscience, Division of Fundamental AgriScience Research,Research Faculty of Agriculture, Hokkaido University

Research aims

Methods

Isomerization and epimerization of carbohydrates are important reactions not only for carbohydrate metabolism butalso for the production of rare sugars from abundantlyavailable sugars. From among carbohydrate isomerases andepimerases, cellobiose 2-epimerase (CE) is the sole enzymecatalyzing the epimerization of oligosaccharides. Structuralanalysis of CE revealed that it forms a superfamily togetherwith several monosaccharide isomerases and epimerases(Fig. 1).1)This superfamily includes many proteins with unknownfunctions, and thus novel enzymes may be found amongthem. In this study, to establish the efficient synthesis ofcarbohydrates using sugar isomerases and epimerases, mutational analysis of CE and functional analysis of an enzyme with unknown functions were carried out.

1. Analysis of mutant cellobiose 2-epimerase fromRhodothermus marinus (RmCE)Arg66, Tyr124, Asn196, His200, Glu262, Tyr307,His320, and Trp322, which are situated around the substrate-binding site of RmCE, were substituted with Alausing Primestar Mutagenesis Basal Kit (Takara Bio). Themutant enzymes were produced in Escherichia coli BL21(DE3) using the pET22b (Merck)-derived expression plasmid as wild type enzyme.2) The mutant enzymes producedwere purified to homogeneity by Toyopearl DEAE 650Mand Toyopearl Butyl 650M column chromatographies. Thepurified mutant RmCEs were incubated with sophorose(Glcβ1-2Glc), laminaribiose (Glcβ1-3Glc), cellobiose(Glcβ1-4Glc), and gentiobiose (Glcβ1-6Glc). A reactionmixture (5 μL) containing 11.3 μM enzyme, 20 mM substrate, and 20 mM sodium phosphate buffer (pH 7.0) wasincubated at 60°C for 24 h. The reaction products were analyzed on thin layer chromatography (TLC), in which2-propanol/1-butanol/water (12/3/4) was used as a developing solvent. The chromatogram was visualized with a detection reagent (acetate/sulfate/anisaldehyde=100/2/1).

Fig. 1. Phylogenetic tree of CE and related enzymes.
AKI, aldose-ketose isomerase; AGE, acylglucosamine 2-epimerase.
Marme_2490 is enclosed in a box.

2. Functional analysis of a protein with unknownfunctions showing structural similarity to CERecombinant Marme_2490 protein from Marinomonasmediterranea was produced in E. coli, and its biologicalfunctions were analyzed. The Marme_2490 gene was obtained by PCR using the genomic DNA of M. mediterraneaas the template. The obtained gene was cloned into pET23a(Merck), and the recombinant protein harboring His-tag atthe C-terminal was produced in E. coli BL21 (DE3). The E.
coli transformant harboring the expression plasmid wascultured in 1 L of LB broth containing 100 μg/mL ampicillin at 37°C, and the protein expression was induced by 0.1mM isopropyl β-d-thiogalactopyranoside. The inductionculture was incubated at 16°C for 20 h. The recombinantprotein was purified from the cell-free extract by the Nichelating column chromatography. Purified protein was incubated with various carbohydrates (40 mμ) at 37°C. Thereaction products were analyzed on TLC, and a developing32Table 1.

Kinetic parameters of Marme_2490 and related enzymes

D-Mannose

D-Lyxose

D-Talose

Enzymekcat(s−1)

Km(mM)

kcat/Km(s−1mM−1)

kcat(s−1)

Km(mM)

kcat/Km(s−1mM−1)

kcat(s−1)

Km(mM)

kcat/Km(s−1mM−1)

Marme_2490EcAKISeAKITfMI

32925.323.3788

16.7108134115

19.70.2340.1746.85

64.413.5N.D.
63.342.3405N.D.
537

1.520.033N.D.
0.118

0.319N.D.
N.D.
N.D.

27.8N.D.
N.D.
N.D.

0.0115N.D.
N.D.
N.D.

EcAKI, E. coli aldose-ketose isomerase; SeAKI, Salmonella enterica aldose-ketose isomerase; TfMI, Thermobifida fusca MI.

bated with various carbohydrates, and the reaction productswere analyzed. This protein showed high isomerization activity to d-mannose, indicating that Marme_2490 is a typeof d-mannose isomerase (MI) (Fig. 2). The kcat/Km value ofthis enzyme for d-mannose was 2.9–113-fold higher of anyother reported MI (Table 1). Marme_2490 exhibited isomerization activity to d-lyxose, similar to other MIs, but incontrast to other MIs, it had isomerization activity to dtalose (4-epimer of d-mannose) and β1-4 linked oligosaccharides. It generated Manβ1-4Fru, lactulose, and cellobiulose from β1-4mannobiose, lactose, and cellobiose, respectively.
Fig. 2. Screening of substrate of Marme_2490.
Plus and minus indicate with and without enzyme, respectively. Arrows indicate reaction products.

Conclusion

solvent, 2-propanol/1-butanol/water (2/2/1) was used. Reaction products were purified by TLC, and their chemicalstructures were determined by electrospray ionization massspectrometry and nuclear magnetic resonance. In the kinetic analysis, reaction rates were determined based on theamount of d-fructose, d-tagatose, and d-xylulose producedfrom d-mannose, d-talose, and d-lyxose, respectively.

In this study, a MI, Marme_2490, with novel substratespecificity was discovered from M. mediterranea.
Marme_2490 has isomerization activity to disaccharidesubstrates and d-talose, unlike other reported enzymes.
Thus it is considered to be useful for carbohydrate conversion. Molecular basis for the substrate specificity ofMarme_2490 is required to be elucidated for better understanding of carbohydrate isomerases and epimerases.

Results

References

1. Site-directed mutagenesis study of RmCEChange of substrate specificity through the site-directedmutations at the substrate-binding site was expected; however, besides cellobiose, the mutant enzymes did not showepimerization activity to other glucobioses.

1)

2. Biochemical functions of Marme_2490 from M.
mediterraneaRecombinant Marme_2490 produced in E. coli was incu-2)

Fujiwara, T., Saburi, W., Matsui, H., Mori, H., and Yao, M. (2014)Structural insights into the epimerization of β-1,4-linked oligosaccharides catalyzed by cellobiose 2-epimerase, the sole enzymeepimerizing non-anomeric hydroxyl groups on unmodified sugars. J.
Biol. Chem. 289: 3405–3415.
Ojima, T., Saburi, W., Sato, H., Yamamoto, T., Mori, H., and Matsui,H. (2011) Biochemical characterization of a thermophilic cellobiose2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785. Biosci. Biotechnol. Biochem. 75: 2162–2168.