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Noda Institute for Scientific Research GRANT2013 Young Investigator Research Grant

Isolation, identification, and characterization of a novelpolysaccharide degradation enzyme for synthesis ofnovel oligosaccharidesMasahiro NakajimaDepartment of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science

Research aimsA recent study identified a novel phosphorylase that actson sophorose (Glc-β-1,2-Glc) in reverse reaction (OGP),thereby providing a simpler enzymatic method for the synthesis of β-1,2-glucan and β-1,2-glucooligosaccharides(Sopns) from sophorose. Sophorose, an expensive sugar,can be replaced by glucose as a starting material for thesynthesis of Sopns. Although polymers are easily producedat an early stage of this reaction, the accumulation of Sopnsis considerably slower when using glucose as a starting material, compared to sophorose. The addition of a β-1,2glucan degrading enzyme to the reaction system wouldlikely accelerate oligosaccharide synthesis; however, suchan enzyme has yet to be identified. The aims of this studyare described below:1. Develop a method for the large-scale synthesis ofβ-1,2-glucan using OGP.
2. Isolate and identify an enzyme that degrades β-1,2glucan, using the synthesized polymer.
MethodsWe first developed a method for large-scale synthesis ofβ-1,2-glucan. As depicted in Fig. 1, two steps were com-Fig. 1. A schematic of 1,2-β-glucan synthesis.
The parenthesis represents an intermediate of the reaction.

bined to construct a one-pot reaction system: The phosphorolysis of sucrose by sucrose phosphorylase (SP),1) generating fructose and glucose 1-phosphate (Glc1P), and the utilization of this Glc1P for β-1,2-glucan synthesis by OGP.
The optimal synthesis conditions were determined bymeasuring the levels of β-1,2-glucan production at an earlystage of the reaction, using thin layer chromatography(TLC). The glucose concentration was optimized first, inthe presence of fixed concentrations of sucrose and inorganic phosphate (1.0 M and 0.1 M, respectively), followedby the SP to OGP ratio. Large-scale synthesis reactions(2 L) were then prepared and incubated for 12 days untilthe available sucrose in the media was spent. Reaction enzymes were removed from the mixture using the anion-exchange resin DEAE cellulose, and the solution was treatedwith yeast to remove unnecessary sugars. β-1,2-Glucan wasprecipitated by mixing the reaction solution with an equalvolume of ethanol, and the resulting precipitate was dried.
To identify β-1,2-glucan-degrading enzymes, five bacterial species were cultured using purified β-1,2-glucan as asole carbon source: Listeria innocua, which possesses theogp gene used in this study; Enterococcus faecalis, whichpossesses an ogp homolog, and whose genome sequence isavailable; E. coli; Chitinophaga arvensicola, the only bacterium previously reported to exhibit hydrolytic activity onβ-1,2-glucan2); and Chitinophaga pinensis, a species relatedto C. arvensicola, and whose genome sequence is available.
Culture filtrates and cell free extracts were assayed for degradation of β-1,2-glucan by TLC and detection of reducingactivity. The β-1,2-glucan degrading enzyme was purifiedusing three chromatography steps. Peptides extracted fromthe purified enzyme by in-gel digestion using trypsin orchymotrypsin were analyzed by LC/MS/MS. A pool of peptide sequences was constructed by de novo sequencingusing PEAKS online, and high confidence peptides wereselected from this pool. A candidate gene was selected byperforming a BLAST search of the selected peptides. Theselected gene was labelled with a His-tag sequence and expressed in E. coli. The recombinant gene product was purified by nickel column chromatography and assayed for substrate specificity and degradation of β-1,2-glucan.

42ResultsLarge-scale synthesis of β-1,2-glucanWe first compared β-1,2-glucan synthesis in the presenceof various concentrations of glucose, at an early stage ofthe reaction. β-1,2-glucan production increased in a glucose-dependent manner up to a concentration of 0.5 M. Asglucose concentrations greater than 0.5 M failed to influence β-1,2-glucan production, 0.5 M was utilized as the optimal glucose concentration for the synthesis reaction.
Next, we assessed the effects of various SP to OGP ratioson β-1,2-glucan synthesis. Reducing the SP concentrationto one-fourth the concentration of OGP did not significantlyaffect the rate of β-1,2-glucan synthesis. As a result, a 1 : 4ratio of OGP to SP was utilized. A large-scale reaction wasthen performed using the optimized conditions. The reaction was carried out for 12 days and yielded over 800 mMof β-1,2-glucan (glucose concentration equivalent). Afterprecipitation, the final amount of purified β-1,2-glucan wasapproximately 167 g from 1 L of the reaction mixture.
Purification and identification of a β-1,2-glucandegrading enzymeOf the bacterial species tested, only C. arvensicola exhibited significant growth in media containing β-1,2-glucanas a sole carbon source. The degrading activity of β-1,2glucan was significantly greater in the cell free extract thanin the culture filtrate. As a result, the bacterial cell free extract was utilized to purify and identify the putative β-1,2glucan-degrading enzyme. After separation by SDS-PAGE,a 45-kDa band was identified that was present only in fractions that exhibited hydrolytic activity on β-1,2-glucan.
Peptides were extracted from this band by treatment withtrypsin or chymotrypsin and analyzed by LC/MS/MS. Mascot analysis failed to obtain a confident candidate for aβ-1,2-glucan degrading enzyme from C. pinensis, a relatedbacterium whose genome is available. We therefore generated a de novo sequence using the two results of LC/MS/MS analysis after trypsin and chymotrypsin treatment.
Seven peptide sequences were common between the twodigests and were selected for use in a BLAST search. Fourof the seven peptides exhibited identity to the protein, SGL.
The recombinant SGL degraded β-1,2-glucan, in vitro, withsufficient velocity to release oligosaccharides with DP of2–5 as end-products. These results indicate that SGL is indeed a β-1,2-glucan-degrading enzyme.
ConclusionWe generated a large-scale method for production ofβ-1,2-glucan from sucrose and glucose. SGL was identifiedfrom the cell free extract of C. arvensicola cultured withβ-1,2-glucan as a sole carbon source. While in vitro analyses verified that SGL is capable of degrading β-1,2-glucan,the amino acid sequence of SGL suggests it does not belong to any known glycoside hydrolase (GH) family andmay therefore comprise a new family of GHs.
References1)2)

Nakai, H., Kitaoka, M., Svensson, B., and Ohtsubo, K. (2013) Recent development of phosphorylases possessing large potential foroligosaccharide synthesis. Curr. Opin. Chem. Biol. 17: 301–309.
Mendoza, N.S., and Amemura, A. (1983) (1→2)-β-d-Glucanhydrolyzing enzymes in Cytophaga arvensicola. J. Ferment. Technol. 61: 473–481.