Journal of Agricultural and Food Chemistry
ARTICLE
also observed in the glucan synthesized by the triple mutant
(S642N/E643N/V644S)-containing mutation, with reduction of
the quantity of R-(1f6) linkages to 40% and increased amounts of
R-(1f3) and R-(1f4) linkages (Table 4; Figure 3). The less
water-soluble glucans of S642N/E643N/V644S and V532P/
V535I/S642N/E643N/V644S contained relatively high propor-
tions of nonreducing ends (12.0 and 13.9%, respectively), due to
branching through both the R-(1f3) and R-(1f4) linkages. The
data suggest that both mutations in conserved regions II and IV of
DSRBCB4 transformed the DSRBCB4 dextransucrase from linear
dextran formation into a branched and less water-slouble glucan
formation.43 A similar result was reported with amylosucrase. In
amylosucrase, the D394, next to the catalytically important H392
and D393 (transition state stabilizer), was shown to be involved in
the correct positioning of the glucosyl residue at this site.44 Mutant
D394A displayed changes in the product formation specificity
(mono- and oligosaccharides) from sucrose.45
The significant changes observed in the triple and combinative
mutant enzymes, with respect to activity for sucrose, linkages,
and solubility of oligosaccharide and glucan synthesized, strongly
support the hypothesis that residues in conserved region IV are
involved in acceptor substrate binding in glucansucrases. Many
mutansucrases possess the same triplet amino acids SEV in
region IV as DSRBCB4 mutant S642N/E643N/V644S, and
instead of synthesizing dextran, mutansucrase synthesizes mu-
tans. Furthermore, the higher amount of R-(1f3) glucosidic
linkages synthesized by mutated DSRBCB4 could also be
explained by the region IV replaced in this study.
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In summary, we demonstrate that the oligosaccharide product
and glycosidic linkage specificity of DSRBCB4 can be modified
by rational site-directed mutagenesis, while maintaining high
transglycosylation efficiency, resulting in excellent product
yields. Enzyme engineering allowing the synthesis of tailor-made
polysaccharides and oligosaccharides for a range of applications
is in progress.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: dmkim@chonnam.ac.kr. Phone: þ62825301844. Fax:
þ62825301849.
Funding Sources
This work was partially supported by Priority Research Centers
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and
Technology (Grant 2010-0029626).
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charide development in Japan. Pure Appl. Chem. 2002, 74, 1245–1251.
(18) Buchholz, K.; Seibel, J. Isomaltooligosaccharides. In Oligosac-
charides in Food and Agriculture; Eggleston, G., Cote, G. L., Eds.; Oxford
University Press: Washington, DC, 2003.
’ ACKNOWLEDGMENT
We are thankful to the Korea Basic Science Institute Gwang-Ju
Branch for the DNA sequencing and NMR analysis.
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(Palatinose (R)): a review of biological and toxicological studies. Food
Chem. Toxicol. 2002, 40, 1375–1381.
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