6
Y. Wang et al.
[2] Ibrahim MA, Rodenburg DL, Alves K, et al. Minor diterpene
Since we used crude extract from cells expressing
glycosides from the leaves of stevia rebaudiana. J. Nat. Prod.
2014;77:1231–1235.
UGT76G1 and AtSUS1, the inclusion of traces of
endogenous UDP and UDP-glucose in the reaction
mixtures was unavoidable. However, combining such
an in situ system for regeneration of UDP-glucose with
glucosyl transfer catalyzed by stevia UDP-glucosyl-
transferase was investigated in this study for in vitro
glycosylation of stevioside into rebaudioside A.
Purification and immobilization of the UGT76G1 and
AtSUS1 can probably increase the production
efficiency, which will outline the course of future work.
[3] Kolb N, Herrera JL, Ferreyra DJ, Uliana RF. Analysis of sweet
diterpene glycosides from Stevia rebaudiana: improved HPLC
Method. J. Agric. Food Chem. 2001;49:4538–4541.
[4] Brandle JE, Starratt AN, Gijzen M. Stevia rebaudiana: its agri-
cultural, biological, and chemical properties. Can. J. Plant Sci.
1998;78:527–536.
[5] Ceunen S, Geuns JM. Steviol glycosides: chemical diversity,
metabolism, and function. J. Nat. Prod. 2013;76:1201–1228.
[6] Clos JF, DuBois GE, Prakash I. Photostability of rebaudioside A
and stevioside in beverages. J. Agric. Food Chem.
2008;56:8507–8513.
[7] Wölwer-Rieck U, Tomberg W, Wawrzun A. Investigations on
the stability of stevioside and rebaudioside A in soft drinks. J.
Agric. Food Chem. 2010;58:12216–12220.
Conclusion
[8] Goyal SK, Samsher, Goyal RK. Stevia (Stevia rebaudiana) a
bio-sweetener: a review. Int. J. Food Sci. Nutr. 2010;61:1–10.
[9] Chatsudthipong V, Muanprasat C. Stevioside and related com-
pounds: therapeutic benefits beyond sweetness. Pharmacol Ther.
2009;121:41–54.
[10] Andress S. U.S. Food and Drug Administration, Agency
Response Letter GRAS Notice No. GRN 000252, CFSAN/
Office of Food Additive Safety. Whole Earth Sweetener Com-
pany LLC: Chicago, IL (2008).
UDP-glucosyltransferase UGT76G1 from S. rebaudi-
ana and sucrose synthase AtSUS1 from A. thaliana
were coexpressed in E. coli. Coupling the activities of
these two recombinant enzymes resulted in the efficient
synthesis of rebaudioside A from stevioside. It is note-
worthy that the recombinant sucrose synthase AtSUS1
produced high levels of UDP-glucose so that a small
amount of UDP could be used instead of UDP-glucose
to initiate the reaction, which can considerably increase
the cost effectiveness of the system.
[11] Brandle JE, Starratt AN, Gijzen M. Stevia rebaudiana: its agri-
cultural, biological, and chemical properties. Can. J. Plant Sci.
1998;78:527–536.
[12] Carakostas MC, Curry LL, Boileau AC, Brusick DJ. Overview:
the history, technical function and safety of rebaudioside A, a
naturally occurring steviol glycoside, for use in food and bever-
ages. Food Chem. Toxicol. 2008;46:S1–S10.
Author contributions
[13] Scipioni GP, Ferreyra DJ, Acuña MG, Schmalko ME. Rebau-
dioside A release from matrices used in a yerba maté infusion. J.
Food Eng. 2010;100:627–633.
[14] Prakash I, DuBois GE, Clos JF, Wilkens KL, Fosdick LE.
Development of rebiana, a natural, non-caloric sweetener. Food
Chem. Toxicol. 2008;46:S75–S82.
[15] Ye F, Yang R, Hua et al. Modification of stevioside using
transglucosylation activity of Bacillus amyloliquefaciens α-amy-
lase to reduce its bitter aftertaste. LWT – Food Sci. Technol.
2013;51:524–530.
Y.L. and M.Y. conceived and designed the study.
Y.W., L.L.C., and Y.Y.L. performed the experiments
and analyzed the data. Y.W. and Y.L. wrote the manu-
script. M.Y., K.Q.C., and N.H. reviewed and com-
mented on the manuscript. Y.L. and L.X. supported in
the finance. All authors have read and approved of the
manuscript.
[16] Abelyan VA, Balayan AM, Ghochikyan VT, Markosyan AA.
Transglycosylation of stevioside by cyclodextrin glucanotrans-
ferases of various groups of microorganisms. Appl. Biochem.
Microbiol. 2004;40:129–134.
[17] Jaitak V, Kaul VK, Bandna, et al. Simple and efficient enzy-
matic transglycosylation of stevioside by β-cyclodextrin glucan-
otransferase from Bacillus firmus. Biotechnol. Lett.
2009;31:1415–1420.
Disclosure statement
No potential conflict of interest was reported by the
authors.
Funding
[18] Li S, Li W, Xiao Q, Xia Y. Transglycosylation of stevioside to
improve the edulcorant quality by lower substitution using corn-
starch hydrolyzate and CGTase. Food Chem. 2013;138:
2064–2069.
[19] Humphrey TV, Richman AS, Menassa R, Brandle JE. Spatial
organisation of four enzymes from Stevia rebaudiana that are
Involved in steviol glycoside synthesis. Plant Mol. Biol.
2006;61:47–62.
[20] Richman A, Swanson A, Humphrey T, et al. Functional geno-
mics uncovers three glucosyltransferases involved in the synthe-
sis of the major sweet glucosides of Stevia rebaudiana. Plant J.
2005;41:56–67.
[21] Xiong AS, Yao QH, Peng RH, et al. A simple, rapid, high-fidelity
and cost-effective PCR-based two-step DNA synthesis method for
long gene sequences. Nucleic Acids Res. 2004;32:e98.
[22] Hoover DM, Lubkowski J. DNAWorks: an automated method
for designing oligonucleotides for PCR-based gene synthesis.
Nucleic Acids Res. 2002;30:e43.
This work was financially supported by the National
High Technology Research and Development Program
of China [grant number 2012AA022101], NSFC [grant
number 21106068], Natural Science Foundation of
Jiangshu Province of China [grant number
BK2011801], Doctoral Fund of Ministry of Education
of China [grant number 20113221120002], and Natural
Science Foundation of the Higher Education Institu-
tions of Jiangsu Province, China [grant number
10KJB530004].
References
[1] Wölwer-Rieck U. The leaves of Stevia rebaudiana (Bertoni),
their constituents and the analyses thereof: a review. J. Agric.
Food Chem. 2012;60:886–895.
[23] Bradford MM. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of pro-
tein-dye binding. Anal. Biochem. 1976;72:248–254.