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Enzymatic synthesis and structural characterization of theanderose through transfructosylation reaction catalyzed by levansucrase from Bacillus subtilis CECT 39 Laura Ruiz-Aceituno, Maria Luz Sanz, Blanca de las Rivas, Rosario Muñoz, Sofia Kolida, Maria Luisa Jimeno, and F Javier Moreno J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03092 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017
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Journal of Agricultural and Food Chemistry
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Enzymatic synthesis and structural characterization of theanderose through
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transfructosylation reaction catalyzed by levansucrase from Bacillus subtilis CECT 39
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Laura Ruiz-Aceitunoa, Maria Luz Sanzb, Blanca de las Rivasc, Rosario Muñozc, Sofia
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Kolidad, Maria Luisa Jimenoe, F. Javier Moreno*a
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a
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(UAM+CSIC), Nicolás Cabrera 9, 28049 Madrid, Spain
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b
Instituto de Química Orgánica General (CSIC), Juan de la Cierva 3, 28006, Madrid, Spain
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c
Instituto de Ciencia y Tecnología de Alimentos y Nutrición, ICTAN (CSIC), Juan de la
Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), CEI
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Cierva 3, 28006 Madrid, Spain
11
d
12
UK
13
e
14
Spain
OptiBiotix Health plc, Innovation Centre, Innovation Way, Heslington, York YO10 5DG,
Centro de Quimica Organica “Lora Tamayo” (CSIC), Juan de la Cierva 3, 28006, Madrid,
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*Corresponding author:
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[email protected] 18
Tel (+34) 91 0017948
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Fax (+34) 91 0017905
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Abstract
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This work addresses the high-yield and fast enzymatic production of theanderose, a
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naturally-occurring carbohydrate, also known as isomaltosucrose, whose chemical structure
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determined by NMR is α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2)-β-D-
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fructofuranose. The ability of isomaltose to act as an acceptor in the Bacillus subtilis CECT
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39 levansucrase-catalyzed transfructosylation reaction to efficiently produce theanderose in
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the presence of sucrose as a donor is described by using four different sucrose:isomaltose
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concentration ratios. The maximum theanderose concentration ranged from 122.4 to 130.4
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gL-1, was obtained after only 1 hour and at a moderate temperature (37°C), leading to high
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productivity (109.7-130.4 gL-1h-1) and yield (up to 37.3%) values. The enzymatic synthesis
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was highly regiospecific, since no other detectable acceptor reaction products were formed.
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The development of efficient and cost-effective procedures for the biosynthesis of
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unexplored but appealing oligosaccharides as potential sweeteners, such as theanderose,
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could help to expand its potential applications which are currently limited by their low
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availability.
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Keywords: theanderose; isomaltosucrose; transfructosylation; sweetener; non-cariogenic;
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isomaltose; Bacillus subtilis CECT39.
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Journal of Agricultural and Food Chemistry
1. Introduction
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Theanderose is a non-reducing trisaccharide whose chemical structure is α-
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glucopyranosyl-(1→6)-α-glucopyranosyl-(1→2)-β-fructofuranoside. It is also known as
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isomaltosucrose,1,2 fructosylated isomaltose or isomaltosylfructoside1 and glucosyl-sucrose.
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3
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and honeys5 although at very low concentrations (less than 0.3%) which makes its isolation
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from natural sources on a large scale very unfeasible. Theanderose has been used as a
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quality marker to indicate authenticity of cane sugar based on the fact that it cannot be
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detected in beet sugar,4 although Abe et al.6 have recently described the minor presence of
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theanderose in beet sugar.
It is a naturally-occurring carbohydrate found in sugar-rich products such as cane sugar4
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Although there is scarce information on potential uses of theanderose, several beneficial
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properties have been so far attributed to its intake, such as non-cariogenicity,1, 3, 7, 8 pleasant
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taste, suitable sweetness (theanderose is the main component of the sweetener
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theandeoligo9), viscosity and humectancy, as well as a low-caloric value.10 All these
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properties could boost the interest in theanderose as a promising ingredient, for instance, in
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food products (as sugar-free confectionery or energy-reduced products), cosmetics and
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pharmaceuticals, as a sweetener, taste-improving agent, stabilizer, growth-promoting agent
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for bifidobacteria, or mineral-absorption-promoting agent,10 as long as convenient and cost-
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efficient methods can be developed for its synthesis.
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Nowadays, there is an urgent trend for reformulation to reduce free sugar content in
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foods and beverages based on advice by expert panels and regulatory bodies,11 as well as by
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the on-going implementation of governmental taxes on free sugar-sweetened beverages.12,13
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The use of high-potency sweeteners to substitute free sugars is considered a feasible, cost-
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effective and efficient strategy to reformulate free sugar-sweetened foodstuffs.14 In this
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context, there is an increasing interest in seeking natural sweeteners to provide clean flavor
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profiles and to avoid controversy over perceived health related concerns of artificial
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sweeteners.
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Theanderose can be enzymatically synthesized by using different types of glycoside
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hydrolases and starting substrates. Sucrose (α-glucopyranosyl-(1→2)-β-fructofuranoside) is
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transglucosylated to produce theanderose together with other isomaltofructosides by using
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α-glucosidases (EC 3.2.1.20) from Bacillus sp. SAM16063 or spinach15 with yields ranging
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from 10.6 to 20% (as compared to the initial substrate concentration) and requiring a
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minimum of 4 hours of enzymatic reaction. Theanderose can also be synthesized by
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transfructosylation, catalyzed mainly by microbial β-fructofuranosidases (EC 3.2.1.26) or
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levansucrases
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glucopyranose) as acceptor, and sucrose as donor. Kitahata8 and Fujita et al.1 used β-
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fructofuranosidase from Arthrobacter sp. K-1, and Nakada et al.10 used the enzyme
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produced by Bacillus sp. V230, obtaining yields of up to 28%, whereas levansucrases from
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Bacillus subtilis var Saccharolyticus7 or B. subtilis NCIMB 1187116 catalyzed the
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production of theanderose with a similar yield (18%). Additionally, a levansucrase derived
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from B. subtilis Marburg strain efficiently synthesized the trisaccharide erlose17, an isomer
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of theanderose. A specific levansucrase, obtained from B. subtilis CECT 39, has been
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successfully used to produce bioactive carbohydrates, such as lactosyl-oligofructosides18
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and lactosucrose19. Considering the promiscuous acceptor specificity of this specific
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levansucrase, together with the high-efficiency synthesis and stereo-specificity of the
(EC
2.4.1.10)
and
using
isomaltose
(α-glucopyranosyl-(1→6)-α-
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Journal of Agricultural and Food Chemistry
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reported acceptor-products, this enzyme could be a useful tool to efficiently produce
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theanderose.
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Therefore, this work addresses the high-yield and rapid production, as well as the
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structural characterization of theanderose synthesized by transfructosylation using the
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recombinant levansucrase from B. subtilis CECT 39.
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2. Materials and methods
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2.1. Reagents
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Reagents used for chromatographic analysis, including pure standards of sucrose were
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obtained from Sigma-Aldrich (St Louis, MO, USA). Isomaltose was purchased from
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Carbosynth (Compton, UK). Theanderose was a kind gift of Dr. Côté (USDA, Peoria, IL,
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USA).20 All other chemicals were of analytical grade. Ultrapure water produced in-house
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with a laboratory water purification system (Milli-Q Synthesis A10, Millipore, Billerica,
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MA, USA) was used throughout.
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2.2. Production, purification and activity assay of recombinant levansucrase enzyme
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Levansucrase (EC 2.4.1.10) from Bacillus subtilis CECT 39 (ATCC 6051) was
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overproduced in Escherichia coli and purified as previously indicated by Díez-Municio et
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al.18 The total activity of levansucrase was expressed as the amount of free glucose, while
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the amount of formed fructose was measured for the determination of the hydrolytic
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(fructosidase) activity. The transfructosylation activity (transferred fructose) was defined as
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the difference between the amount of released glucose and fructose. In consequence, the
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levansucrase expressed a total activity of 2.9 units per milligram (U mg-1), where 1 unit is 5 ACS Paragon Plus Environment
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defined as the amount of enzyme releasing 1 µmol of glucose per minute under the assayed
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conditions (that is, 37°C and a sucrose concentration of 100 g L-1 at pH 6.0 in 50 mM
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potassium phosphate buffer). The fructosidase activity was 1 U mg-1, where 1 unit is
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defined as the amount of enzyme releasing 1 µmol of fructose per minute under the assayed
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conditions. Finally, the transfructosylation activity was 1.9 U mg-1, where 1 unit is defined
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as the amount of enzyme required to transfer 1 µmol of fructose per minute at other
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molecules under the assayed conditions. Enzyme activity measurements were repeated
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three times, and the experimental error was