Enzymatic Synthesis and Structural Characterization of Theanderose

Nov 13, 2017 - Instituto de Química Orgánica General (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain. § Instituto de Ciencia y Tecnología de Alim...
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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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

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d

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UK

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e

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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]

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Tel (+34) 91 0017948

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Fax (+34) 91 0017905

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Page 2 of 34

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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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