Subscriber access provided by READING UNIV
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
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.
Page 1 of 34
Journal of Agricultural and Food Chemistry
1
Enzymatic synthesis and structural characterization of theanderose through
2
transfructosylation reaction catalyzed by levansucrase from Bacillus subtilis CECT 39
3 4
Laura Ruiz-Aceitunoa, Maria Luz Sanzb, Blanca de las Rivasc, Rosario Muñozc, Sofia
5
Kolidad, Maria Luisa Jimenoe, F. Javier Moreno*a
6
a
7
(UAM+CSIC), Nicolás Cabrera 9, 28049 Madrid, Spain
8
b
Instituto de Química Orgánica General (CSIC), Juan de la Cierva 3, 28006, Madrid, Spain
9
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
10
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,
15 16
*Corresponding author:
17
[email protected] 18
Tel (+34) 91 0017948
19
Fax (+34) 91 0017905
1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
20
Page 2 of 34
Abstract
21
This work addresses the high-yield and fast enzymatic production of theanderose, a
22
naturally-occurring carbohydrate, also known as isomaltosucrose, whose chemical structure
23
determined by NMR is α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2)-β-D-
24
fructofuranose. The ability of isomaltose to act as an acceptor in the Bacillus subtilis CECT
25
39 levansucrase-catalyzed transfructosylation reaction to efficiently produce theanderose in
26
the presence of sucrose as a donor is described by using four different sucrose:isomaltose
27
concentration ratios. The maximum theanderose concentration ranged from 122.4 to 130.4
28
gL-1, was obtained after only 1 hour and at a moderate temperature (37°C), leading to high
29
productivity (109.7-130.4 gL-1h-1) and yield (up to 37.3%) values. The enzymatic synthesis
30
was highly regiospecific, since no other detectable acceptor reaction products were formed.
31
The development of efficient and cost-effective procedures for the biosynthesis of
32
unexplored but appealing oligosaccharides as potential sweeteners, such as theanderose,
33
could help to expand its potential applications which are currently limited by their low
34
availability.
35 36
Keywords: theanderose; isomaltosucrose; transfructosylation; sweetener; non-cariogenic;
37
isomaltose; Bacillus subtilis CECT39.
2 ACS Paragon Plus Environment
Page 3 of 34
38
Journal of Agricultural and Food Chemistry
1. Introduction
39
Theanderose is a non-reducing trisaccharide whose chemical structure is α-
40
glucopyranosyl-(1→6)-α-glucopyranosyl-(1→2)-β-fructofuranoside. It is also known as
41
isomaltosucrose,1,2 fructosylated isomaltose or isomaltosylfructoside1 and glucosyl-sucrose.
42
3
43
and honeys5 although at very low concentrations (less than 0.3%) which makes its isolation
44
from natural sources on a large scale very unfeasible. Theanderose has been used as a
45
quality marker to indicate authenticity of cane sugar based on the fact that it cannot be
46
detected in beet sugar,4 although Abe et al.6 have recently described the minor presence of
47
theanderose in beet sugar.
It is a naturally-occurring carbohydrate found in sugar-rich products such as cane sugar4
48
Although there is scarce information on potential uses of theanderose, several beneficial
49
properties have been so far attributed to its intake, such as non-cariogenicity,1, 3, 7, 8 pleasant
50
taste, suitable sweetness (theanderose is the main component of the sweetener
51
theandeoligo9), viscosity and humectancy, as well as a low-caloric value.10 All these
52
properties could boost the interest in theanderose as a promising ingredient, for instance, in
53
food products (as sugar-free confectionery or energy-reduced products), cosmetics and
54
pharmaceuticals, as a sweetener, taste-improving agent, stabilizer, growth-promoting agent
55
for bifidobacteria, or mineral-absorption-promoting agent,10 as long as convenient and cost-
56
efficient methods can be developed for its synthesis.
57
Nowadays, there is an urgent trend for reformulation to reduce free sugar content in
58
foods and beverages based on advice by expert panels and regulatory bodies,11 as well as by
59
the on-going implementation of governmental taxes on free sugar-sweetened beverages.12,13
60
The use of high-potency sweeteners to substitute free sugars is considered a feasible, cost-
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 34
61
effective and efficient strategy to reformulate free sugar-sweetened foodstuffs.14 In this
62
context, there is an increasing interest in seeking natural sweeteners to provide clean flavor
63
profiles and to avoid controversy over perceived health related concerns of artificial
64
sweeteners.
65
Theanderose can be enzymatically synthesized by using different types of glycoside
66
hydrolases and starting substrates. Sucrose (α-glucopyranosyl-(1→2)-β-fructofuranoside) is
67
transglucosylated to produce theanderose together with other isomaltofructosides by using
68
α-glucosidases (EC 3.2.1.20) from Bacillus sp. SAM16063 or spinach15 with yields ranging
69
from 10.6 to 20% (as compared to the initial substrate concentration) and requiring a
70
minimum of 4 hours of enzymatic reaction. Theanderose can also be synthesized by
71
transfructosylation, catalyzed mainly by microbial β-fructofuranosidases (EC 3.2.1.26) or
72
levansucrases
73
glucopyranose) as acceptor, and sucrose as donor. Kitahata8 and Fujita et al.1 used β-
74
fructofuranosidase from Arthrobacter sp. K-1, and Nakada et al.10 used the enzyme
75
produced by Bacillus sp. V230, obtaining yields of up to 28%, whereas levansucrases from
76
Bacillus subtilis var Saccharolyticus7 or B. subtilis NCIMB 1187116 catalyzed the
77
production of theanderose with a similar yield (18%). Additionally, a levansucrase derived
78
from B. subtilis Marburg strain efficiently synthesized the trisaccharide erlose17, an isomer
79
of theanderose. A specific levansucrase, obtained from B. subtilis CECT 39, has been
80
successfully used to produce bioactive carbohydrates, such as lactosyl-oligofructosides18
81
and lactosucrose19. Considering the promiscuous acceptor specificity of this specific
82
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-
4 ACS Paragon Plus Environment
Page 5 of 34
Journal of Agricultural and Food Chemistry
83
reported acceptor-products, this enzyme could be a useful tool to efficiently produce
84
theanderose.
85
Therefore, this work addresses the high-yield and rapid production, as well as the
86
structural characterization of theanderose synthesized by transfructosylation using the
87
recombinant levansucrase from B. subtilis CECT 39.
88 89
2. Materials and methods
90 91
2.1. Reagents
92
Reagents used for chromatographic analysis, including pure standards of sucrose were
93
obtained from Sigma-Aldrich (St Louis, MO, USA). Isomaltose was purchased from
94
Carbosynth (Compton, UK). Theanderose was a kind gift of Dr. Côté (USDA, Peoria, IL,
95
USA).20 All other chemicals were of analytical grade. Ultrapure water produced in-house
96
with a laboratory water purification system (Milli-Q Synthesis A10, Millipore, Billerica,
97
MA, USA) was used throughout.
98 99
2.2. Production, purification and activity assay of recombinant levansucrase enzyme
100
Levansucrase (EC 2.4.1.10) from Bacillus subtilis CECT 39 (ATCC 6051) was
101
overproduced in Escherichia coli and purified as previously indicated by Díez-Municio et
102
al.18 The total activity of levansucrase was expressed as the amount of free glucose, while
103
the amount of formed fructose was measured for the determination of the hydrolytic
104
(fructosidase) activity. The transfructosylation activity (transferred fructose) was defined as
105
the difference between the amount of released glucose and fructose. In consequence, the
106
levansucrase expressed a total activity of 2.9 units per milligram (U mg-1), where 1 unit is 5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 34
107
defined as the amount of enzyme releasing 1 µmol of glucose per minute under the assayed
108
conditions (that is, 37°C and a sucrose concentration of 100 g L-1 at pH 6.0 in 50 mM
109
potassium phosphate buffer). The fructosidase activity was 1 U mg-1, where 1 unit is
110
defined as the amount of enzyme releasing 1 µmol of fructose per minute under the assayed
111
conditions. Finally, the transfructosylation activity was 1.9 U mg-1, where 1 unit is defined
112
as the amount of enzyme required to transfer 1 µmol of fructose per minute at other
113
molecules under the assayed conditions. Enzyme activity measurements were repeated
114
three times, and the experimental error was
