Biotechnology Advances xxx (xxxx) xxx–xxx

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Inulin and its enzymatic production by inulosucrase: Characteristics,

structural features, molecular modifications and applications
⁎
Dawei Nia, Wei Xua, Yingying Zhua, Wenli Zhanga, Tao Zhanga, Cuie Guanga, Wanmeng Mua,b,
a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China
b
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, Jiangsu 214122, China

A R T I C LE I N FO A B S T R A C T

Keywords: Inulin, a natural fructan, cannot be hydrolyzed by digestive enzymes in the human body and plays a role as a
Inulin dietary fiber and prebiotic. Due to its versatile physicochemical properties and physiological functions, inulin
Inulosucrase has been widely applied in food, pharmaceuticals, and many other fields. The microorganism-derived inulin-
Characteristics forming enzyme inulosucrase (ISase) (EC: 2.1.4.9) can biosynthesize higher-molecular-weight inulin than plants
Structure
using sucrose as the sole substrate, and the enzyme also shows transfructosylation activity toward other sac-
Molecular modification
charide acceptors. In this article, the properties, functions, and applications of inulin are overviewed. The
Applications
biosynthesis of inulin by ISase is addressed, including ISase characteristics, structural features, molecular
modifications and applications.

1. Introduction Due to the extensive plant sources of inulin, corresponding studies

Fructan, a versatile homopolysaccharide, has numerous applications
due to its superior physiochemical properties (water-retaining, thick-
ening, gel-forming, etc.) and significant physiological functions (anti-
cancer, anti-oxidant, anti-pathogenic, immunostimulatory, etc.). Inulin
and levan are two types of fructans. Inulin is abundant in plants, par-
ticularly in Compositae, and has been categorized as ‘Generally
Recognized as Safe’ (GRAS) for use in food since 2002 (Flores et al.,
2016). The Food and Drug Administration (FDA) approved inulin as a
dietary fiber ingredient used to improve the nutritional value of man-
ufactured food products in June 2018 (https://www.fda.gov/
FoodGuidances). The global inulin market demand was 246.5 k tons in
2013 and is expected to exceed 400 kt to reach USD 2.35 billion by
2020 (https://www.radiantinsights.com/). Because the β-(2, 1) bonds of
inulin cannot be hydrolyzed by enzymes secreted by the human body
and can be fermented only by bacteria existing in the colon, inulin has
dietary fiber and prebiotic functions (Roberfroid and Slavin, 2000). The
ability to form gels, improve sensory quality and regulate texture makes
inulin useful for application as a functional ingredient in many foods
(Morris and Morris, 2012; Shoaib et al., 2016). Furthermore, the pro-
ducts derived from inulin via biosynthesis (Fig. 2) or chemical mod-
ification (Stevens et al., 2001) have also been reported to possess fa-
vorable properties or functions and show potential for a variety of
applications.
investigating its properties and functions have been more in-depth than
those investigating levan. Traditionally, hot water diffusion is used to
separate inulin from plants, but recently, some modern technologies
have been applied to improve the yield and purity of inulin extraction
(Zhu et al., 2016). In contrast, levan exists in only few plants, such as
Agropyron cristatum and Dactylis glomerata, in small quantities (Srikanth
et al., 2015). Fortunately, levan-type fructans can be biosynthesized by
levansucrase (LSase) (EC: 2.1.4.10) in many microorganisms, including
Gram-positive and Gram-negative bacteria. Compared to LSase, ISase
has been found in fewer microorganisms, one of which is a fungus,
Aspergillus sydowi IAM 2544 (Kawai et al., 1973), and others are Gram-
positive bacteria.
Sucrose is abundant in nature, easy to obtain and inexpensive.
Recently, health issues resulting from excessive absorption of sucrose
have received attention. Hence, exploitation of functional sugars using
sucrose as feedstock is of practical significance and has attracted many
researchers. LSase and ISase belong to the glycoside hydrolase 68
(GH68) family (http://www.CAZy.org), whose members transform the
fructosyl moiety of sucrose to synthesize levan-type and inulin-type
fructan, respectively. Therefore, the application of LSase and ISase can
expand diversity in resource utilization and produce functional sac-
charides from sucrose. Compared with plant-derived inulin, whose
polymerization degree (DP) varies from 2 to 60 (Flores et al., 2016) and
molecular weight (Mw) is no > 104 Da, most microbial inulins possess

⁎
Corresponding author at: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China.
E-mail address: wmmu@jiangnan.edu.cn (W. Mu).

https://doi.org/10.1016/j.biotechadv.2019.01.002
Received 8 August 2018; Received in revised form 4 January 2019; Accepted 4 January 2019
0734-9750/ © 2019 Published by Elsevier Inc.

Please cite this article as: Ni, D., Biotechnology Advances, https://doi.org/10.1016/j.biotechadv.2019.01.002
 D. Ni et al.

Table 1
Comparison of ISases form different microorganisms.
Microorganisms GenBank Proteins Mw (kDa) Optimized pH Optimized Thermostability (T1, Ca2+ DP Mw (kDa) References
accession temperature (°C) T2, L)

B. agaradhaerens WDG185 ATN45518.1 Recombinant (B. 47.3a 6.0–10.0 60 ND No effect 3–25 3 Kralj et al., 2018
subtilis)
L. citreum CW28 AAO25086.1 Native and 170b 6.5 45 35 °C, 420 min, 50% Effect ND 2.6–3.4 del Moral et al., 2008; Olivares-Illana et al.,
Recombinant (E. coli) 50 °C, 30 min, 0 1.35–1.6× 106 2003; Olivares-Illana et al., 2002; Ortiz-Soto
et al., 2004
W. confusa MBFCNC-2(1) ADB27748.1 Recombinant (E. coli ND ND ND ND ND ND ND Malik, 2012; Malik et al., 2015
and B. subtilis)
Bacillus sp. 217C-11 ND Native 45b 7–8 45–50 ND ND 10–18 ND Wada et al., 2003
L. reuteri 121 OJI11288.1 Recombinant (E. coli) 87a 5–5.5 50 ND Effect ND > 107 van Hijum et al., 2002

2
L. reuteri TMW1.106 CAL25302.1 Native ND ND ND ND ND ND ND Schwab and Gänzle, 2006
S. mutans GS-5 AAA88584.1 Recombinant (E. coli) 70b 4.5 30 ND ND ND 7 × 107 Aduse-Opuko et al., 1998
L. gasseri DSM 20243 ND Recombinant (E. coli) ND 4.5–5.5 50 ND Effect ND ND Anwar et al., 2010
L. gasseri DSM 20604 ACZ67286.1 Recombinant (E. coli) 63b 5.5 35 50 °C, 180 min, 84% Effect ND 5.858 × 106 Ni et al., 2018
S. mutans BHT ND Native ND ND ND ND ND 8 ND Ebisu et al., 1975
S. mutans JC-1 ND Native ND ND ND ND ND 27 ND Ebisu et al., 1975
S. mutans JC-2 ND Native ND ND ND ND ND ND 2 × 107 Rosell and Birkhed, 1974
S. viridochromogenes EFL36273.1 Native ND ND ND ND ND ND 2.5 × 107 Frasch et al., 2017
DSM40736
L. johnsonii NCC 533 2YFR_A Recombinant (E. coli) 63a 7.0 55 ND Effect 4 × 107 Anwar et al., 2008
A. sydowi IAM 2544 CAB89083.1 Native ND ND ND ND ND ND 2 × 107 Kawai et al., 1973

ND, not described.

a
Predicted value according to the amount of amino acids.
b
Actual value based on the SDS-PAGE, (T1, T2, L): (incubation temperature, incubation time, left activity compared to original activity).
Biotechnology Advances xxx (xxxx) xxx–xxx
D. Ni et al.
Fig. 1. Production of inulin.
higher Mws (> 106 Da) (Table 1). Regrettably, whether the distinction
in Mw between plant-derived and IS-derived inulin will generate dif-
ferent physiochemical properties or physiological function remains
unclear.
To date, fifteen ISase-producing bacterial strains have been char-
acterized; ten of these have a known ISase sequence (Fig. 1), and the
crystal structure of L. johnsonii NCC 533 ISase was resolved in 2011
(Pijning et al., 2011). To explore the relationships among amino se-
quences, structures and functions, rational engineering has been re-
quired. The applications of ISase are mainly focused on generating
novel oligosaccharides utilizing its transfructosylation ability. In this
review, the applications of inulin are summarized, and major attention
has been applied to the biosynthesis of inulin by ISase, including its
characteristics, structural features, molecular modifications and appli-
cations.
2. Synopsis of inulin
2.1. Structure and definition
The fructosyl molecules of inulin-type and levan-type fructan
backbones are connected by β-(2, 1) and β-(2, 6) glycosidic bonds,
respectively, and there is a glucose at the nonreducing end of the chain
(Srikanth et al., 2015). Most inulin is linear. Sporadically, small num-
bers of vegetal and microbial inulins have branches connected by β-(2,
6) linkages (Lopez et al., 2003; van Hijum et al., 2006), but it remains
unknown how these branches form. According to differentiation based
on DP, inulin-type fructan can be divided into fructooligosaccharides
(DP ≤ 10) and inulin (DP > 10) (Morris and Morris, 2012).
2.2. Occurrence in nature
Inulin-type fructan in plants mainly acts as a carbohydrate reserve
for use during dormancy, regrowth after defoliation and sprouting in
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the spring (Ritsema and Smeekens, 2003). In addition to playing a role
in energy storage, inulin in plants plays an essential part in abiotic
stress tolerance, and its osmoregulatory function can protect plants
from drought, salt and cold stresses (Apolinário et al., 2014; Ritsema
and Smeekens, 2003). It has been hypothesized that the molecular
mechanisms connected with increased protection against these stresses
allow inulin to maintain the integrity of lipid bilayers and prevent
membrane damage by interacting with phospholipids (Demel et al.,
1998; Vereyken et al., 2001).
Usually, inulin is extracted commercially from chicory (Van Laere
and Van den Ende, 2002). In addition to chicory, elecampane (Toma
et al., 2001), dandelion (Schütz et al., 2006), agave (Lopez et al.,
2003),dahlia (Bunim et al., 1937) and Jerusalem artichoke (Wei et al.,
2007) are optional alternatives due to their abundant content. Cer-
tainly, some common crops such as onion, banana and wheat have
inulin, but due to their low content, separation and purification tech-
nologies have not been extensively considered (Drabińska et al., 2016).
Currently, the major approach for inulin extraction from plants is pre-
cipitation by ethanol (Zhu et al., 2016), prior to which the plants should
be pretreated (washed, peeled, cut, dried, milled, sieved and boiled)
(Flores et al., 2016) (Fig. 1). However, this approach is considered
uneconomical as well as unsuitable on an industrial scale, so some
modern technologies have attempted to use alternative approaches for
inulin extraction, such as sonication (Wei et al., 2007; Milani et al.,
2011), microwave (Lou et al., 2009) and pulsed electric field processing
(Zhu et al., 2013). Microbial inulin is not applied in industry, so its
production and purification are performed in the laboratory by ethanol
precipitation, and then it is freeze-dried (Kawai et al., 1973; Anwar
et al., 2010; Kralj et al., 2018). The fundamental diversity among dif-
ferent sources of inulin is the DP, which is determined by the plant
source and influenced by growing conditions, weather, harvesting time
and storage conditions (Flores et al., 2016).
D. Ni et al.
2.3. Physicochemical properties and applications
Thus far, the solubility (Naskar et al., 2010), thermal behavior
(Kawai et al., 2011), crystal morphology (André et al., 1996), rheolo-
gical properties (Kumar et al., 2015) and stability (Chiavaro et al.,
2007) of various sources of inulin have been studied in detail. All of
these characteristics are determined by the average DP and actual size
distribution, so different sources of inulin exhibit various but regular
traits (Mensink et al., 2015a). In summary, based on its outstanding
capabilities, including retaining water, thickening, forming micro-
crystals, forming gel, modifying rheological behavior and improving
mouthfeel, inulin has been used as an additive in bread, pasta, con-
fectioneries, sauces, desserts, cheese, yogurt, ice cream and many other
foods, serving as a prebiotic, fat replacer or texture modifier, among
other roles (Drabińska et al., 2016; Karimi et al., 2015; Mensink et al.,
2015a; Meyer et al., 2011; Morris and Morris, 2012; Shoaib et al.,
2016). In addition to applications in food, inulin can be used in the
pharmaceutical field to deliver drugs (Mensink et al., 2015b) and in the
chemistry field to produce biodegradable compounds via chemical
modification (Stevens et al., 2001).
2.4. Metabolism and physiological functions
The anomeric C2 of fructosyl in inulin has the beta conformation, so
it is resistant to hydrolyzation by enzymes in the digestive system,
which are specific for alpha linkages (Roberfroid and Slavin, 2000).
Some clinical studies have suggested that inulin cannot be digested in
the upper gastrointestinal tract and small intestines (Knudsen and
Hessov, 1995) and passes into the large bowel, where it is selectively
fermented by a limited number of potentially beneficial bacteria, sti-
mulating bifidobacterial and lactobacilli growth while controlling pa-
thogens (E. coli and clostridia) at low levels (Gibson et al., 1995). Inulin
is ultimately transformed in the colon into short-chain fatty acids,
which can be metabolized and absorbed by many parts of the body
(Flamm et al., 2001). Therefore, various important physiological
functions based on inulin as a dietary fiber and prebiotic have been
identified, such as anti-obesity, anti-diabetes, anti-hypertension, anti-
oxidant, and anti-colon cancer functions and the ability to control in-
flammatory bowel disease, relieve constipation, promote colonic ab-
sorption of minerals, regulate glucose and lipid metabolism, regulate
the endocrine system and stimulate the immune system, etc., all of
which have been reviewed (Roberfroid, 1993; Roberfroid, 2007;
Schaafsma and Slavin, 2015; Shoaib et al., 2016; Vogt et al., 2015).
2.5. Downstream mediators
The applications of inulin are numerous, and its enormous potential
to produce valuable downstream mediators has been given more at-
tention recently (Fig. 2). For example, high-fructose syrup and fructo-
oligosaccharides, which are considered low-calorie and healthy sweet-
eners, as well as prebiotics, can be enzymatically biosynthesized from
inulin by inulinase (exoinulinase and endoinulinase, respectively) (EC:
3.2.1.7) (Flores et al., 2016; Singh et al., 2018). Furthermore, fructo-
oligosaccharides have the potential to be used in infant nutrition as a
replacement for human milk oligosaccharides (Akkerman et al., 2018).
Difructose dianhydrides (DFAs) comprise two fructose residues forming
two reciprocal glycosidic linkages (García-Moreno et al., 2008). DFA I
and DFA Ш are two types of more common DFAs and can be produced
from inulin by inulin fructotransferase (IFTase) (DFA I-forming) (EC:
4.2.2.17) and (DFA Ш-forming) (EC: 4.2.2.18), respectively. DFAs are
functional sweeteners. Taking DFA Ш as an example, its energy is only
1/15 that of sucrose, but it possesses half the relative sweetness (Zhao
et al., 2011). It can promote the absorption of minerals, flavonoids and
immunoglobulin G (Wang et al., 2015). Rare sugar is scarce in nature
and has promising potential applications (Zhang et al., 2017), and the
bioconversion from inulin to rare sugar has been attempted in the forms
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of sorbitol (Wei et al., 2001), mannitol (Saha, 2006) and D-psicose
(Song et al., 2017).
Some nonsaccharide substances can also be produced from inulin,
such as bioethanol (Chi et al., 2011; Zhang et al., 2010), citric acid (Liu
et al., 2010), L-lactic acid (Ge et al., 2009) and signal cell oil (Chi et al.,
2011). Single cell oil, namely, microbial oil, attracts much attention due
to its roles as a functional oil and feedstock for biodiesel production
(Huang et al., 2013). However, these downstream products are mainly
produced in the laboratory, and their industrial production and com-
mercial applications deserve further research.
3. Biosynthesis of inulin
3.1. Inulin-forming enzymes
Plant- and microorganism-derived inulin is biosynthesized from
sucrose by different enzymes. In plants, a two-step catalyzed reaction
generates mature inulin, and each step is catalyzed by a distinct enzyme
belonging to the GH32 family, specifically sucrose: sucrose 1-fructo-
syltransferase (1-SST EC: 2.4.1.99) and fructan: fructan 1-fructosyl-
transferase (1-FFT EC: 2.4.1.100) (Livingston et al., 2009). 1-SST
transfers the fructose moiety of a sucrose molecule to another sucrose
molecule to yield 1-kestose and a molecule of glucose, and then the
produced fructans, of which 1-kestose is the initial one, are used as
fructosyl donors and acceptors by 1-FFT to catalyze a transfructosyla-
tion reaction (Edelman and Jefford, 1968). 1-FFT can also use sucrose
as a fructosyl acceptor and is highly specific for β-(2, 1) linkage hy-
drolysis and formation to guarantee inulin elongation (Edelman and
Jefford, 1968; Vergauwen et al., 2003). In contrast to plants, inulin-
producing microorganisms synthesize inulin using a single enzyme,
ISase, which can generate inulin using sucrose as the sole substrate
(Fig. 1).
3.2. Microbial sources of ISases
ISase and LSase are two types of fructosyltransferases (FSases) that
can synthesize inulin-type and levan-type fructans. To date, many
LSases have been experimentally identified from Gram-positive and
Gram-negative bacteria in either native or recombinant forms, as de-
scribed by recent reviews (Hill et al., 2017; Li et al., 2015a; Öner et al.,
2016; Srikanth et al., 2015). Since 2017, novel LSases have been
identified in various microorganisms, including Clostridium arbusti
SL206 (Li et al., 2017), Bacillus licheniformis ANT 179 (Xavier and
Ramana, 2017), Brenneria goodwinii (Liu et al., 2017), Clostridium
acetobutylicum (Gao et al., 2017), Bacillus subtilis CECT 39 (Ruiz-
Aceituno et al., 2017), Leuconostoc mesenteroides Lm 17 (Iliev et al.,
2018) and Brenneria sp. EniD312 (Xu et al., 2018). However, currently,
only fifteen microorganisms have been identified as harboring the
ability to synthesize ISase, and fourteen of them are Gram-positive
bacteria (Table 1), while one is a fungus, Aspergillus sydowi IAM 2544
(Kawai et al., 1973). The enzyme activity, DP and Mw of the produced
inulin vary when sucrose is used as substrate, and some reaction con-
ditions, such as the pH, temperature, metal ions and sucrose con-
centration, have significant effects on these properties. Thus far, most
research probing the enzymatic properties of ISases has focused on
recombinant enzymes, but information regarding the primary enzy-
matic properties of native ISases, such as the optimized pH and opti-
mized temperature, etc., is insufficient to understand and apply them
(Table 1).
3.3. Enzymatic reaction and activity
ISase possesses three main crucial functions: cleaving sucrose,
transferring fructosyl to another sucrose, and extending the inulin
chain; hence, three different and sequential reaction processes will
occur when ISase utilizes sucrose as substrate (Fig. 2). First, sucrose is
D. Ni et al.
Fig. 2. The ISase reaction process and its application.
cleaved into a molecule of glucose and an ISase-fructosyl intermediate,
and then, if water serves as a fructosyl acceptor, fructose will be re-
leased, and a sucrose hydrolysis reaction occurs. In contrast, if another
sucrose acts as an acceptor, a transfructosylation reaction occurs, pro-
ducing 1-kestose. The next step is generating long-chain inulin with
produced fructooligosaccharides as fructosyl acceptors; thus, a poly-
merization reaction occurs. Ozimek et al. discovered that ISase can also
catalyze a disproportionation reaction (Ozimek et al., 2006a).
Generally, three types of enzyme activities are defined to describe
ISase. The amount of glucose in the reaction system is identical to the
donor sucrose; hence, it represents total activity. The amount of fruc-
tose signifies how much sucrose is hydrolyzed, so it is regarded as hy-
drolysis activity. The amount of glucose minus the amount of fructose,
corresponding to total activity minus hydrolysis activity, gives the
amount of fructose used for transfructosylation and is the transfructo-
sylation activity.
4. Enzymatic properties of ISase
The fundamental significance of ISase application is the ability to
obtain higher-Mw inulin than that from plants. Therefore, the condi-
tions controlling inulin production have been studied and optimized.
The properties of all reported ISases and corresponding products are
summarized in Table 1, and detailed information will be discussed next.
4.1. pH
Normally, most microbial ISases exhibit maximal total activity in a
slightly acidic or neutral environment (4.5–7.0) (Table 1), except the
ISase from Bacillus sp. 217C-11, which is optimally active under neutral
and alkaline conditions (7.0–8.0) and is stable at pH 6.0–9.0 but un-
stable below pH 5.0 (Wada et al., 2003). Recently, Kralj et al. identified
a novel ISase from B. agaradhaerens WDG185 that displayed a broad
optimal pH spectrum ranging from 6.0 to 10.0 (Kralj et al., 2018). Some
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studies exploring the effects of pH on transfructosylation and hydrolysis
activities separately showed that these activities reached peak values in
the same pH range for ISases such as those obtained from L. gasseri DSM
20604 (pH 5.5) (Ni et al., 2018a) and L. johnsonii NCC 533 (pH 4.5–7.0)
(Anwar et al., 2008).
4.2. Temperature
The optimal temperature for ISase varies from 30 to 60 °C, and half
of ISases fall in the range of 40–45 °C (Table 1). B. agaradhaerens
WDG185 and S. mutans GS-5 ISases are at two extremes, exhibiting the
highest and lowest optimal temperatures: 60 °C and 30 °C, respectively.
Similar to some LSases (Hettwer et al., 1995; Liu et al., 2017; Visnapuu
et al., 2011), the optimal transfructosylation and hydrolysis tempera-
tures of ISases are usually different. In a case study of L. gasseri DSM
20604 ISase, 45 °C and 25 °C were most suitable for hydrolysis and
transfructosylation, respectively (Ni et al., 2018b). This phenomenon in
which low temperature promotes polymerization reactions and high
temperature favors sucrose hydrolysis frequently occurs with LSases (Li
et al., 2015b). However, in contrast, L. johnsonii NCC 533 ISase tends to
hydrolyze sucrose at 40 °C and generate inulin at 55 °C (Anwar et al.,
2008).
Thermostability is a significant feature when adapting enzymes to
industrial application. Nevertheless, information concerning the ther-
mostability of ISases remains limited, and merely two from L. citreum
CW28 (del Moral et al., 2008; Olivares-Illana et al., 2002) and L. gasseri
DSM 20604 (Ni et al., 2018a) have been studied for their thermo-
stability. The half-life of L. citreum CW28 ISase at 35 °C was 420 min
(del Moral et al., 2008), and it was inactivated completely after 30 min
at 50 °C. L. gasseri DSM 20604 IS was fairly stable at ≤50 °C but became
labile once the temperature exceeded 55 °C. Compared to reported heat-
resistant LSases (Ammar et al., 2002; Ni et al., 2018b), ISase thermo-
stability needs to be improved to satisfy industrial applications. Iden-
tification of the properties of known ISases, exploration of novel
D. Ni et al.
microbial sources and rationally designed mutagenesis are promising
approaches for discovering thermostable enzymes or mutants.
4.3. Metal ions
According to the crystal structure of L. johnsonii NCC 533 ISase,
there is a Ca2+ binding site in the active center domain (Pijning et al.,
2011), which implies that the presence of Ca2+ may have positive ef-
fects on enzyme activity and/or stability and even the Mw of bio-
synthesized inulin. Correspondingly, some studies indeed revealed that
Ca2+ can enhance catalytic activity. For example, compared to adding
1 mM CaCl2 to the reaction mixture, the absence of CaCl2 results in 79%
decreased activity for L. reuteri 121 ISase (van Hijum et al., 2002).
Anwar and coworkers isolated two ISases from L. gasseri and found that
Ca2+ can counteract the inhibiting effect of EDTA on enzyme activity,
which indicates that Ca2+ is required for L. gasseri ISases to display
their highest activity (Anwar et al., 2010). Recently, our group dis-
covered that Ca2+ can increase the total activity of L. gasseri DSM
20604 slightly, but Mn2+ can improve it by 157% (Ni et al., 2018a).
This phenomenon suggests that the optimal metal ions for diverse
sources of ISases may vary with each individual. Nevertheless, B.
agaradhaerens WDG185 ISase is an exception in which Ca2+ has no
influence on its activity because of the lack of Ca2+ binding sites.
Ozimek et al. built a mutant of the Ca2+ binding site of L. citreum CW28
ISase, which resulted in severe reductions in its activity and thermo-
stability (Ozimek et al., 2005) (Table 1). The sucrose-bound and 1-
kestose-bound crystal structures showed that the Ca2+ binding site is
fully occupied only in the presence of 5 mM CaCl2 (Pijning et al., 2011).
Regrettably, there are no reports describing the effects of ion con-
centration on ISase activity, thermotolerance or structural stability,
which are worth researching. Inversely, heavy metal ions such as Cu2+
and Hg2+ inhibit activity (Ni et al., 2018b; van Hijum et al., 2003;
Wada et al., 2003).
4.4. Kinetic parameters
According to many previous studies (Anwar et al., 2010; Anwar
et al., 2008; van Hijum et al., 2003), with the exception of hydrolysis
activity, total and transfructosylation activities did not follow normal
Michaelis-Menten kinetics because these activities were not saturated
by sucrose, resulting in high standard errors with curve fits, although
the Hill equation was more applicable for them. The Hill equation is
used under the condition that the binding of a substrate to an enzyme is
often enhanced if there are already other substrates present on the same
enzyme. This special behavior has been ascribed to a reaction process in
which many oligosaccharides are produced during the early period of
the reaction, and they may be used as more suitable acceptors than the
growing polymer (Anwar et al., 2010; Anwar et al., 2008). Hill-type
kinetics are based on the hypothesis that there is more than one sub-
strate binding site in the enzyme or that it is a multimeric protein
(Anwar et al., 2008; van Hijum et al., 2003). Indeed, according to the
three-dimensional structure of L. johnsonii NCC 533 ISase with bound
sucrose, there are two sucrose binding sites in the enzyme (Pijning
et al., 2011); this may explain the Hill-type kinetics of this enzyme.
However, there are exceptions for enzymes that follow Michaelis-
Menten kinetics rather than the Hill equation. Ortiz-Soto et al. revealed
that L. citreum CW28 ISase showed Michaelis-Menten kinetics (Ortiz-
Soto et al., 2004); thereafter, del Moral et al. constructed four truncated
ISases (IslA-IslA4) from L. citreum CW28 and found that all of the forms
exhibited Michaelis-Menten kinetics, except IslA3, which had 757
truncated amino acids at the C-terminus (del Moral et al., 2008). Re-
cently, B. agaradhaerens WDG185 ISase was identified, and its trans-
fructosylation and total activities followed Michaelis-Menten kinetics
with Km values for sucrose of 1.4 ± 0.2 and 2.2 ± 0.2, respectively
(Kralj et al., 2018).
Michaelis-Menten kinetics fit the vast majority of LSases
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Biotechnology Advances xxx (xxxx) xxx–xxx
(Inthanavong et al., 2013; Lu et al., 2014; Nakapong et al., 2013; Öner
et al., 2016), except for the enzymes from B. amyloliquefaciens (Tian and
Karboune, 2012), L. reuteri 121 (van Hijum et al., 2004), Zymomonas
mobilis 113S (Andersone et al., 2004) and L. sanfranciscensis TMW 1.392
(Tieking et al., 2005). Although the catalytic mechanism of FSase has
been generally accepted as a “ping-pong” mechanism, whether the
different kinetics result from different polysaccharide extension pro-
cesses remains unknown.
4.5. Sucrose and enzyme concentrations
All research probing the effects of sucrose concentration on ISase
activity has shown that a high concentration of sucrose is beneficial for
transfructosylation activity and that low concentrations tend to pro-
mote hydrolysis (Kralj et al., 2018; Ni et al., 2018a; Ortiz-Soto et al.,
2004; Peña-Cardeña et al., 2015; Wada et al., 2003). Taking L. reuteri
121 as an example, its transfructosylation activity reaches 90% of total
activity when the sucrose concentration is 1.7 M (Ozimek et al., 2006a).
A similar phenomenon is also detected for LSase (Li et al., 2015b) and
can even be used to control the Mw of produced levan (Abdel-Fattah
et al., 2005; González-Garcinuño et al., 2017). A high substrate con-
centration may impede water molecules from entering the active site,
rendering the enzymes able to enhance transfructosylation activity and
reduce hydrolysis activity (Chuankhayan et al., 2010).
Regarding the enzyme concentration, Peña-Cardeña et al. found that
ISase hydrolysis activity increased, and the transfructosylation product
was mainly fructooligosaccharide instead of polymer when the enzyme
concentration increased (Peña-Cardeña et al., 2015). When the enzyme
dosage exceeded 4.5 U/mg sucrose at 300 g/L sucrose, biosynthesized
inulin decreased, but fructose present in the reaction mixture increased
gradually (Ni et al., 2018b), which indicates that high enzyme con-
centrations conflict with transfructosylation reactions.
4.6. Mw of microbial inulin
High Mw is the most obvious feature of microbial inulin relative to
vegetal inulin. As shown in Table 1, most microorganisms can produce
inulin with Mws of > 106 Da, the highest of which is S. mutans GS-5,
which can produce inulin with a Mw of 7 × 107 Da (Aduse-Opuko et al.,
1989). There are also four ISases producing low-Mw inulins like vegetal
inulin. Resolving the formation mechanism of high-Mw inulin and
probing the potential advantages of high-Mw inulin will be important
for the production and application of microbial inulin.
5. Crystal structure and molecular modification
5.1. Overall structures
Ten amino acid sequences of ISases are available in the NCBI da-
tabase, and these share 17% to 98% identity with each other (Table S1).
The primary structure of ISase can be divided into three regions: the
variable N- and C-termini and catalytic core domain. The N- and C-
termini usually have a signal peptide and a cell-wall anchoring motif,
respectively. They likely do not contribute to catalysis and are averse to
intracellular overexpression and enzyme accumulation in E. coli.
Therefore, N- and/or C-terminal truncation strategies usually have been
used to study ISases, including the only ISase crystal structure (Anwar
et al., 2010; Anwar et al., 2008; Pijning et al., 2011).
Pijning et al. resolved the crystal structure of ISase from L. johnsonii
NCC 533, which was truncated at both the N- and C-termini with re-
sidues 145–708 remaining. Additionally, structures with bound sugar
were obtained by soaking the protein crystals in high concentrations of
sucrose or 1-kestose (Pijning et al., 2011). The catalytic core domain
adopts the five-bladed β-propeller fold, a common feature of Clan GH-J
enzymes, and each blade comprises four antiparallel β-strands. All of
these strands establish the funnel-shaped catalytic pocket (Fig. 3). The
D. Ni et al. Biotechnology Advances xxx (xxxx) xxx–xxx

Fig. 3. Overall structure of ISase from L. johnsonii NCC533. (A) Top view; (B) bottom view. The five-bladed β-propellers of the catalytic core domain are shown in red,
magenta, cyan, orange and yellow for β-propeller 1, β-propeller 2, β-propeller 3, β-propeller 4 and β-propeller 5, respectively, from the N-terminus to the C-terminus.
The N-terminus and C-terminus are shown in pink and blue, respectively. The loop connecting the N-terminus and β-propeller 1 is shown in green. Space structures
were generated by the program PyMOL. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

sequences of β-sheets are highly conserved among ISases, and the se-
quence variation is mainly in the loops joining the β-strands of the β-
propeller. A clamp-like loop (residues 210–255) runs half the cir-
cumference of the propeller from the N-terminus to β-propeller 1. This
structural property is consistent with known LSases.
5.2. Active and binding sites
Until now, four LSase structures have been analyzed by X-ray
crystallography, but only one ISase structure has been analyzed. The
active sites are located in the middle-upper part of the catalytic pocket
and comprise two aspartic acids and a glutamic acid, which function as
a nucleophile, transition-state stabilizer and general acid/base, re-
spectively (Fig. 4A).
According to the crystal structure of ISase with bound sucrose
(Fig. 4A), there are six residues interacting with the fructosyl moiety of
sucrose at the −1 subsite. The −1 subsite is the binding site of the
fructosyl moiety that is transferred to the growing fructan polymer. The
fructosyl moiety is specifically bound through many hydrogen bonds.
Based on a sequence alignment of bacterial ISases (Fig. 5), five residues
in the −1 subsite are completely conserved, with Ser340 being replaced
by an alanine in the B. agaradhaerens WDG185 and S. viridochromogenes
DSM40736 ISases. The conservation of +n (≥1) subsites is weaker
than that of the −1 subsite, in which both fructosyl and glucosyl can
bind at +n subsites. The ISase-sucrose complex shows that Glu522 and
Arg542 form hydrogen bonds with O3, O4 and O2 of the glucosyl unit
at the +1 subsite. However, Arg542 transforms into a conformation
that forms a hydrogen bond with the glucosyl unit at the +2 instead of
the +1 subsite when ISase binds with 1-kestose. The other amino acid
forming a hydrogen bond at the +2 subsite is Arg545, which is shown
with an alternative conformation, and only one can form a hydrogen
bond with the glucosyl unit (Fig. 4B). Hence, it can be speculated that
Arg542 and Arg545 at the +2 subsite may play a significant role in
elongation of the product.
Metal ions, particularly Ca2+, can improve the activities of FSases
from Gram-positive bacteria (Ortiz-Soto et al., 2004), which can be
ascribed to the Ca2+ binding sites of these enzymes. Elucidation of the
three-dimensional structures of two LSases and one ISase from Gram-
positive bacteria provides a visual tool for Ca2+ binding sites. As shown
in Fig. 4C and Fig. 5, highly conserved residues encircling the Ca2+
binding site form a hydrogen bond network that involves the residue at
the +1 subsite, Glu522. Therefore, Ca2+ may have a hypothetical
impact on stabilizing the adjacent area and influencing the donor and
acceptor to bind and undergo catalysis.
5.3. Catalytic mechanism
Because of the similarity of the catalytic domains of ISase and LSase
(Fig. S1 and Fig. S2), their catalytic mechanisms may share some of the
same steps and are often discussed together (Ozimek et al., 2006b). The
FSase catalytic process involves two steps, sucrose cleavage and fructan
elongation; therefore, these two distinct steps in the mechanism are
considered to illuminate the reaction process. In brief, LSase and ISase
adopt a ping-pong (double-displacement) mechanism to cleave sucrose,
resulting in the formation of the enzyme-fructosyl intermediate (Fig. 2)
(Chambert et al., 1974). Processive and nonprocessive mechanisms
have been introduced to explain the synthesis of high-Mw and low-Mw
levan (Ozimek et al., 2006a; Raga-Carbajal et al., 2015). However, al-
though some site-directed mutants change the product spectra of FSases
(He et al., 2018; Homann et al., 2007; Li et al., 2011; Ozimek et al.,
2006a), the specific roles of these amino acids responsible for poly-
saccharide formation from a structural perspective remain largely un-
explored, especially for ISase. One exception is that Seibel's group has
revealed the relevant residues of Bacillus megaterium LSase, which dis-
tinctly influence the polysaccharide product spectrum, providing a
novel perspective on polysaccharide synthesis mechanisms (Strube
et al., 2011). Therefore, elucidation of the amino acid residues that
control polysaccharide elongation, e.g., by site-directed mutagenesis,
may give meaningful clues regarding regulation of the Mw of the pro-
duced inulin.
5.4. Molecular modification
Site-directed mutagenesis studies have been summarized in Table 2.
Ozimek et al. demonstrated the essential role of the catalytic triad early
on; they changed these residues into their amide counterparts and
found that their activities decreased by at least 10,000-fold (Ozimek
et al., 2004). Soon after, this group revealed that Asp520 of the L. reuteri
121 ISase plays a significant role in Ca2+ binding (Ozimek et al., 2005).
In addition, they constructed four single-site mutants of L. reuteri 121
ISase at the −1 subsite, W271 N, W340 N, R423H and R423K, which
are completely conserved in the GH 68 family. The results showed that
the activities of these mutants decreased dramatically, and the size
distribution of the biosynthesized products (Table 2) was obviously
affected (Ozimek et al., 2006a). Thereafter, Anwar et al. studied fifteen
single-site and four multiple-site mutants, selecting amino acid residues

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D. Ni et al. Biotechnology Advances xxx (xxxx) xxx–xxx

Fig. 4. Active and binding sites. (A) Active site of L. johnsonii NCC 533 ISase in complex with sucrose (PDB: 2YFS); (B) active site of L. johnsonii NCC 533 ISase in
complex with 1-kestose (PDB: 2YFT); (C) Ca2+ binding site. Residues labeled with red are the three active sites. Ca2+ and water are shown as red and green spheres,
respectively. Yellow and red broken lines indicate hydrogen bonds and metal interactions, respectively. The space structures were generated by the program PyMOL.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

that are conserved among ISases but are different in LSases, to probe
whether these amino acid residues control the type of glucosidic bond
in the synthesized polysaccharide. However, these mutants only altered
their activities or the product spectra without influencing the glucosidic
bond (Anwar et al., 2012). Other molecular modification studies of
ISase were conducted with L. citreum CW28, and some mutants speci-
fically could synthesize inulin or FOS, such as S425A, R618K and
R696K (Rodríguez-Alegría et al., 2010). Mutations based on crystal
structures were designed to probe the importance of the 1B—1C loop,
and the results showed that the residues in this loop play a role in
enzyme activity and acceptor binding (Pijning et al., 2011).
6. Application of ISase
The application of ISase not only reflects its hydrolysis but its po-
tential as a transfructosylation tool to biosynthesize promising sac-
charides. The α-(2, 1) glycosidic bond between the sugars in subsites
−1 and + 1 can be cleaved by Glu524 acting as a general acid in this
step (Fig. 4A). Therefore, theoretically, saccharides containing a su-
crose unit and ending with the fructosyl can be used by ISase as fruc-
tosyl donors, which has been proven because melibiose and raffinosyl-
oligofructosides (RFOS) are detected in the reaction mixture when
raffinose is used as a starting substrate (Díez-Municio et al., 2016)
(Fig. 2). Melibiose is an indigestible disaccharide possessing note-
worthy physiological functions such as promoting calcium absorption,
increasing the growth of bifidobacteria and improving the induction of
oral tolerance (Xu et al., 2017); hence, taking advantage of the
hydrolysis activity of ISase to produce melibiose is a valuable approach.
RFOS belong to heterofructooligosaccharides, which have potential
applications in the functional food, pharmaceutical and cosmeceutical
fields (Gimeno-Perez et al., 2014). In addition, ISase is utilized to
transfer fructosyl of sucrose to maltose to synthesize maltosylfructo-
sides with erlose [α-D-glucopyranosyl-(1, 4)-α-D-glucopyranosyl-(1, 2)-
β-D-fructofuranoside] and neoerlose [β-D-fructofuranosyl-(2, 6)-α-D-
glucopyranosyl-(1, 4)-α-D-glucopyranose] as the principal products
(Díez-Municio et al., 2013), which have promising properties to allow
them to serve as substitute sweeteners and probiotics (Gimeno-Perez
et al., 2014; Hodoniczky et al., 2012). Compared to maltose, xylose is
the more competitive acceptor to obtain the fructosyl moiety of sucrose.
The predominant transfructosylation product is xylofructoside, with
only a little fructooligosaccharide and inulin being produced
(Rodríguez-Alegría et al., 2010).
The transfructosylation reaction to synthesize fructosyl derivatives
from sucrose and acceptors with FSase has been studied mainly in
LSase. For example, lactosucrose, theanderose, erlose, acarbose-fruc-
toside and many other fructosyl-saccharide derivatives have been
formed by using lactose (Choi et al., 2004; Han et al., 2009; Li et al.,
2015a; Park et al., 2005; Wu et al., 2015), isomaltose (Ruiz-Aceituno
et al., 2017), maltose (Canedo et al., 1999), acarbose (Nam et al., 2009)
and other saccharides (Seibel et al., 2006) as acceptors, respectively. In
addition to saccharides, some aromatic and aliphatic alcohols can act as
fructosyl acceptors to obtain derivatives with promising applications
due to their versatile properties (Kang et al., 2009; Kim et al., 2000;
Mena-Arizmendi et al., 2011). However, there has been no report

8
D. Ni et al. Biotechnology Advances xxx (xxxx) xxx–xxx

(caption on next page)

9
D. Ni et al. Biotechnology Advances xxx (xxxx) xxx–xxx

Fig. 5. Multiple sequence alignment of ISases. The full names and GenBank accession numbers are listed as follows: Lajo: Lactobacillus johnsonii NCC 533 (2YFR_A),
Baag: Bacillus agaradhaerens WDG185 (ATN45518.1), Leci: Leuconostoc citreum CW28 (AAO25086.1), Weco: Weissella confusa MBFCNC-2(1) (ADB27748.1), Lare 121:
Lactobacillus reuteri 121 (OJI11288.1), Lare TMW: Lactobacillus reuteri TMW1.106 (CAL25302.1), Stmu: Streptococcus mutans GS-5 (AAA88584.1), Laga: Lactobacillus
gasseri DSM 20604 (ACZ67286.1), and Stvi: Streptomyces viridochromogenes DSM40736 (EFL36273.1). The red and green arrows are amino acids at the −1 subsite,
and the red arrows represent active sites. The blue and black arrows indicate the amino acids at the +1 and + 2 subsites, respectively. The red circles are Ca2+
binding sites. The alignment was prepared using the program ESPript. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

Table 2
Molecular modification of ISases.
Microorganisms mutants Results References

L. citreum CW28 S425A It produces only FOS. Rodríguez-Alegría et al., 2010

L499F It produces FOS and inulin but becomes severely inactive.
R618K It produces only FOS.
R696K It produces only inulin.
L. reuteri 121 D249N They result in a 105 time reduction of total activity. Ozimek et al., 2004
D404N
E503Q
D520A/N Activities and thermostability are severely reduced, and the Ca2+ dependency becomes decreased at lower Ozimek et al., 2005
temperatures.
W271 N It produces more FOS (DP > 10) and polymer. Ozimek et al., 2006
W340 N It produces only kestose and nystose.
R423H No activity
R423K It produces more FOS (DP > 10) and polymer but fails to produce FOS (DP 6–10).
G416E Activities are increased prominently. Anwar et al., 2012
W486 L
A538S
N543S It produces only FOS up to DP6.
L. johnsonii NCC 533 N301S/A Activities are reduced to different degrees. Pijning et al., 2011
N305S/A
Δ301–305

describing the acceptor reaction of ISase to utilize alcohols. This ab-
sence may be attributable to the fact that ISase and LSase have different
abilities to transfer fructosyl to alcohols; in other words, alcohols may
not be used as acceptors by ISase in double substrate systems. Although
the active domains in ISase and LSase are highly overlapping (Fig. S2),
their acceptor actions exhibit different traits. The amino acid residues,
sequence regions or space conformations controlling the different ac-
ceptor action behaviors have yet to be revealed; this information will
provide deep insight into both enzymes.
belong to Lactobacillus, so whole-cell fermentation to produce inulin
should be a focus. Lactobacillus is a safe food-grade microorganism.
Therefore, improving ISase expression levels in Lactobacillus by reg-
ulating the ISase biosynthesis pathway or homologous expression is a
promising strategy to facilitate the industrial application of this en-
zyme. Additionally, probing whether microorganism-derived high-Mw
inulin possesses distinctive physicochemical properties or physiological
functions will promote its application in the food field.
Acknowledgements

7. Conclusions
This work was supported by the Support Project of Jiangsu Province
(No. 2015-SWYY-009), the Research Program of the State Key
There is no doubt that functional ingredients, functional oligo-
Laboratory of Food Science and Technology, Jiangnan University (No.
saccharides in particular, will occupy an important position in the
SKLF-ZZA-201802 and SKLF-ZZB-201814), the National First-Class
modern food industry and human diet, and enzymatic biosynthesis has
Discipline Program of Food Science and Technology (No.
gradually developed into the predominant route for the future pro-
JUFSTR20180203) and the Postgraduate Research and Practice
duction of functional ingredients. This article summarized the appli-
Innovation Program of Jiangsu Province (NO. KYCX18_1782).
cations of inulin as a functional ingredient in foods and many other
fields. More attention has focused on the enzymatic production of high-
Appendix A. Supplementary data
Mw inulin with ISase. At present, fifteen microorganisms have been
shown to possess ISase, and the crystal structure of L. johnsonii NCC 533
Supplementary data to this article can be found online at https://
ISase has already been resolved. In addition, based on sequence-struc-
doi.org/10.1016/j.biotechadv.2019.01.002.
ture-function relationships, some site-directed mutagenesis has been
attempted to change the product spectra and/or enzymatic activities.
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