Galacto-oligosaccharide synthesis using immobilized β-galactosidase

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Publication date: 2014
Citation for published version (APA): Benjamins, F. (2014). Galacto-oligosaccharide synthesis using immobilized β-galactosidase. [Thesis fully internal (DIV), University of Groningen]. [S.n.].
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Galacto-oligosaccharides (GOS) are carbohydrates generated from glucose and galactose
generally described by the formula Galn-Glc, where n = 2 – 20. However, disaccharides
(n = 1) with linkages other than β-D-Gal(1→4)-D-Glc (lactose) are often considered GOS
also (Voragen, 1998). Beside these structures generated from glucose and galactose also
Galn structures are considered GOS. Their presence in lactose derived GOS however, is
generally rather low (Coulier, et al., 2009).
GOS exhibit prebiotic functionality (Boehm and Stahl, 2007; Depeint, et al., 2008) which
means that they are not digested and selectively stimulate the growth of beneficial
bacteria in the colon, thereby improving the health of the host (Gibson and Roberfroid,
1995). Moreover, GOS have been reported to have potential as anti-infective against
enteric infections. Other beneficial effects that have been attributed to GOS include
enhanced mineral absorption, prevention of allergies and reduction of gut inflammation
(Tzortzis and Vulevic, 2009; Vulevic, et al., 2008). GOS are applied in several food
applications like yoghurt, bakery products and beverages. However, the main applications
of GOS are infant milk formula, follow-on milk formula and infant and toddler nutrition
(Playne and Crittenden, 2009; Torres, et al., 2010).
4
GOS differ structurally from human milk oligosaccharides (HMOs) which are, besides
glucose and galactose, generated from N-acetyl-glucosamine, fucose and syalic acid.
Tomarelli et al. (Tomarelli, et al., 1954) identified a disaccharide consisting of galactose
and N-acetylglucosamine, derived from porcine mucin, as a Bifidus factor and this
structure is found also as the building block for HMOs. This Bifidus factor was
discovered by György et al. (György, et al., 1954) in 1954 and is a metabolic substrate
for desired bacteria in the composition of the intestinal microbiota with health benefits for
breast-fed infants (Bode, 2012). To date, more than 200 species of oligosaccharides from
human milk have been identified differing in composition, linkage type and length (Kunz,
et al., 2000).
GOS molecules contain β1→4, β1→6, β1→3 linkages in various combinations, but also
the occurrence of β1→2 and even 1↔1 linkages has been described. The linkage types
and length of the GOS molecules that are formed largely depend on the source of the β-
galactosidase used for the synthesis of GOS (Coulier et al., 2009; Torres et al., 2010 ;
Fransen, et al., 1998).
This introduction provides an overview of historical and current literature on the use of β-
galactosidases for the synthesis of (galacto-)oligosaccharides. A brief description and
background of the enzyme (β-galactosidase) and the mechanism for synthesis are
provided, as well as an overview of the current literature in the field of GOS synthesis by
β-galactosidases. Subsequently, the industrial applications of β-galactosidases are
discussed. Because of the importance of stable enzymes and the need to reduce the
production cost, immobilization of β-galactosidases will be further addressed. In addition,
5
the possibilities and potential of this technology for oligosaccharide synthesis are
discussed.
1.2 Enzyme
Enzymes that are used for research or industrial purposes are in general, derived from
bacteria, yeasts or moulds. An overview of some commercially available β-galactosidase
preparations is shown in Table 1. Despite the differences in source organism, pH and
optimum temperature, all enzymes listed in Table 1 catalyze the hydrolysis of lactose into
glucose and galactose. The preponderance of the Kluyveromyces genus can be attributed
to the fact that they are safe and highly productive (Fonseca, et al., 2008).
Table 1. Commercially available sources of β-galactosidase
Brand name Manufacturer Organism pHopt Topt [°C]
Maxilact DSM, The Netherlands Kluyveromyces lactis 6.5 40 Tolerase DSM, The Netherlands Aspergillus oryzae 4.0 40 β-galactosidase Megazyme International Ireland Ltd. Aspergillus niger 4.5 60 β-galactosidase Megazyme International Ireland Ltd. Kluyveromyces lactis 6.5 45 Lactozym Novozymes, Denmark Kluyveromyces lactis 6.0 48 Lactoles L3 Amano Enzyme Inc., Japan Bacillus circulans 6.0 65 Lactase F Amano Enzyme Inc., Japan Aspergillus oryzae 5.0 55 β-galactosidase Worthington Biochemicals Inc., UK Escherichia coli 6.0-8.0 37 β-galactosidase Sigma-Aldrich, USA Escherichia coli 6.0-8.0 37 β-galactosidase Sigma-Aldrich, USA Bos taurus 4.3 37 β-galactosidase Sigma-Aldrich, USA Aspergillus oryzae 4.5-5.5 50 L017P Biocatalysts, UK Aspergillus oryzae 4.5-5.5 55 Ha-lactase Chr. Hansen, Denmark Kluyveromyces lactis 6.5 40 Lactase NL Enzeco, USA Kluyveromyces lactis 6.5 40 Fungal lactase Enzeco, USA Aspergillus oryzae 4.0 -5.5 55 GODO-YNL2 GODO SHUSEI Co., Ltd., Japan Klyuveromyces lactis 6.5 40 Biolactase F Kerry Ingredients and Flavours, Ireland Aspergillus oryzae 4.5 55 Biolactasa NTL Biocon, Spain Bacillus circulans 6.0 65 Biolactase L Kerry Ingredients and Flavours, Ireland Kluyveromyces lactis 6.0 40 GODO-YNL2 DuPont Danisco, Denmark Kluyveromyces lactis 6.5 40 Lactase 100 Specialty Enzymes & Biotechnologies
Co., USA Aspergillus oryzae 4.5-5.5 55
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glycoside hydrolases). This enzyme catalyzes the hydrolysis of terminal non-reducing β-
D-galactose residues in β-D-galactosides. The preferred natural substrates for these
enzymes include lactose, non-lactose disaccharides (e.g. allo-lactose) and polymeric
galactans (Balasubramaniam, et al., 2005; van Casteren, et al., 2000). In some cases β-
galactosidases act on sphingolipids, glycoproteins, muco-polysaccharides and
gangliosides (Hahn, et al., 1997; Kobayashi, et al., 1986; Mahoney, 2003) and few β-
galactosidases show no activity towards lactose (Chantarangsee, et al., 2007; van Laere,
et al., 2000) or are inhibited by lactose (Li, et al., 2001). Whereas the IUB-MB
nomenclature of these enzymes is based on substrate specificity and/or mechanism, the
CAZy database (http://www.cazy.org/Glycoside-Hydrolases.html) distinguishes enzymes
based on their amino acid sequence similarities (Henrissat, 1991). The group of
glycoside hydrolases (GH) consists of 118 families, containing 5 families that display β-
galactosidase activity (GH 1, GH2, GH35, GH42 and GH59). Additionally GH 98 maybe
considered a β-galactosidase albeit very specific (CAZy 2010).
Except the GH98 family, the other GH families with β-galactosidase activity display a
retaining mechanism (further explained in paragraph 2.2. See also Figure 1). GH98
family enzymes operate with an inverting mechanism (Figure 2). The majority of
enzymes listed in Table 1 belong to the GH2 family. The β-galactosidase from Bos taurus
belongs to the GH35 family.
7
1.3.1 Mechanism of hydrolysis of lactose
In humans the lactase activity is at its maximum immediately after birth (Shukla, 1975)
where the β-galactosidase enzyme is present in the brush border of the small intestine
facilitating the absorption of the monosaccharides into the bloodstream. Besides its
presence in humans, this enzyme can be found in a wide variety of organisms (Chang, et
al., 2009; Shukla, 1975). In the CAZy database glycoside hydrolases are described as a
widespread group of enzymes which hydrolyze the glycosidic bond between two or more
carbohydrates or a carbohydrate and a non-carbohydrate moiety, indicating the versatility
of these enzymes. Two models for the catalytic mechanism of the hydrolysis of the
glycosidic linkage are described in the literature (Henrissat, et al., 1995; Sinnott and
Souchard, 1973). Two invariant glutamic acid residues in the enzyme active center are
directly involved in the catalytic mechanism, acting as a proton donor and a nucleophile /
base (Gebler, et al., 1992; Vasella, et al., 2002; Wallenfels and Malhotra, 1961; White
and Rose, 1997). Figure 1 schematically displays the hydrolysis of lactose by the action
of a retaining β-galactosidase.
Figure 1. Mechanism of β-galactosidase catalyzed hydrolysis of lactose
Due to the chirality of the substrate involved, a distinction can be made between either a
retaining or an inverting mechanism. The latter is depicted in Figure 2, showing the
difference between both mechanisms.
Figure 2. Glycosydic linkage hydrolysis via inverting mechanism
The attack of the nucleophilic water molecule in the inverting mechanism and cleavage of
the glycosidic linkage take place simultaneously, resulting in inversion of the
configuration around the anomeric carbon of galactose. In contrast, the retaining
mechanism operates through multiple steps, including an enzyme-galactose complex
intermediate. This intermediate forces nucleophiles to attack from the side opposite to the
bond between the base group in the enzyme’s catalytic center and the galactose moiety.
Figure 1 schematically shows that retention of the anomeric configuration is actually
achieved by double anomeric inversion; firstly in the formation of the covalent enzyme-
glycosyl intermediate, followed by another inversion after nucleophilic attack of water.
10
Besides the hydrolytic action of β-galactosidases, the transgalactosylational activity of
these enzymes was recognized many decades ago (Wallenfels and Malhotra, 1961).
Transgalactosylation occurs through the mechanisms described previously. Instead of
water being the nucleophile, a glycoside molecule acts as an acceptor molecule for the
glycoside intermediate, thus yielding an oligosaccharide (Figure 3) (Gänzle, 2012; Otieno,
2010). The transglycosylational activity of (β-)galactosidases has been studied quite
extensively. β-Galactosidases from different sources have been characterized and studied
for their ability to synthesize (galacto-) oligosaccharides (see Table 2). In reality, the two
types of reaction, namely hydrolysis and transgalactosylation can occur simultaneously in
one reaction mixture. Besides lactose, the synthesized galacto-oligosaccharides can be
hydrolysed as well. However, the ratio of synthesis / hydrolysis is largely dependent on
the enzyme used and on the reaction conditions chosen. These enzyme properties are of
great importance when selecting an enzyme for either hydrolytic or synthetic reactions.
The yield of desired transglycosylation products of a kinetically controlled reaction is
independent of the enzyme concentration. The time to reach this yield, however, is
inversely proportional to the enzyme concentration (Kasche, et al., 1984). When given
sufficient time, the thermodynamically controlled hydrolysis reaction will eventually
yield glucose and galactose (Gosling, et al., 2009; Nakanishi, et al., 1983).
11
O
H
HO
H
O
H
H
OHH
OH
HO
O
HO
H
H
HO
H
H
OHH
HO
O
HO
H
H
HO
H
H
OHH
HO
12
1.3.3 Combinations of lactose and various acceptors
In the previous paragraphs, the activity of β-galactosidases in lactose as a preferred
substrate is described, but many β-galactosidases have the ability to transfer the sugar
moieties to another sugar or alcohol, resulting in the formation of oligosaccharides
(Adamczak, et al., 2009; Albayrak and Yang, 2002d; Berger, et al., 1995b; Li, et al.,
2009a; Li, et al., 2010; Mozaffar, et al., 1989; Takayama, et al., 1996), glycoconjugates
and alkylglycosides (Bankova, et al., 2006; Bridiau, et al., 2006; Menzler, et al., 1997;
Vic, et al., 1997). The efficiency of this transglycosylating activity strongly depends on
the source of the β-galactosidases and the conditions applied during the reaction (Boon, et
al., 2000; Gekas and Lopez-Leiva, 1985; Mahoney, 1998; Prenosil, et al., 1987a;
Prenosil, et al., 1987b). The types of oligosaccharides formed or the nature of the formed
glycosidic linkages also strongly depend on the source of enzyme. For instance, A. oryzae
β-galactosidase was shown to synthesizes many oligosaccharides with β-D-(1→6)
glycosidic bonds (Toba, et al., 1985), while the β-galactosidase from B. circulans
produces mainly β-D-(1→3) and β-D-(1→4) bonds (Coulier, et al., 2009). However,
Vetere and Paoletti have shown that the preference for the linkage position of the latter
enzyme is dependent on pH and temperature during oligosaccharide formation (Vetere
and Paoletti, 1996a; Vetere and Paoletti, 1996b). Zeng et al. additionally reported that the
regioselectivity of B. circulans β-galactosidase was greatly dependent on the nature of the
acceptor. Replacement of p-nitrophenyl-β-D-galactoside by p-nitrophenyl-β-D-
galactosaminide changed the regioselectivity from predominantly β-D-(1→3) linkages to
β-D-(1→6) linked disaccharides, caused by a more favorable orientation of the acceptor
in the hydrophobic binding locus in the active site (Zeng, et al., 2010; Zeng, et al., 2000).
13
Several glycoconjugates can be synthesized using the transglycosylating properties of β-
galactosidases. In the following section, a number of examples from the literature are
discussed. Lactose can be combined with various receptor compounds to obtain specific
(galacto-) oligosaccharides like N-acetyllactosamine (Vetere and Paoletti, 1996a; Vetere
and Paoletti, 1996b) and N-acetylglucosamine containing oligosaccharides (Takayama, et
al., 1996). Benzyl-D-xylopyranoside was shown to be a suitable acceptor for the synthesis
of galactosyl-xylopyranoside-O-Benzyl (Guisán, et al., 1993), as well as 2-hydroxybenzyl
alcohol and related compounds. Compounds that were structurally related to 2-
hydroxybenzyl alcohol were also shown to be suitable acceptors. Remarkably, the
adsorption of 3-aminobenzyl alcohol on silica yielded 96% acceptor conversion. Both O-
and N-galactosylated products were obtained.
The modification of drugs by means of glucosylation is one approach that can be taken to
prolong pharmacological activity and reduce adverse effects. Bridiau et al. chose this
approach in their examination of the acceptor properties of the drugs guaifenesin and
chlorphenesin with K. lactis β-galactosidase (Bridiau, et al., 2006). Galactosylation of the
latter compound was likewise carried out by Scheckermann et al., who also carried out
the galactosylation of chloramphenicol by using A. oryzae β-galactosidase
(Scheckermann, et al., 1997). Both studies showed that chlorphenesin was a moderate
acceptor for the galactose moiety. Acceptor conversion was approximately 15% in the
case of (Bridiau, et al., 2006), who adsorbed the acceptor molecules to various solid
supports. Scheckermann et al. used cosolvents to anticipate to the hydrophobic properties
of these compounds and achieve higher yields. Although higher yields (approx. 12.5%)
14
were obtained using acetonitrile as a cosolvent, the enzyme stability was better with
dioxane.
β-D-Galactopyranosyl-(1→6)-β-D-galactopyranosyl-(1→4)-β-D-fructopyranose, together
with the α- and β- fructofuranosidic variants were derived from lactulose using a β-
galactosidase from K. lactis (Martinez-Villaluenga, et al., 2008). The synthesis of
lactulose from mixed solutions of lactose and fructose using β-galactosidases from
different sources was reported by several authors (Adamczak, et al., 2009; Kim, et al.,
2006; Mayer, et al., 2010). Cryo-protective galactosyl-trehalose trisaccharides were
produced using a β-galactosidase (Kim, et al., 2008). Although glycosyltransferases are
in general more regioselective (Berger and Rohrer, 2003; Zigova, et al., 1999), the use of
expensive activated sugars can be a major drawback for their application. The
transglycosylation reaction catalyzed by β-galactosidases does not require activated
sugars and is thus cheaper, but less selective, yielding a variation of reaction products. If
high purity of the desired compound is required, additional downstream processing is
needed.
1.3.4 Non-aqueous reaction media
A combination of water-miscible organic solvents and water can also be used as the
reaction medium. Bankova et al. demonstrated transglycosylation activity of A. oryzae β-
galactosidase in the presence of DMSO or DMF. The activity, however, was
demonstrated to be supressed. The most plausible reason for this was lowering of the
dielectric constant in the presence of miscible organic solvents, which increases the
electrostatic interactions between polar and charged residues. Due to this the flexibility of
the protein is affected and the accessibility of substrates to the active site is reduced. The
15
presence of the water immiscible solvents iso-propanol and iso-butanol yielded
oligosaccharides as well as alkylglycosides. Increasing the iso-butanol concentration to
50%, yielded 6.7% trisaccharides and 14.5% isobutylglycosides. Due to their bipolar
character, the synthesized products could possibly be applied in pharmaceutical, chemical
or cosmetic industries as emulsifiers and / or surfactants (Bankova, et al., 2006; Carretti,
et al., 2007). Sauerbrei and Thiem conducted transglycosylation reactions with A. oryzae
and E. coli β-galactosidase in aqueous solutions containing up to 50% acetonitrile.
Besides o- and p-nitrophenyl glycosides, they also synthesized galactosylated L-serine
(β-Gal-L-Ser) (Sauerbrei and Thiem, 1992). Pérez-Sánchez et al. demonstrated a change
in regioselectivity for B. circulans β-galactosidase from β-D-(1→4) linkages to β-D-
(1→6) linkages when a 2M concentration of glycerol derived solvents was used during
the synthesis of disaccharides using p-nitrophenyl-β-D-galactopyranoside and N-acetyl-
glucosamine as substrates. This phenomenon was explained by a molecular modeling
study and it was found that the three-dimensional arrangement between GlcNAc and the
water-solvent mixture in the active site of the enzyme, favors the β-D-(1→6) linkage
(Pérez-Sánchez, et al., 2011). These investigations show the usability and wide
applicability of β-galactosidases as catalysts for numerous reactions. Moreover, above
mentioned research reports show that β-galactosidases can be used for the synthesis of a
large amount of compounds besides galacto-oligosaccharides. Obviously, when lactose is
used in combination with other substrates, also regular galacto-oligosaccharides are
formed as part of the reaction mixture.
The solubility of lactose is another factor that needs to be considered. Being already
poorly soluble in water, the solubility of lactose in organic solvents is even lower. On the
16
other hand, water miscible solvents help lower the water activity and may therefore
contribute to a change in kinetic properties, thereby shifting more towards synthetic
activity.
The industrial enzymatic synthesis of galacto-oligosaccharides is, in most cases, carried
out in batch wise operation using stirred-tank reactor systems (Friesland Foods Domo,
2007; GTC Nutrition, 2009; Yakult Pharmaceutical Industry Co., Ltd., 2010). In the
literature, however, many other reactor systems have been described, in most cases
concerning immobilized β-galactosidases. Continuous oligosaccharide production on
laboratory or pilot scale using a packed bed reactor (PBR) systems have been described
quite extensively (Albayrak and Yang, 2002c; Albayrak and Yang, 2002d; Mozaffar, et
al., 1986; Nakkharat and Haltrich, 2007; Sheu, et al., 1998; Shin, et al., 1998a; Torres
and Batista-Viera, 2012b; Zheng, et al., 2006). These PBR systems are usually equipped
as a column system containing a fixed bed consisting of enzyme immobilized on solid
support. Given the tendency of lactose to crystallize at high concentrations these PBR
systems are forced to operate at substrate concentrations between 5 and 15% (w/v), which
is relatively low compared to the concentrations used in the batch systems. Crystallization
of lactose in a PBR causes severe problems and should therefore be avoided. In so called
enzyme-membrane reactors the enzymes are either retained by membranes (Czermak, et
al., 2004; Das, et al., 2011; Ebrahimi, et al., 2006; Engel, et al., 2008; Foda and López-
Leiva, 2000) or immobilized on the membrane surface (Bakken and Hill, 1992;
Chockchaisawasdee, et al., 2005; Güleç, et al., 2010; Prenosil and Hediger, 1985;
Pruksasri, 2007). Enzyme membrane reactors can facilitate the removal of unreacted
17
substrate and inhibiting monosaccharides. Another elegant solution for the latter case was
cell surface engineering of yeast, for which the authors reported the immobilization of a
β-galactosidase on the outer cell membrane of yeast cells. By doing so, the β-
galactosidases could synthesize galacto-oligosaccharides. Simultaneously, the resulting
glucose was utilized by the yeast as a carbon source preventing the inhibition of the
enzyme (Li, et al., 2009b).
Enzyme membrane systems, just like the PBR systems, can be susceptible to issues like
blocking when undissolved substrate is present.
1.3.6 High substrate conditions
In order to favour the synthesis of galacto- oligosaccharides formation, the synthesis is
generally performed at higher substrate concentrations (Boon, et al., 1999; Boon, et al.,
2000; Nakkharat and Haltrich, 2007; Neri, et al., 2009a; Neri, et al., 2009b; Neri, et al.,
2009c; Park, et al., 2008). High substrate concentrations lower the water activity and
facilitate the saturation of the enzyme with nucleophilic molecules other than water.
Lactose, the natural substrate for β-galactosidases and a regenerable raw material, readily
available in large quantities, however has poor dissolving properties at low temperatures
(Machadoa, et al., 2000; McSweeney and Fox, 2009; Walstra, et al., 2006). For…