University of Groningen Galacto-oligosaccharide synthesis ... · Souchard, 1973). Two invariant...
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University of Groningen
Galacto-oligosaccharide synthesis using immobilized β-galactosidaseBenjamins, Frédéric
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1.
Introduction
1.1 Background
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).
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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,
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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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1.3 Enzyme classification
β-galactosidase is systematically called β-D-galactoside galactohydrolase ((EC 3.2.1.23,
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.
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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.
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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.
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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.
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1.3.2 Mechanism of transglycosylation
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).
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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
OH
HO
H
HO
H
H
OHH
OH
HO
Glc
Enzyme-Galactose complex
O
HO
H
H
HO
H
H
OHH
HO
Enzyme-Galactose complex
O-
O
OO
H
OO
O-
O
OHO
O-
O
OO
O-
O
O
HO
H
H
HO
H
H
OHH
HO OH
HO
H
O
H
H
OHH
OH
HO
O
HO
H
H
HO
H
OHH
HO O
HO
H
H
O
H
H
OHH
HO O
HO
H
O
H
H
OHH
Trisaccharide
OH
Figure 3. β-galactosidase catalyzed oligosaccharide formation
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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).
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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%)
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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
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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
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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.
1.3.5 Reactors
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
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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 the
synthesis of GOS, it is therefore desirable that the reaction can be performed at high
temperature, thus necessitating the use of the enzymes that are stable at elevated
temperatures. While the stabilizing properties of highly concentrated sugar solutions have
been described (Arakawa and Timasheff, 1982; Back, et al., 1979), β-galactosidases from
hyperthermophilic micro-organisms might also offer a solution to overcome this hurdle
(Bruins, et al., 2003; Hansson, et al., 2008; Ji, et al., 2005; Petzelbauer, et al., 2001).
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However, β-galactosidases from these organisms may have the benefits of very high
stability at elevated temperatures and thus very high substrate concentrations, but this
does not necessarily mean a higher yield of GOS, as also becomes clear from Table 2.
Many enzymes have been used to synthesize GOS; Table 2 provides a comprehensive,
yet not exhaustive, overview of the enzymatic synthesis of galacto-oligosaccharides by β-
galactosidases from various sources. Distinction was made in terms of enzyme source,
whether derived from moulds, bacteria, yeast or other sources. Besides GOS synthesis
solely from lactose, occasionally some examples of combined substrates are mentioned.
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Table 2. Overview of GOS production using free enzymes
(Substrate is lactose unless stated otherwise)
Enzyme source Lactose
[g.L-1]
T
[°C]
pH
Max. GOS
[%]
t
[h]
P
[g-1.L-1.h-1] Ref.
BACTERIA
Bacillus circulans 52.3 60 6.0 - a 23 - (Yanahira, et al., 1995)
Bacillus circulans 171b 30c 4.5 2b 60 - (Kamerke, et al., 2012)
Bacillus circulans β-galactosidase II 45.6 40 6.0 6 0.25 10.9 (Mozaffar, et al., 1984)
Bacillus circulans 250d 15 5.0 7d 3 2.3 (Vetere and Paoletti, 1996b)
Bacillus circulans 221.6 50 5.0 12.5 - - (Boon, et al., 2000)
Bacillus circulans 248e 40 7.0 13e 15 - (Usui, et al., 1993)
Bacillus circulans 342 40 6.0 15.36 5 10.5 (Li, et al., 2010)
Bacillus circulans AJ1284f 300 30 - 19.0 16 1.2 (Onishi, et al., 1995)
Bacillus circulans 200 30 6.0 24g 12 4.0 (Mozaffar, et al., 1985)
Bacillus circulans 250h 4 6.6 30.6 2 30.6 (Das, et al., 2011)
Bacillus circulans 171i 40 6.0 37.8i 4 - (Li, et al., 2010)
Bacillus circulans 100 45 - 39.7 - - (Pruksasri, 2007)
Bacillus circulans β-galactosidase I 45.6 40 6.0 41 3.3 5.6 (Mozaffar, et al., 1984)
20
Bacillus circulans 200 40 6.6 42 - - (Gosling, et al., 2011)
Bacillus circulans 400 - 5.5 49.4 6.5 30.4 (Rodriguez-Colinas, et al., 2012)
Bacillus circulans 100 50 6.6 55 30 1.8 (Gosling, et al., 2009)
Bacillus circulans 300j 40 6.0 56.0j - - (Li, et al., 2009a)
Bacillus megaterium AJ1272f 300 30 19.3 16 1.2 (Onishi, et al., 1995)
Bacillus thiaminolyticus AJ1366f 300 30 24.3 16 1.5 (Onishi, et al., 1995)
Bacillus sp. 330 50 5.0 34 5 22.4 (Cheng, et al., 2006a)
Bacillus sp. 360 55 5.5 43 9 17.2 (Cheng, et al., 2006b)
Bacillus stearothermophilus 180 37 6.5 23 ~7k 5.9 (Placier, et al., 2009)
Bifidobacterium infantis RW-8120l 400 50 - 13.2 6 8.8 (Roy, et al., 2002)
Bifidobacterium infantis HL95m 300 60 7.5 20 10 6.0 (Hung and Lee, 2002)
Bifidobacterium pseudolongum 300 55 7.5 26.8 24 3.4 (Rabiu, et al., 2001)
Bifidobacterium longum BCRC 15708 400 45 6.8 32.5 10 13.0 (Hsu, et al., 2007)
Bifidobacterium bifidum BB12 300 55 7.5 37.6 24 4.7 (Rabiu, et al., 2001)
Bifidobacterium adolescentis 300 55 7.5 43.1 24 5.4 (Rabiu, et al., 2001)
Bifidobacterium bifidum NCIMB 41171 450 40 6.2 43.8 6 32.8 (Goulas, et al., 2007)
Bifidobacterium angulatum 300 55 7.5 43.8 24 5.5 (Rabiu, et al., 2001)
Bifidobacterium bifidum DSM 20215m 200 37 6.0 44.0 20 4.4 (Jørgensen, et al., 2001)
Bifidobacterium bifidum NCIMB 41171 400 40 6.4 47n 24 7.8 (Goulas, et al., 2009)
Bifidobacterium infantis 300 55 7.5 47.6 24 6.0 (Rabiu, et al., 2001)
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Bifidobacterium bifidum NCIMB 41171l 390 55 6.8 50o 15 13.0 (Osman, et al., 2010)
Bifidobacterium bifidum NCIMB 41171 BbgIVm 430 65 6.8 54.8 8 35.1 (Osman, et al., 2012)
Clavibacter michiganensis ATCC492f 300 30 18.3 16 1.1 (Onishi, et al., 1995)
Enterobacter cloacae B5p 275 50 7.0 55 12 11.5 (Lu, et al., 2009)
Enterobacter agglomerans B1 125 50 7.5 38 8 5.9 (Lu, et al., 2007)
Enterococcus faeccium MTCC5153 330 37 7.0 - 24 - (Badarinath and Halami, 2011)
Escherichia coli 171q 30 7.3 31.5q 96 0.56 (Reuter, et al., 1999)
Lactobacillus sakei Lb790 215 37 6.5 41 3 29.4 (Iqbal, et al., 2011)
Lactobacillus plantarum 205 37 6.5 41 - - (Iqbal, et al., 2010)
Lactobacillus acidophilus R22 205 30 6.5 38.5 - - (Nguyen, et al., 2007)
Lactobacillus reuteri 205 37 6.5 38 - - (Splechtna, et al., 2006)
Lactobacillus fermentum K4m 400 45 6.5 37 9 16.4 (Liu, et al., 2011)
Lactobacillus bulgaricus L3 190 45 - 32s 18 3.4 (Lu, et al., 2010)
Lactobacillus pentosus KUB-ST10-1 205 30 6.5 31 - - (Maischberger, et al., 2010)
Lactobacillus sp. 205 37 6.0 30 - - (Nguyen, et al., 2007; Splechtna, et al., 2007b)
Pyrococcus furiosusa 282 80 5.0 6.8 - - (Bruins, et al., 2003)
Pyrococcus furiosuss 700 95 5.0 44.6 105 3 (Hansson, et al., 2008)
Pyrococcus furiosusa 700 95 5.0 40 56 5 (Hansson and Adlercreutz, 2001)
Pyrococcus furiosusa 270 70 - 32 - - (Petzelbauer, et al., 2001)
Rhizobium meliloti AJ2823f 300 30 14.0 16 0.88 (Onishi, et al., 1995)
22
Streptococcus thermophilus 700 50 6.5 40 - - (Smart, 1991)
Streptococcus thermophilus DN-001065 - - - 35 - - (Ji, et al., 2005; Perrin, et al., 2000)
Sulfolobus solfataricusm 600 80 6.0 53 48 6.6 (Park, et al., 2008)
Sulfolobus solfataricusm 700 85 6.5 37.0 2 129.5 (Hansson and Adlercreutz, 2001)
Sulfolobus solfataricusm 270 70 - 26 - - (Petzelbauer, et al., 2001)
Sulfolobus solfataricusm
342t 65 7.0 47.7p 72 2.3 (Reuter, et al., 1999)
Thermotoga maritimam 500 80 6.0 18.6 5 18.2 (Ji, et al., 2005)
Thermus aquaticus YT-1 160 70 4.6 32.4 24 2.2 (Akiyama, et al., 2001; Berger, et al., 1995b)
Thermus sp. Z-1 301 70 6.8 39.9 4 30.0 (Akiyama, et al., 2001)
Vibrio metschnikovii AJ2804f 300 30 14.0 16 0.88 (Onishi, et al., 1995)
YEASTS
Apiotrichum humicola ATCC14438f 300 30 - 13.3 16 0.83 (Onishi, et al., 1995)
Candida bombicola 9016u 400 28 7.0 30 4 30 (Petrova V.Y. and Kujumdzieva, 2010)
Cryptococcus laurentii IFO609f 300 30 - 14.3 16 0.89 (Onishi, et al., 1995)
Cryptococcus laurentii OKN-4l 100 30 3 32.5v 33 0.98 (Ozawa, et al., 1991)
Cryptococcus laurentii OKN-4 2.5 50 5.0 44 29 0.04 (Ohtsuka, et al., 1990)
Geotrichum amycelium ATCC56046f 300 30 - 19.0 16 1.2 (Onishi, et al., 1995)
Kluyveromyces fragilis AJ4060f 300 30 - 12.0 16 0.75 (Onishi, et al., 1995)
Kluyveromyces fragilis 300 40 6.5 13w 0.5 117 (Adamczak, et al., 2009)
Kluyveromyces fragilis 221.6 40 6.5 (Boon, et al., 2000)
23
Kluyveromyces fragilis 9016u 400 28 7.0 25.0 4 25.0 (Petrova V.Y. and Kujumdzieva, 2010)
Kluyveromyces lactis 221.6 40 7.3 4.5 6 1.7 (Boon, et al., 2000)
Kluyveromyces lactis 300 40 6.5 8.2w 0.3 73.8 (Adamczak, et al., 2009; Martinez-Villaluenga,
et al., 2008)
Kluyveromyces lactis 200 37 6.8 13 48 0.54 (Burvall, et al., 1979)
Kluyveromyces lactis 250 50 6.5 14.05x 5 7.0 (Martinez-Villaluenga, et al., 2008)
Kluyveromyces lactis 200y 37 6.75 16.5 60 0.55 (Pocedičová, et al., 2010)
Kluyveromyces lactis 400 40 7.0 24.8 4 24.8 (Chockchaisawasde, et al., 2005)
Kluyveromyces lactis BP4z 250 50 6.5 25.6 4 16.0 (Padilla, et al., 2012)
Kluyveromyces lactis O1z 250 50 6.5 26.3 4 16.4 (Padilla, et al., 2012)
Kluyveromyces lactis BP6z 250 50 6.5 27.4 4 17.1 (Padilla, et al., 2012)
Kluyveromyces lactis C2z 250 50 6.5 27.9 4 17.4 (Padilla, et al., 2012)
Kluyveromyces lactis BP5z 250 50 6.5 27.9 4 17.4 (Padilla, et al., 2012)
Kluyveromyces lactis BP8z 250 50 6.5 30.1 4 18.8 (Padilla, et al., 2012)
Kluyveromyces lactis BP1z 250 50 6.5 30.2 4 18.9 (Padilla, et al, 2012)
Kluyveromyces lactis BP3z 250 50 6.5 31.4 4 19.6 (Padilla, et al., 2012)
Kluyveromyces lactis BP2z 250 50 6.5 31.5 4 19.7 (Padilla, et al., 2012)
Kluyveromyces lactis O2z 250 50 6.5 31.7 4 19.9 (Padilla, et al., 2012)
Kluyveromyces lactis C1z 250 50 6.5 31.9 4 19.9 (Padilla, et al., 2012)
Kluyveromyces lactis BP7z 250 50 6.5 32.0 4 20.0 (Padilla, et al., 2012)
24
Kluyveromyces lactis CECT1961T z 250 50 6.5 32.4 4 20.3 (Padilla, et al., 2012)
Kluyveromyces lactis 250 40 6.5 39.5 48 2.1 (Montilla, et al., 2012)
Kluyveromyces lactis 400 40 6.8 40.7 22 7.4 (Rodriguez-Colinas, et al., 2011)
Kluyveromyces lactis 400 40 6.8 42.6 22 7.7 (Rodriguez-Colinas, et al., 2011)
Kluyveromyces lactisae 400 40 6.8 44.2 6 29.5 (Rodriguez-Colinas, et al., 2011)
Kluyveromyces lactis 250 40 6.5 50.5 3 42.1 (Cardelle-Cobas, et al., 2009)
Kluyveromyces marxianus t1u 400 28 7.0 5.0 24 0.83 (Petrova V.Y. and Kujumdzieva, 2010)
Kluyveromyces marxianus 909u 400 28 7.0 5.0 4 5.0 (Petrova V.Y. and Kujumdzieva, 2010)
Kluyveromyces marxianus 903u 400 28 7.0 12.5 4 12.5 (Petrova V.Y. and Kujumdzieva, 2010)
Kluyveromyces marxianus CCT7082aa,ab 500 45 7.0 12.8 3 16.0 (Manera, et al., 2011)
Kluyveromyces marxianus CCT7082aa,ac 500 45 7.0 14.0 3 23.3 (Manera, et al., 2011)
Kluyveromyces marxianus CCT7082aa 500 45 7.0 14.4 3 24 (Manera, et al., 2011)
Kluyveromyces marxianus CCT7082aa,ad 500 45 7.0 15.0 3 25 (Manera, et al., 2011)
Kluyveromyces marxianus CCT7082aa 500 45 7.0 16.6 3 27.6 (Manera, et al., 2011)
Kluyveromyces marxianus Var. lactis OE-20 100 30 7.0 25.1 18 1.4 (Kim, et al., 2001)
Kluyveromyces marxianus O4z 250 50 6.5 35.0 4 21.9 (Padilla, et al., 2012)
Kluyveromyces marxianus 330 50 6.5 35.0 3 38.5 (Cheng, et al., 2006a)
Kluyveromyces marxianus 905u 400 28 7.0 37.5 24 6.25 (Petrova V.Y. and Kujumdzieva, 2010)
Kluyveromyces marxianus 906u 400 28 7.0 37.5 24 6.25 (Petrova V.Y. and Kujumdzieva, 2010)
Kluyveromyces marxianus O3z 250 50 6.5 41.8 4 26.1 (Padilla, et al., 2012)
25
Kluyveromyces marxianus t3u 400 28 7.0 42.5 4 42.5 (Petrova V.Y. and Kujumdzieva, 2010)
Kluyveromyces marxianus 904u 400 28 7.0 50.3 4 50.3 (Petrova V.Y. and Kujumdzieva, 2010)
Lipomyces NKD-14 200 30 6.5 50.0 144 0.69 (Munehiko, et al., 1988)
Rhodotorula minuta IFO879f 300 30 - 22.3 16 1.4 (Onishi, et al., 1995)
Rhodotorula minuta IFO879 200 60 6.0 44.0 2 44.0 (Onishi, et al., 1995)
Rhodotorula lactose IFO1423 100 30 6.5 40.0 72 0.56 (Munehiko, et al., 1988)
Saccharopolyspora rectivirgula 600 70 7.0 41 - - (Nakao, et al., 1994)
Sirobasidium magnum CBS6803f 300 30 21.0 16 1.3 (Onishi, et al., 1995)
Sirobasidium magnum CBS6803 200 60 5.0 36.0 24 1.5 (Onishi and Tanaka, 1997)
Sirobasidium magnum CBS6803 200 60 6.0 36.8 2 36.8 (Onishi and Yokozeki, 1996)
Sporobolomyces singularis 200 45 3.5 38.3 50 1.53 (Ishikawa, et al., 2005)
Sporobolomyces singularis 180 50 5.0 50 23 3.9 (Cho, et al., 2003a; Cho, et al., 2003b)
Sporobolomyces singularisl 100 24 3.75 50 0.69 – 0.52 72-96 (Gorin, et al., 1964)
Sporobolomyces singularisl 50 25 6.0 72 70 0.51 (Shin, et al., 1995)
Sterigmatomyces elviae CBS8119f 300 30 24.7 16 1.5 (Onishi, et al., 1995)
Sterigmatomyces elviae CBS8119ae 360 60 6.0 37.5 20 6.8 (Onishi, et al., 1995)
Sterigmatomyces elviae CBS8119 200 60 6.0 45.5 2 45.5 (Onishi and Yokozeki, 1996)
Sterigmatomyces elviae CBS8119af 400 30 6.0 54 68 3.17 (Onishi and Tanaka, 1998)
Sterigmatomyces elviae CBS8119af 360 - - 64.4 - - (Onishi and Tanaka, 1998)
26
MOULDS
Aspergillus aculeatus 285 60 6.5 30 7 12.2 (Cardelle-Cobas, et al., 2008a; Cardelle-Cobas,
et al., 2008b)
Aspergillus aculeatus 150 60 6.5 19.0x 5 5.7 (del-Val, et al., 2001)
Aspergillus aculeatus 285 60 6.5 - a - - (Cardelle-Cobas, et al., 2008a; Cardelle-Cobas,
et al., 2009)
Aspergillus aculeatus 450ag 60 6.5 28 24 5.25 (Cardelle-Cobas, et al., 2008a; Cardelle-Cobas,
et al., 2008b)
Aspergillus nidulans 72ah 37 5.0 50 8 - (Nakai, et al., 2010)
Aspergillus nigerao 90ai 65 4.5 50 240 1.8 (Yamashita, et al., 2005)
Aspergillus oryzae 379.6 40 4.5 31 5 23.5 (Iwasaki, et al., 1996)
Aspergillus oryzae 342t 60 4.8 35.5t 96 1.3 (Reuter, et al., 1999)
Aspergillus oryzae 150aj 40 5.5 21.2 24aj 1.3 (Bankova, et al., 2006)
Aspergillus oryzae 221.6 40 4.5 9.6x 6 3.5 (Boon, et al., 2000)
Aspergillus oryzae 100 40 6.5 13.7w 0.3 41.1 (Adamczak, et al., 2009)
Aspergillus oryzae 300 37 4.8 - a - 8 (Toba, et al., 1985)
Aspergillus oryzae 422 40 4.5 28 10 11.8 (Vera, et al., 2011)
Aspergillus oryzae 475 47.5 4.5 29 10 13.8 (Vera, et al., 2012)
Aspergillus oryzae 221.6 40 4.5 47.9 - - (Boon, et al., 2000)
Aspergillus oryzae 167 47 4.5 22.8 1.4 27.2 (Chen, et al., 2002)
27
Aspergillus oryzae 253ak 50 4.5 31.1 3.5 22.5 (Chen, et al., 2002)
Aspergillus oryzae 330 30 - 21 3 23.1 (Cheng, et al., 2006a)
Paecilomyces aerugineusal 300 50 5.0 19.7 4 14.8 (Katrolia, et al., 2011)
Penicillium funiculosum Cellulase 50 40 5.0 20 6 1.67 (Shin and Yang, 1996)
Penicillium simplicissimum 600 50 6.5 30.5 5 36.6 (Cruz, et al., 1999)
Penicillium sp. KFCC 10888 400 55 4.0 40.5 96 1.69 (In and Chae, 1998)
Talaromyces thermophilus 200 40 6.5 50 8 12.5 (Nakkharat, et al., 2006)
Trichoderma harzianum 150 30 7.0 32.1 300 - 500 0.16 – 0.096 (Prakash, et al., 1987)
OTHER
Bos taurus -am 45 6.5 75 - - (Zeng, et al., 2010)
Rhynchophorus palmarum 136.8an 37 6.0 43an 20 0.22 (Yapi, et al., 2007)
Scopulariopsis sp. ATCC44206 400 45 5.0 20.2 12 6.7 (dos Santos, et al., 2009)
a Total GOS yield was not reported, a structural analysis of the formed GOS was performed; b 171 g.L-1 lactose + 56.6 g.L-1 UDP-Glucose, yielding UDP-GOS; c Microwave assisted heating; d 250 g.L-1 lactose + 50 g.L-1 N-acetylglucosamine, only N-acetyllacosamine yield was reported; e 248 g.L-1 lactose + 160 g.L-1 N-acetylgalactosamine, GalNAc-oligosaccharide yield was reported; f Whole cell fermentation and simultaneous GOS synthesis; g Trisaccharide yield was reported; h 20% lactose w/w in whey permeate; i 171 g.L-1 lactose + 110.5 g N-acetylglucosamine, GlcNAc-oligosaccharide yield was reported; j 300g.L-1 lactose + 300 g.L-1 sucrose, lactosucrose yield was reported;
k Derived from figure 4 in (Placier, et al., 2009); l Whole cells;
28
m Recombinant, expressed in Escherichia coli; n Highest yield of 4 iso-forms of β-galactosidase from Bifidobacterium bifidum NCIMB 41171; o Derived from Fig. 2 in (Osman, et al., 2010) ; p Freeze thawed cells; q 171 g.L-1 lactose + 110.5 g.L-1 N-acetylglucosamine, oligosaccharide yield expressed as % of initial donor concentration; r Trisaccharides and higher; s 342 g.L-1 lactose + 221 g.L-1 N-acetylglucosamine, oligosaccharide yield expressed as % of initial donor concentration; t Mutant, expressed in Escherichia coli; u Crude cell preparations, toluene treated; v 171 g.L-1 lactose + 110.5 g.L-1 N-acetylglucosamine, oligosaccharide yield expressed as % of initial donor concentration; w Derived from Fig. 1, (Adamczak, et al., 2009) x Trisaccharides; y UF whey permeate; z Crude cell extract, organism extracted from artisanal cheese; aa Permeabilized cells (isopropanol); ab Pressurized in CO2; ac Pressurized in propane, 250 bar, 6 hour; ad Pressurized in n-butane, 10 bar, 1 hour; ae Toluene treated resting cells; af Fermentation system; ag 450 g.L-1 lactulose, yielding 6’-galactosyllactulose (15%) and HRTOS (13%); ah 72 g.L-1 mannose + 40 mM p-NPαGal, yielding galactosylmannosides; ai Substrate was 90% w/v galactose; aj
150 g.L-1 lactose in 20% iso-butanol, yielding 6.68% GOS and 14.5% alkylglycosides; ak Whey permeate; al Recombinant, expressed in Pichia pastoris; am 5’-O-β-D-Galactosyl-floxuridine was formed from 20 mM floxuridine (acceptor) and 10mM O-nitrophenol-β-D-galactoside (galactose donor); an Substrate was 138.6 g.L-1 lactose + 10 g.L-1 2-phenylethanol, yielding phenylethylgalactoside (yield is expressed as amount of 2-phenylethylglycoside); ao α – Galactosidase.
29
1.4 Sources of β-galactosidases
As becomes clear from Table 2, many β-galactosidases from numerous sources can be
utilized to synthesize galacto-oligosaccharides. In this paragraph a selection of β-
galactosidases from different microbial sources will be further discussed.
1.4.1 Fungal β-Galactosidases
Fungal β-galactosidases have been investigated thoroughly. Especially β-galactosidases
from the Aspergillus genera are described in many publications.
Aspergillus oryzae β-galactosidase
The β-galactosidase from A. oryzae has been known for quite some time already. Several
enzyme manufacturers produce commercial enzyme preparations derived from this
organism. Given its stability at low pH values, the β-galactosidase from A.oryzae is also
applied in food supplements. The enzyme can be ingested in tablet form after the
consumption of lactose containing products; this β-galactosidase, active at the pH value
in the stomach, can hydrolyze lactose into its monosaccharides and thus prevent
discomfort resulting from lactose intolerance. Besides its good hydrolyzing properties,
this particular enzyme possesses high transglycosylating capacity as well (Table 2).
Reuter et al. achieved a 35.5% yield of N-acetylated oligosaccharides using lactose and
N-acetylglucosamine as substrates (Reuter, et al., 1999). Chen and co-workers (Chen, et
al., 2002) applied this enzyme in whey permeate, yielding 31.1% of GOS based in total
saccharide content, a value equal to that of Iwasaki (Iwasaki, et al., 1996), who reported a
31% yield.
30
Figure 4. GOS yield as function of temperature and lactose concentration using β-galactosidase from A.
oryzae. GOS yield is expressed as percentage of the total solids (i.e. substrate) (labels refer to following
references: a: (Adamczak, et al., 2009), b: (Bankova, et al., 2006), c: (Chen, et al., 2002), d: (Boon, et al.,
2000), e: (Chen, et al., 2002), f: (Cheng, et al., 2006a), g: (Reuter, et al., 1999), h: (Iwasaki, et al., 1996), i:
(Vera, et al., 2011), j: (Vera, et al., 2012).
The effect of high substrate concentrations on the yield of GOS is well-known. GOS
synthesis using the β-galactosidase from A. oryzae is no exception to this. Figure 4 shows
the GOS yield reported by several authors using various conditions. Clearly, GOS yields
are higher at high lactose concentrations in combination with elevated temperatures. In
general substrate concentrations of 100 – 475 g.L-1 are used within a temperature range of
45 – 50 °C under slightly acidic conditions (pH 4.5). GOS yields are reported to be
around 30%, based on total carbohydrates. Given the acidic pH optimum of this enzyme
it could be well applied for GOS synthesis in acid whey or other applications with a low
pH.
31
Other fungal β-galactosidases
Besides the β-galactosidase from A. oryzae only a limited number of fungi derived β-
galactosidases are mentioned in literature. Another Aspergillus species that was
investigated because of its trangalactosylating activity was A. aculeatus. The actual
enzyme preparation (Pectinex SL, Novozymes) is a pectinase preparation used as
maceration aid for fruit and vegetable processing. A number of authors showed the
preparation possessed β-galactosidase side-activity. Cardelle-Cobas et al. synthesized
30% GOS starting from a 285 g.L-1 lactose solution (Cardelle-Cobas, et al., 2008a). Some
β-galactosidases from the genera of Penicillium have shown some nice results with GOS
yields ranging from 20 – 40 % based on total carbohydrates (Cruz, et al., 1999; In and
Chae, 1998; Shin and Yang, 1996). Lesser known species in terms of β-galactosidase
activity, like Talaromyces thermophilus, Trichoderma harzianum and Paecilomyces
aerugineus also showed the ability of GOS synthesis (Katrolia, et al., 2011; Nakkharat, et
al., 2006; Prakash, et al., 1987). In general, it can be concluded that fungi derived β-
galactosidases have relatively low hydrolysis / transglycosylation ratios, which is
beneficial for GOS synthesis.
1.4.2 Yeast β-Galactosidases
Taxonomically, yeasts are classified in the kingdom of Fungi. They are well recognized
for their excellent productive capacities. Here we describe a selection of β-galactosidases
derived from yeasts and their ability to synthesize galacto-oligosaccharides.
32
Kluyveromyces spp. β-galactosidases
An example of an elaborately described β-galactosidase is the enzyme derived from
Kluyveromyces spp. This enzyme has been on the market for decades and is
manufactured, for instance, by DSM under the name Maxilact® since 1978 (DSM, 2013).
Besides its use as a lactase, the enzyme readily synthesizes oligosaccharides at higher
substrate concentrations. These conditions also contribute to increased heat stability of
the enzyme. Whereas the enzyme will rapidly become inactivated in diluted aqueous
systems, the presence of high concentrations of substrate enables the use of higher
temperatures. Table 2 contains over 20 references of GOS synthesis using β-galactosidase
from Kluyveromyces sp. GOS levels between 30 – 50% are achieved using β-
galactosidase from K. lactis. The highest yield was reported by Cardelle-Cobas et al.,
who achieved a GOS level of 50,5%, starting from 250 g.L-1 lactose (Cardelle-Cobas, et
al., 2009; Martínez-Villaluenga, et al., 2008). The activity of this enzyme is increased in
the presence of K+ and especially Mg2+ ions (Jurado, et al., 2006), which can contribute
to more efficient use of enzyme.
Sporobolomyces singularis β-galactosidases
This particular yeast was discovered in 1962 by Phaff and Do Carmo Sousa, who isolated
this species from bark beetle frass (Phaff and Do Carmo-Sousa, 1962). The β-
galactosidase from S. singularis showed good transglycosylation properties, as was
shown by Gorin et al. (Gorin, et al., 1964). Synthesis of 4’-galactosyllactose and a
tetrasaccharide (O-β-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-O-β-D-
galactopyranosyl-(1→4)-D-Glucose) during fermentation yielded 50% GOS (combined
33
yield) and showed a strong preference for β-D-(1→4) linkages. Vigorous growth of the
yeast was observed at a pH value of 6, but remarkably no GOS were formed at this pH
value. This could indicate the catabolism of substrate to provide the energy required for
growth. Many years later, Shin et al. optimized the culture conditions for the production
of GOS. The optimized medium contained 5% lactose and 0,75% yeast extract, yielding
72% GOS in 70 hours fermentation (Shin, et al., 1995). Sakai et al. achieved a GOS yield
of 40% with immobilized cells of S. singularis. The authors indicate that the isolation
process for this enzyme is rather complicated, thereby justifying their choice for using
whole cells (Sakai, et al., 2008).
Other yeast β-galactosidases
Besides the yeast sources mentioned in the previous paragraphs, many other species have
also been investigated for their transgalactosylational properties. Cryptococcus laurentii,
Rhodotorula minuta, Sirobasidium magnum, Sterigmatomyces elviae and
Saccharopolyspora rectivirgula, were reported to have high transglycosylation properties
with GOS yields of 44.0%, 44.0%, 36.8%, 64.4% and 41.0%, respectively (Nakao, et al.,
1994; Ohtsuka, et al., 1990; Onishi and Yokozeki, 1996; Onishi and Yokozeki, 1996;
Onishi and Tanaka, 1998). In general, yeast β-galactosidases seem to outperform the
fungal versions in terms of oligosaccharide production yield.
1.4.3 Bacterial β-Galactosidases
Bacterial β-galactosidases have a profound reputation with respect to oligosaccharide
synthesis. In this paragraph we highlight the GOS production with β-galactosidase from a
number of well-described sources.
34
Bacillus circulans β-galactosidase
The β-galactosidase from Bacillus circulans was shown to have strong
transgalactosylational properties. Mozaffar and Nakanishi (Mozaffar, et al., 1984)
isolated two iso-forms from the crude cell extract of B. circulans. Both β-galactosidases
had transgalactosylational properties, however β-gal I produced significantly higher
amounts of galacto-oligosaccharides, even at low substrate levels. Vetere et al. isolated a
third iso-form from B. circulans crude cell extract, yet with different properties (Vetere
and Paoletti, 1998). Much later Song et al. isolated 4 different β-galactosidase iso-forms
from B. circulans. All iso-forms showed both hydrolytic and synthetic activity; however
large differences with respect to these properties were found (Song, et al., 2011). In
contradiction to the general observation that high substrate concentrations generally
favour transglycosylation, thus increasing the GOS yield, Gosling et al. reported 55%
GOS on total solids starting from a 100 g.L-1 lactose solution, showing the strong
synthetic properties of B. circulans β-galactosidase (Gosling, et al., 2009; Gosling, et al.,
2011). Song et al. demonstrated that the four iso-forms present in a commercial enzyme
preparation are derived from one initially formed β-galactosidase. During fermentation
also low protease activity is present, which causes truncation of the initial enzyme. A
specific domain (DS) domain in the C-terminal peptide region appeared to be essential
for the repression of GOS synthesis (Song, et al., 2011; Song, et al., 2013). Cleavage of
this part of the enzyme results in lower hydrolysis / transglycosylation ratios for the
truncated enzymes. Eventually, the obtained GOS mixture will be the result of the
combined activity of these iso-forms.
35
Bifidobacterium spp. β-galactosidase
The synthesis of GOS using β-galactosidase from Bifidobacteriaceae was reported by a
number of authors (Table 2). Several authors postulated the idea that GOS, produced by
β-galactosidases derived from Bifidobacteriaceae, would possess improved prebiotic
properties. Since the synthesized GOS is determined to be digested by Bifidobacteriaceae
in the large intestine, GOS structures like these would be more compatible to the
metabolism of these microorganisms (Gibson and Rastall, 2006; Rabiu, et al., 2001).
These GOS are manufactured with the aim of a higher selectivity towards specific
bacterial groups (Depeint, et al., 2008; Tzortzis, 2011). GOS synthesis using whole cells
of B. infantis, yielded a modest 13.2% GOS. Using a recombinant or the native β-
galactosidase from the same organism resulted in higher yield of 20% and 47.6%,
respectively (Hung and Lee, 2002; Rabiu, et al., 2001). Jørgensen et al. (Jørgensen, et al.,
2001) obtained a higher oligosaccharide yield after truncation of a β-galactosidase from
B. bifidum DSM 20215. The enzyme exhibited a 4-fold increase in transglycosylation
activity, compared to the native enzyme, yielding 44% GOS on total solids. This effect is
highly similar to the above mentioned truncation of B. circulans, again indicating that
structural elements of the native enzyme can hamper large structures to move into and
away from the active site. On the other hand B. bifidum (strain NCIMB 41171) whole
cells were used by Osman et al., showing GOS yields of approximately 50% can be
achieved by using another strain under different conditions (Osman, et al., 2010). Still,
the highest GOS yields using Bifidobacteriaceae were obtained by the same authors, who
reported 54.8% GOS on total solids by the action of a recombinant B. infantis β-
galactosidase (Osman, et al., 2012). At first, one may assume that the best way of GOS
36
synthesis would be to use free enzyme, since that seems to results in the highest GOS
yields. Whereas diffusion limitation could be a possible disadvantage, whole cells are
much cheaper than free enzyme. In contrast, adding more whole cells, i.e. the same
equivalent in activity units, can give the results similar to free enzyme synthesis. In fact,
the commercial prebiotic product Bimuno® is produced using permeated cells of B.
bifidum NCIBM 41171, showing whole cell synthesis could be an attractive option from
an economical point of view (Tzortzis, 2011).
Lactobacillus spp. β-galactosidase
Another genera of colonic microorganisms found in the human intestinal tract,
Lactobacilli, were also investigated for their synthetic properties. With substrate
concentrations starting from ~ 200 g.L-1 lactose, yields between 31% and 41% GOS were
reported (Iqbal, et al., 2010; Iqbal, et al., 2011; Liu, et al., 2011; Lu, et al., 2010;
Maischberger, et al., 2010; Splechtna, et al., 2006; Splechtna, et al., 2007b).
Similarly to K. lactis β-galactosidases, it was found for β-galactosidases from several
lactic acid bacteria that their activity is also enhanced in the presence of Mg2+ ions.
(Garman, et al., 1996). In analogy with GOS produced with β-galactosidases derived
from Bifidobacteriaceae, GOS obtained with Lactobacillus enzymes may also exhibit
specific selectivity.
Escherichia coli β-galactosidase
The β-galactosidase from E. coli was extensively studied by Wallenfels and co-workers
(Wallenfels and Malhotra, 1960). This protein has a tetrameric structure with a molecular
weight of 464 kDa (Matthews, 2005). This structure was shown to be essential for the
37
enzyme’s functioning. The β-galactosidase from E. coli has been the subject of many
studies aiming to elucidate either the mechanism of action or the amino acids involved in
catalysis. The essential amino acid for catalysis is Glu537, which is involved in the
cleavage of the glycosidic linkage and formation of the covalent enzyme-galactosyl
intermediate. In this step of the reaction cycle also the aglycon (glucose in the case of
lactose as substrate) moiety is released, facilitated by Glu461, modulated by His418
together with a magnesium ion (Juers, et al., 2009). The β-galactosidase from
Escherichia coli was shown earlier to have a strong preference for β-D-(1→6) linkages,
making it useful in the synthesis of compounds where this type of linkage is desired
(Reuter, et al., 1999). This phenomenon was observed by others as well (Ajisaka, et al.,
1987; Kuhn, et al., 1955). Additionally, lactose was preferably converted into its allo-
isomer. This type of transglycosylation partially follows a deviant pathway compared to
the mechanism described earlier (Figure 3). Huber et al. distinguished between direct and
indirect transglycosylation. The latter being the generally accepted mechanism for
transglycosylation, the former is also described as migration of the galactose moiety from
the 4 to 6 position of the glucose without prior release of glucose (Huber, et al., 1976).
Juers et al. showed that His418 plays an important role in binding glucose as an acceptor,
indicating the importance of this amino acid for allo-lactose formation (Juers, et al.,
2009). At high glucose concentrations (i.e. in an advanced phase of the hydrolysis
reaction) both direct and indirect transglycosylation occur. Since E. coli β-galactosidase
is able to hydrolyze a wide variety of β-D-galactopyranosides with different aglycones,
eventually the formed allo-lactose is likely to be hydrolyzed to glucose and galactose,
since it is a better substrate to the enzyme than lactose (Wallenfels and Malhotra, 1961).
38
β-Galactosidases from extremophiles
The natural substrate for β-galactosidases is lactose, a disaccharide with rather poor
dissolving properties in water (37.2 g.100 g-1 at 60 °C) (Machadoa, et al., 2000). Since it
is well recognized that the kinetically controlled formation of oligosaccharides is
favoured at high substrate concentrations, research has been addressed to the use of β-
galactosidases from hyperthermophiles, i.e. organisms capable of flourishing at
temperatures above 80 °C. The use of these enzymes would enable very high substrate
concentrations, favouring the yield of the transglycosylation reaction. Organisms that
have been studied include Pyrococcus furiosus, Sulfolobus solfataricus, Thermotoga
maritime (Ji, et al., 2005), Thermus thermophiles (Fourage, et al., 2000; Gu, et al., 2009)
and Thermus aquaticus (Berger, et al., 1995b). The highest yields in terms of GOS were
reported for β-galactosidases derived from the former two organisms. The recombinant
mutant enzyme from P. furiosus yielded 44.6% of GOS on total solids (700 g.L-1 lactose)
at 95 ° C (Hansson, et al., 2008). S. solfataricus β-galactosidase achieved 53 % of GOS
on total solids (600 g.L-1 lactose) at a temperature of 80°C (Park, et al., 2008). The high
thermal stability of β-galactosidases from extremophiles makes them potentially
interesting for GOS synthesis. Their resistance to high temperatures allows for high
substrate concentrations. An additional benefit of these highly thermostable enzymes
could be that they are easily sterilized in the immobilized form. Conversely, formation of
undesired colour compounds due to increased Maillard reaction could be a drawback.
Additionally, enzymes with a lower temperature optimum may possess a higher
transglycosylation / hydrolysis ratio, producing more GOS as a percentage of the offered
substrate. Moreover, starting with very high lactose concentrations in combination with
39
only moderate conversion could result in undesired crystallization of residual lactose in
the final product upon cooling or concentration.
1.5 Industrial applications of β-galactosidase
In view of the previous the selection of a specific β-galactosidase depends on the
application and desired properties of the final product. The food industry predominantly
applies β-galactosidases for the hydrolysis the lactose in dairy products. This contributes
to the digestibility, taste and organoleptical properties and improved processing of dairy
products (Shukla, 1975). Lactose free products give people suffering from lactose
intolerance the opportunity to consume dairy products without the undesired discomfort
associated with this disorder. Needless to say β-galactosidases with a high hydrolysis /
transglycosylation ratio are preferred for this application. Although the mechanism of
transglycosylation by a β-galactosidase was described by Wallenfels (Wallenfels and
Malhotra, 1960) in the 1960s (and earlier for α-galactosidase by Blanchard and N. Albon
(Blanchard and Albon, 1950)), large industrial production of GOS became visible only in
the end of the 1980s (Yakult Honsha Co. Ltd., 2012). Following the introduction of the
concept of prebiotics by Gibson and Roberfroid (Gibson and Roberfroid, 1995), a
growing interest in prebiotics and the recognition of their functionality boosted the
application of GOS in infant nutrition. The Japanese company Yakult has established the
production of galacto-oligosaccharides since 1989 (Yakult Honsha Co. Ltd., 2012).
Together with their activities on probiotics they are considered pioneers in the field.
Nowadays the Dutch dairy cooperation FrieslandCampina is one of the largest
manufacturers of galacto-oligosaccharides worldwide. Table 3 provides an overview of
40
commercially available GOS products and, if known, the sources of the enzymes that are
used to produce these products. Although not all of the enzymes sources used for
commercial GOS production are known, in general the enzymes mentioned in Table 3
possess good transglycosylation properties in order to achieve high GOS yields.
Galactooligosaccharides are applied mainly in infant nutrition but other food applications
are also known. Three manufacturers have obtained the GRAS (Generally Recognized As
Safe) status for their products by the American Food and Drug Administration (FDA).
41
Table 3. Commercially available GOS products.
Product Manufacturer GOS
(%)
GRAS status
notification
Organism / enzyme Ref.
Promovita® GOS First Milk Ingredients 35 No unknown
Bimuno® Clasado Ltd. 50 No Bifidobacterium bifidum
NCIMB 41171
(Goulas and Tzortzis, 2007;
Vulevic, et al., 2008)
Vivinal® GOS FrieslandCampina Domo 60 Yes Bacillus circulans (Friesland Foods Domo, 2007)
Oligomate® 55N Yakult Pharmaceutical Industry Co., Ltd 55 Yes Sporobolomyces
singularis /
Kluyveromyces lactis
(Yakult Pharmaceutical Industry
Co., Ltd., 2010)
Cup Oligo Kowa Company Ltd. / Nisshin Sugar 701 No Cryptococcus laurentii (Hartemink, et al., 1997; Osamu, et
al., 1986; Osamu, et al., 1987a;
Osamu, et al., 1987b)
Profile GOSTM Kerry Dairy Ingredients - No Bacillus circulans (Kerry Inc., ; Rodriguez-Colinas,
et al., 2012)
FloraidTM GOS Wright Agri Industries Limited, UK 39 Yes Aspergillus oryzae (International Dairy Ingredients,
2013)
Sunoligo L500 SamYang - No Unknown (SamYang Genex, 2012)
42
PurimuneTM GTC nutrition 901 Yes Bacillus circulans (GTC Nutrition, 2009)
GOS BaoLingBao Biology No Unknown (BaoLingBao Biology Co., 2012 )
GOS-570-S /
GOS-270P
New Francisco Biotechnology Co. 57
27
No Unknown (New Francisco Biotechnology
Corporation, 2012)
GOS 60L Quantum High Biological Co., Ltd. No Unknown (Quantum High Biological Co.,
Ltd., 2012)
1 Additional downstream processing was applied to increase the GOS content after synthesis (e.g. removal of monosaccharides)
43
Being a mixture of numerous oligosaccharides, the prebiotic effect of a GOS product is
the result of the prebiotic index (PI) (Sanz, et al., 2005) of all components together. As
stated before, GOS for selective stimulation of specific, desired intestinal bacteria species
have been developed. The composition of the human gut flora is known to shift during
life (Saunier and Doré, 2002). Therefore it is likely that in the coming years, this
information could influence the selection for a β-galactosidase for GOS manufacturing.
Not only will the transgalactosylation rate and stability be decisive, but also on the
specificity in terms of reaction products and their ability to selectively promote specific
gut bacteria.
1.6 Immobilized β-galactosidase
1.6.1 Introduction
Often, in industrial production, the parameters such as temperature, pH, substrate
concentration and ionic strength are chosen to optimize the product yield. These
conditions, however, may well be outside the range of those in the natural habitat of the
organisms that produce the enzyme (Hernandez and Fernandez-Lafuente, 2011; Iyer and
Ananthanarayan, 2008). These harsh conditions thus may lead to the denaturation and/or
inactivation/inhibition of enzymes. Enzyme immobilization can improve the stability of
the enzyme and may also lead to reduced inhibition (Mateo, et al., 2004). For instance,
the stabilization of enzymes by multi point attachment on epoxy supports was described.
This resulted in enhanced resistance to unfolding, which in turn decreases denaturation
and inactivation of the enzyme (Garcia-Galan, et al., 2011; Mateo, et al., 2000a; Mateo,
et al., 2002c). The β-galactosidases of E. coli (Roth and Rotman, 1975), K. lactis
44
(Pereira-Rodriguez, et al., 2012), Thermus sp. (Pessela, et al., 2003) and many other
micro-organisms are multimeric enzymes. These enzymes comprise of individual sub
units in a specific conformation. The enzyme’s activity is dependent on the conformation
of these subunits and dissociation of subunits leads to loss of activity. Strategies to
prevent this have been developed, enabling the immobilization and stabilization of
multimeric enzymes (Bolivar, et al., 2010; Fernandez-Lafuente, 2009). It is therefore
very important to understand the properties of the selected β-galactosidase in order to
choose a suitable immobilization strategy. The occurrence of multimeric enzymes is one
of the properties, hindering a general approach for β-galactose immobilization.
Enzyme immobilization is able to improve a number of enzyme characteristics such as
activity, stability and selectivity. It is therefore not surprising that the main goal of
enzyme immobilization is to maximize the number of reuse cycles and subsequently
reducing the enzyme cost contribution to the cost price of the product. Also the use of
immobilized enzymes enables the industry to process in a continuous way, which may
lead to a reduction of production costs of 40% (Katchalski-Katzir, 1993). The
immobilization of β-galactosidases, with the aim of GOS synthesis or lactose hydrolysis,
has been intensively studied in lab scale. Nevertheless, immobilized β-galactosidases
appear only to have been applied on industrial scale for lactose hydrolysis. Table 4
provides an overview of industrial applications of immobilized β-galactosidase used for
lactose hydrolysis and commercially available technologies.
45
Table 4. Overview of industrial application of immobilized β-galactosidase for lactose hydrolysis and commercially available immobilized lactase technologies
Company Country Immobilized enzyme system Capacity
[L.day-1]
Remarks REF
Central del Latte Italy K. lactis β-galactosidase
entrapped in cellulose triacetate
acetate fibres (Dinelli, et al.,
1976)
8000 Developed by SnamProgretti
Batch process
(Champagne, et al., 2010;
Liese, et al., 2006; Zuidam
and Nedovic, 2010)
Drouin Cooperative
Butter Factory
(closed in 1990)
Australia Fungal β-galactosidase on
amphoteric IEX phenol
formaldehyde resin
- Developed by Sunitomo Chemical
Co.
(Champagne, et al., 2010;
Zuidam and Nedovic,
2010)
Valio Finland Valio Hydrolysis Process.
Enzyme bound to adsorption resin
(Valio IML enzyme) (Valio,
2013)
- (Swaisgood, 2003)
Snow Milk
Products Co., Ltd.
Japan Fibrous immobilized β-
galactosidase on wire mesh
cylinder
- (Liese, et al., 2006)
Dairy Crest Wales Valio process - (Swaisgood, 2003)
ULN Condi (France), Dairy Crest (UK),
Kroger (USA)
A. Niger lactase bound to silica
beads to hydrolyse acid whey
Semi-industrial Developed by Corning Glass
(UK).
(Gekas and López-Leiva,
1985)
46
Since little recent information is available about the application of immobilized lactase
with the aim of lactose hydrolysis the references in Table 4 could be somewhat outdated.
For instance, Centrale del Latte di Torino (Italy) reduces lactose in some of their products
by enzymatic hydrolysis (Centrale del Latte di Torino, 2013), while Centrale del Latte di
Brescia (Italy) has chosen to apply microfiltration to reduce the lactose for their range of
improved digestibility products (Centrale del Latte di Brescia, 2013). It remains unclear
however if immobilized enzyme is used for the hydrolysis. The immobilized lactase
technology (IML) developed by Valio is still commercially available, which denotes that
immobilized lactases are used to date (Valio, 2013).
1.6.2 History of immobilized β-galactosidase
Since the first publication on enzyme immobilization by Nelson and Griffin (Nelson and
Griffin, 1916), the research on enzyme immobilization has thrived; the number of
publications on the subject of enzyme immobilization is still increasing. Numerous
enzymes have been immobilized on a vast variety of carrier materials. The earliest
immobilization of β-galactosidase can be dated back to 1969. Sharp and co-workers
immobilized a β-galactosidase on porous DEAE cellulose sheets (Sharp, et al., 1969). In
1971, Olson and Stanley performed a straightforward immobilization of a lactase from
Aspergillus niger on a phenol-formaldehyde resin (Olson and Stanley, 1973) for the
purpose of lactose hydrolysis in a column system. In the beginning of the 1980s
Nakanishi et al. (Nakanishi, et al., 1983) reported for the first time the immobilization of
Bacillus circulans β-galactosidase on Duolite ES-762, Dowex MWA-1 and sintered
alumina by adsorption with glutaraldehyde treatment. Since then, the immobilization of
various β-galactosidases such as from Escherichia coli (Bayramoglu, et al., 2007;
47
Bodalo, et al., 1991; Ladero, et al., 2001), Aspergillus oryzae (Albayrak and Yang,
2002a; Albayrak and Yang, 2002c; Dominguez, et al., 1988; Güleç, et al., 2010; Neri, et
al., 2009a; Torres, et al., 2003a), Kluyveromyces fragilis (Carrara and Rubiolo, 1994;
Roy and Gupta, 2003) and Klyuveromyces lactis (Chockchaisawasdee, et al., 2005;
Gonzalez Siso and Suarez Doval, 1994; Mateo, et al., 2004; Zhou and Chen, 2001; Zhou,
et al., 2003) have been intensively investigated. The applications of immobilized β-
galactosidases in the food industry were reviewed by Grosová, who concluded that the
immobilization of β-galactosidases plays an important role in dairy processing, especially
for hydrolysis of lactose (Grosová, et al., 2008a; Grosová, et al., 2008b). Whereas the
immobilization of β-galactosidases for hydrolysis of lactose has been reported quite
extensively, only one article evaluates the economics of processes using immobilized β-
galactosidases for this particular purpose; Axelsson and Zacchi evaluated the economic
feasibility of lactose hydrolysis in a continuous way by using an immobilized β-
galactosidase. Their calculations showed that the hydrolysis of lactose using immobilized
β-galactosidase was economically feasible, compared to a batch system using free
enzyme. The costs per kilogram of lactose using a plug flow tubular reactor (PFTR) were
23% of the cost of the batch system using free enzyme (Axelsson and Zacchi, 1990). To
date no other economical evaluation of GOS production using β-galactosidases, either
free or immobilized, has been published. From the viewpoint of competitiveness, it is
likely that sensitive business information is preferably not disclosed. Comparing (semi-)
continuous systems to STR-systems using free enzyme can be treacherous. Usually, the
performance of a system is measured by its (volumetric) productivity, expressed in for
example, kg.L-1.h-1 (see also Table 2 and Table 5). Besides this, also space time yield
48
(STY) is often calculated, i.e. the amount of GOS per volumetric unit of the reactor in
time (kg.[h.m3]-1). The values for these parameters can be quite far apart, while the orders
of magnitude of the volumetric productivity are generally closer to each other. The STY
is decisive for the size of a reactor, while the volumetric productivity indicates a value for
the output of a system. For example, Engel et al. reported a very high STY for their
membrane reactor; more than 98 g GOS.[h.cm3]-1 (Engel, et al., 2008). Values of one
order of magnitude lower were found by Albayrak and Yang, who reported 5.8 - 6 g
GOS.[h.cm3]-1 (Albayrak and Yang, 2002c). Calculation of the volumetric productivity
gives 48.4 g GOS.L-1.h-1 and 103.2 g GOS.L-1.h-1 for Engel et al. and Albayrak and Yang,
respectively. Table 5 shows both parameters, if possible. Remarkably, the enzymatic
productivity, i.e. the amount of GOS produced by a certain amount of enzyme, remains
underexposed. Since enzymes often contribute significantly to the cost price of products,
this factor should certainly not be omitted.
1.7 Techniques for β-galactosidase immobilization
Immobilization is generally divided into five method categories. Here we will give a
number of examples of each method for the immobilization of β-galactosidase.
49
Figure 5. Schematic overview of various immobilization techniques
1.7.1 Adsorption
Physical adsorption of proteins in general, and enzymes in particular, was one of the
earliest developed immobilization methods. We can, however, distinct between the
following classes:
Hydrophobic interaction
The basic principle behind this method is the affinity of hydrophobic domains of the
protein with a solid material having similar properties (Cao, 2006). Whereas some amino
acids, including valine and (iso)leucine exhibit a hydrophobic character because of their
low hydrogen substituent content, amino acids like tryptophane and phenylalanine
contain aromatic groups resulting in high hydrophobicity for these amino acids (Betts and
Russell, 2003). Areas that have a rather high hydrophobic amino acid density evidentially
50
interact with enzyme carriers existing of hydrophobic polymers. A good example of the
latter is (poly)styrene, which is used as hydrophobic matrix for carrier manufacturing
(Purolite International Ltd., 2012). Immobilization of enzymes that have large
hydrophobic areas in the vicinity of the active center, carry the risk of losing activity
because of wrongly oriented binding. If known, the distribution of hydrophilic and
hydrophobic areas on the surface of the enzyme molecule should be taken into
consideration when selecting a carrier.
Affinity adsorption
Proteins can contain groups that display specific affinity for, for instance, saccharide
structures (Carbohydrate Binding Domains). This particular affinity can be put to use
when immobilizing these corresponding enzymes. Carriers can be functionalized with
saccharides through covalent coupling and subsequently enzymes can be immobilized
through their specific affinity with the carrier surface. Examples of this type of affinity
immobilization were reported by Velikodvorskaya et al. (Velikodvorskaya, et al., 2010),
who immobilized a fusion protein on cellulose making use of the cellulose-binding
module (CBM) from Anaerocellum Thermophilum which was combined with the lactase
LacA (LacZ) from the thermophilic bacterium Thermoanaerobacter ethanolicus.
Whereas the latter was catalytically active, the former was utilized for the immobilization
of the enzyme on cellulose granules, making use of the interaction between the ligand and
the acceptor. Another method for affinity adsorption of enzymes is based on antibody-
antigen affinity, which can also be used for immobilization purposes. Haider et al.
immobilized polyclonal anti β-galactosidase antibodies (rabbit IgG) on cellulose powder.
The immobilized enzyme showed improved heat stability and storage stability. Also the
51
enzyme was more resistant to urea and the pH dependent activity increased over the
entire range from pH 2 – 9 (Haider and Husain, 2009c). Furthermore the lectin
concanavalin A (a carbohydrate binding protein) was used for the immobilization of A.
oryzae β-galactosidases on ConA layered calcium-alginate-starch beads for lactose
hydrolysis in milk and whey. In the case of concanavalin A and A. oryzae β-
galactosidase, this works quite well because this particular enzyme is known to be
glycated (Nakao, et al., 1987). Haider and Husain reported that the immobilized enzyme
retained 65% of its initial activity after 6 times reuse (Haider and Husain, 2009b). Despite
the ability to reuse the enzyme and improved enzyme properties obtained after affinity
based immobilization, higher costs of ligand development could be a drawback.
Ionic adsorption
Besides hydrophilic and hydrophobic regions, amino acids also carry charged groups like
carboxylic acids. Glutamate and aspartate are examples of amino acids that are usually
negatively charged at physiological pH, while arginine and lysine are positive under the
same conditions (Betts and Russell, 2003). Depending strongly on the pH, these charged
groups can be deployed to immobilize the protein on, for instance, ionic exchange resins
carrying groups with a counter charge. Immobilization through this method can give solid
constructs (Pessela, et al., 2003; Pessela, et al., 2005). Obviously, without additional
modification, these systems are sensitive to changes in pH and ionic strength when used
in processing. Both adsorption and ionic interaction methods offer the advantage of
reusing the carrier when immobilization without additional cross linking is applied.
However, Prenosil et al. reported the adsorption of a β-galactosidase derived from A.
oryzae onto a polyethersulfone membrane followed by additional glutaraldehyde cross
52
linking and regeneration of the membrane by removal of the exhausted biocatalyst using
bleaching agents (Prenosil and Hediger, 1985). Fernandes et al. immobilized a cold-
active β-galactosidase after purification and characterization. DEAE sepharose, an ion
exchange resin, was used as the carrier for the enzyme derived from Pseudoalteromonas
sp. with 60-70% of activity retention. The immobilized enzyme showed an increased
stability compared to the free enzyme (Fernandes, et al., 2002). The obvious advantage of
immobilization by adsorption is its simplicity and possibility of reuse of the carrier.
However, leaching of the enzyme during processing is considered a major disadvantage.
Therefore additional modifications like cross-linking are applied, but that makes reuse of
the carrier more difficult. Examples of β-galactosidase immobilization by adsorption have
been reported by numerous authors and many different carrier materials (Bódalo, et al.,
1991; Carpio, et al., 2000; de Lathouder, et al., 2006; Gaur, et al., 2006; Olson and
Stanley, 1973; Poletto, et al., 2005; Sharp, et al., 1969). When immobilizing β-
galactosidases by any of the above mentioned adsorption techniques, parameters like
time, pH, ionic strength and temperature are of considerable importance. In the first
place enough contact between carrier and enzyme should be allowed to facilitate
sufficient coverage of the carrier surface. Adsorption is enhanced by choosing the right
pH, ionic strength and temperature. Exposure of the desired groups, i.e. hydrophobic or
charged contributes to the attraction of enzyme and carrier surface. In view of the latter,
the enzyme loading also plays an important role. At low enzyme loading, the enzyme
molecules have the tendency to maximise the contact with the carrier. This might
influence the conformation of the enzyme and thus minimalize the activity retention. In
53
general, the formation of a monolayer of enzyme molecules results in the highest activity
retention. Usually this amounts to 2 – 3 mg protein per m2 of carrier surface (Cao, 2006).
1.7.2 Covalent attachment
Binding enzyme to a ready-made and available commercial carrier with activated
functionalities for direct coupling of enzyme is a straightforward method for enzyme
immobilization. Covalent linkage of enzymes can be achieved for instance by coupling of
ε-lysine groups of proteins with carriers carrying epoxide groups. This particular method
offers the possibility to immobilize enzymes in one single step. Besides functionalization
with epoxide groups, often amino groups are applied. These can either be activated by
glutaraldehyde, to yield aldehyde functionality, or reacted together with compounds like
1-Ethyl-3-[3-dimethylaminopropyl]-carbodiimide and the target protein for carboxylic
coupling. The reaction of aldehyde groups with ε-lysine yields relatively unstable imine
bonds, but reduction with e.g. NaBH4, gives stable amide bonds. Examples of
commercially available carriers for covalent attachment include Eupergit C,
Sepabeads, silica and functionalized agarose beads. However, it should be pointed out
that these commercial carriers might be not suitable for the enzyme of choice, regarding
the peculiarities of each enzyme and process. Often, the covalently immobilized enzymes
display higher thermal stability, compared with free enzymes. This is largely due to the
fact that the scaffold of the enzyme is tightened due to multiple point attachment,
resulting in a more rigid enzyme conformation (Mateo, et al., 2002b).
In the case of covalent enzyme immobilization, it is important to pay attention to the
following parameters used for the immobilization such as:
54
Enzyme/carrier ratio.
The right enzyme/carrier ratio is essential for obtaining high retention of enzyme activity.
For Biolacta N5, a β-galactosidase preparation derived from Bacillus circulans, Hernaiz
and Crout reported an optimal enzyme/carrier ratio of 1:20 (mg protein : mg support) for
the immobilization on Eupergit C, a epoxide-activated acrylic carrier (Hernaiz and
Crout, 2000). Torres et al., however immobilized the same enzyme on Sepabeads EC-
EP, also an acrylic carrier with epoxide functionality. They found an optimal ratio of 1:30
(mg protein : mg support), where a higher ionic strength was applied during
immobilization (Torres and Batista-Viera, 2012a). The necessity of a stronger buffer
during immobilization may be explained by the slightly more hydrophobic character of
Sepabeads EC-EP (Mateo, et al., 2002a) .
Ionic strength
As briefly touched upon in the previous paragraph, ionic strength during immobilization
can be of tremendous importance. Carriers that exhibit a more hydrophobic character may
influence the configuration of the enzymes active center negatively. In order to achieve
high enzyme loading, the selected ionic strength should facilitate the maximal adsorption
prior to the covalent enzyme binding, although this might be disadvantageous to the
enzyme activity yield. The detrimental effect caused by the hydrophobic carrier may be
countervailed by an additional step after the initial attachment of the enzyme. A
conventional method is blocking the unreacted functional groups with a hydrophilic
component like glycine or ethanolamine (Mateo, et al., 2002a). This increases the
hydrophylicity of the carrier surface and results in a more advantageous configuration of
55
the enzyme. Additionally, blocking the unreacted groups also prevents further interaction
between the enzyme and the carrier (Mateo, et al., 2000b).
pH and coupling time
As well as the ionic strength, the pH value during immobilization is also an important
parameter. Epoxide groups display higher reactivity at higher pH values. Moreover,
depending on the pH value, the enzymes reactive groups can be either protonated or not,
which, in turn, determines the reactivity of these groups. It is therefore evident that the
pH value during immobilization can have a large impact on the eventual immobilization
yield. Supplementary to this, the use of multiple pH steps during immobilization can
drastically improve the rigidity of the immobilized biocatalyst. The reaction time can
have considerable influence on the development of multi point attachment, especially in
combination with high pH values (Mateo, et al., 2003a; Torres and Batista-Viera, 2012a).
Nature of the carrier
The nature of the carrier can significantly influence the immobilization of an enzyme.
Recently, Bernal et al. showed that multipoint immobilization in mesoporous silica
improved the thermal stability of a B. circulans β-galactosidase (half-life increased 370-
fold compared to the soluble enzyme). This was explained by the pore morphology,
which affected the geometrical congruence for the enzyme (Bernal, et al., 2012). Before
mentioned carrier properties like hydrophobicity or hydrophilicity are also very important
during covalent immobilization. In order to react with the active groups of the carrier the
enzyme first needs to be in close proximity of the carrier surface, where the functional
56
groups are located. Covalent attachment can be divided into two steps; initial adsorption
to the carrier surface followed by the formation of a covalent linkage. Recently, carriers
were developed that make use of this principle. The surface of these Sepabeads is
modified with spacer molecules containing amino groups and epoxide functionality.
Whereas the initial adsorption proceeds via the amino groups, covalent attachment is
facilitated because the enzyme and carrier are in close vicinity (Bolivar, et al., 2009;
Mateo, et al., 2010; Mateo, et al., 2003b; Torres, et al., 2003a; Torres, et al., 2003b).
Pore size
The pore size of the carrier will definitively influence the activity retention and the
performance of the immobilized enzymes. For Eupergit C and Eupergit C250L, the
pore sizes are 10 nm and 100 nm respectively. Whereas, for instance, Penicillin G acylase
has a molecular weight of 80 kDa with a dimension of 70 Å, 50 Å and 55 Å (Tishkov, et
al., 2010), it can enter the Eupergit C carrier easily. In contrast, a β-galactosidase from
Kluyveromyces lactis has the following dimensions 140 Å, 153 Å and 216 Å (Pereira-
Rodriguez, et al., 2012). Bacillus circulans β-galactosidase appears in 4 forms, the largest
with a molecular weight of 189 kDa (Song, et al., 2011). A predicted structure of one of
the truncated forms shows the dimensions to be 90 Å, 65 Å, and 63 Å (Roy, et al., 2010).
It is therefore likely to obtain higher activity retention for these enzymes with Eupergit
C250 L than Eupergit C.
1.7.3 Entrapment
Physical entrapment of enzymes or whole cells can be achieved by cross-linking of
polymeric molecules in a solution containing also the biocatalyst. Probably the most
57
applied and described method for entrapment is cross-linking of sodium alginate with
calcium (Ates and Mehmetoglu, 1997; Barroso Jordão, et al., 2001; Batra, et al., 2005;
Crittenden and Playne, 2002; Dominguez, et al., 1988; Genari, et al., 2003; Haider and
Husain, 2007; Haider and Husain, 2009a; Haider and Husain, 2009b; Li, et al., 2008).
Entrapment methods are often combined with an additional step to strengthen the beads.
Cross-linking and simultaneous attachment to the support with glutaraldehyde is a
popular method (Krajewska, 2004), but the use of another polymer during immobilization
is also applied. A combination of two polymers and cross-linking was reported by
Tanriseven and Dogan (Tanriseven and Dogan, 2007). Lijuan et al. immobilized a lactate
dehydrogenase on carbon nanotubes and subsequently immobilized these in alginate
beads (Lijuan, et al., 2008). Other polymers that are used, whether or not in combination
with alginate, for entrapment or encapsulation of enzymes are chitosan (Azarnia, et al.,
2008; Wentworth, et al., 2004), cellulose acetate (Morisi, et al., 1972), gelatin
(Fuchsbauer, et al., 1996; Numanoglu and Sungur, 2004; Shen, et al., 2011; Tanriseven
and Dogan, 2007), κ-carrageenan, polyacrylamide, latex, agarose (Berger, et al., 1995a;
Berger, et al., 1995b; Khare, et al., 1994; McLarnon-Riches and Robinson, 1998),
cellulose triacetate (Morisi, et al., 1972; Vikartovska, et al., 2007) and polyvinyl alcohol
(Agira, et al., 1993; Batsalova, et al., 1987; Fernandes, et al., 2009; Grosová, et al.,
2008b; Grosová, et al., 2009; Hronska, et al., 2009). In general, immobilization
techniques that involve entrapment are simple and straightforward. On the other hand
they may have the disadvantage of diffusion limitation of substrate and products in and
out of the beads. Recently attempts have been reported that reduce this limitation by
producing lens-shaped particles in which the enzymes are entrapped (Hronska, et al.,
58
2009). Besides diffusion limitation, leakage of enzyme from the beads can also be a
drawback for use of this technique, but cross-linking of the enzyme before entrapment or
attachment to the polymer could reduce this problem.
1.7.4 Cross-linking
Cross-linking of enzymes was already mentioned as an additional step e.g. to fortify
already adsorbed enzymes. The α-galactosidase from Thermus sp. was immobilized on
different supports by Filho et al. Additional glutaraldehyde treatment gave a dramatic
increase in half-life (over 1000-fold) (Filho, et al., 2008). Also cross-linking can be very
useful in the immobilization of multimeric enzymes to prevent dissociation of the enzyme
structure and loss of activity (Fernandez-Lafuente, 2009).
1.7.5 Cross-linked Enzyme Aggregates (CLEAs)
Except from the use for added stability, cross-linking as such can also be used to
immobilize enzymes. Cao et al. described an elegant and simple method for cross-linking
of penicillin acylase after aggregation using different precipitants (Cao, et al., 2000) and
thereby immobilizing the enzyme without the use of a carrier. Figure 6 schematically
shows the formation of CLEAs. Magnetic nanoparticles can be incorporated in the
CLEAs to facilitate the separation of product and catalyst (Talekar, et al., 2012). Also
CLEAs can be co-polymerized for increased operational stability (Sheldon, 2007).
59
Cross-l
inking
Crosslinking &
nanoparticles
Figure 6. Formation of Cross Linked Enzyme Aggregates (CLEAs)
1.8 Desired characteristics of immobilized β-galactosidases
Industrial conditions often demand a lot from the biocatalyst used. In order to optimize
the yield in batch wise processes the temperatures chosen are often near the limits of
tolerance for the particular enzyme.
The desired characteristics of immobilized β-galactosidases depend on the interplay of
various factors. One might state that the envisaged process will determine selection of the
enzyme carrier based on the mechanical properties. Of course this is partially true, but in
spite of suitable mechanical characteristics, the carrier of choice is not necessarily the
right choice for immobilization of the enzyme. Naturally, the opposite also applies. What
would be the best approach? The desired characteristics of an immobilized β-
galactosidase can differ to a large extent as will be explained by means of two examples
both with the aim of lactose conversion.
The automation of processes in the dairy industry is at a well-advanced level and the
processing of milk and whey can be considered as a continuous process. For the
60
enzymatic hydrolysis of lactose in whey or milk a continuous process would be the
readiest choice. As is described in many articles, this process may well be performed by
means of a packed bed reactor (PBR), a column system with a packed bed of
immobilized β-galactosidases (Bayramoglu, et al., 2007; Chen, et al., 2009; Gekas and
López-Leiva, 1985; Gonzalez Siso and Suarez Doval, 1994; Li, et al., 2007; Mariotti, et
al., 2008; Obon, et al., 2000; Petzelbauer, et al., 2002; Roy and Gupta, 2003; Szczodrak,
1999; Zhou, et al., 2003). An enzyme carrier in a PBR should be able to withstand the
pressure drop over the column and will therefore have to be comprised of a rigid matrix.
In general, synthetic or inorganic carrier beads are used for this application. Some
examples are beads made of silica or porous glass. Also, macro-porous poly-methacrylate
(Sepabeads© and Eupergit©) or cross-linked polystyrene (Purolite©) are used, even though
they possess some degree of flexibility. This flexibility is a feature that carrier beads
should have to be able to resist the mechanical stress of the stirring in a Stirred Tank
Reactor (STR). A certain degree of flexibility is needed in order to prevent breaking of
the beads during the process. In general the production of GOS is carried out in a batch
process, usually implemented as a STR. Suitable carrier beads for this are natural
supports, such as agarose, but also the above-mentioned poly-methacrylates (Garcia-
Galan, et al., 2011).
Of course, of even greater importance is the performance of the immobilized enzyme. In
both examples the choice of the enzyme is of indisputable importance. In the first
example, the hydrolysis / transglycosylation ratio should be high enough in order to bring
about cleavage of lactose. Preferably, the enzyme is stable for a long period of time in
order to minimize interruptions of the process. The continuity of the process could be
61
ensured by using a multi-column system. In the second example, transglycosylation
should have the upper hand over hydrolysis. In addition, it is desirable that sufficient
enzyme activity is maintained throughout a significant period of time (or for large
number of batch reactions). Since the GOS synthesis reaction is usually carried out at
high substrate concentrations and associated high temperatures, it is important that the
enzyme is sufficiently heat stable.
Post-immobilization techniques can contribute to enhance the properties of an
immobilized enzyme is. Techniques such as cross-linking with a bi-functional component
(Fernández-Lafuente, et al., 1995; Lopez-Gallego, et al., 2005; Lopez-Gallego, et al.,
2007; Mozaffar, et al., 1988; Olson and Stanley, 1973), or "coating" with a polymer
(Alonso-Morales, et al., 2004; Betancor, et al., 2004; Betancor, et al., 2005; Fernandez-
Lafuente, 2009; Fernández-Lafuente, et al., 1999) can contribute to improve robustness
of the enzyme. In fact, single techniques for the immobilization of enzymes often fall
short when a robust enzyme needs to be obtained (Cao, 2006).
Since the space-time yield varies from enzyme to enzyme, from reactor to reactor, from
enzyme dosage to enzyme dosage, from substrate concentration to substrate
concentration, a fair comparison between the different reaction systems is not possible. In
spite of this observation, Table 5 shows an overview of GOS synthesis using immobilized
enzymes. The reactor system of choice in most cases is a stirred tank reactor (STR) in
which the enzyme is added to a substrate solution and left to react for a designated time.
Although considerably high amounts of oligosaccharides can be obtained in the final
product, the overall productivity in a batch reactor is generally lower than in continuous
reaction systems.
62
Table 5. Overview of GOS production using immobilized enzymes
Enzyme source Type of immobilization Reactor type
Lac. [g / L]
T [°C]
pH [ - ]
Max GOS[ %]
GOS Pvol [g·l-1·h-1]
GOS STY [g.[cm3.h]-1]
tP. [h] Ref.
BACTERIA
Bacillus circulans
Covalent STR 45.6 40 6.0
35 -
- -
(Mozaffar, et al.,
1986) Adsorption PFR 200 48.8 -
Bacillus circulans Adsorption + cross linking STR 45.6 40 6.0 40 - - -
(Mozaffar, et al., 1988; Mozaffar, et
al., 1989)
Bacillus circulans Batch, glutaraldehyde treated enzyme
STR 43.4 40 6.0 40 - - - (Mozaffar, et al.,
1987)
Bacillus circulans Covalent attachment STR 45.6 40 6.0 40.0 4.2 - 192 (Nakanishi, et al., 1983)
Bacillus circulans Adsorption + cross-linking
NFMR 100 45 6.0 40.0 283.3 - 50 (Pruksasri, 2007)
Bacillus sp.
Covalently immobilized on chitosan (THP coupling agent)
STR 360 55 5.0 41 16.4 9 (Cheng, et al.,
2006b)
Lactobacillus reuteri Membrane retention CSTR 200 37 6.0 24 55 - (Splechtna, et al.,
2007a; Splechtna, et
al., 2007b)
Thermus aquaticus
YT-1
Cross-linking + entrapment STR 160 70 4.6 34.8 0.77 72
(Berger, et al.,
1995b)
YEAST
Bullera singularis Adsorption PBR 100 45 4.8 55.0 4.4 0.044 360 (Shin, et al., 1998a; Shin, et al., 1998b)
Bullera singularis Immobilized whole cells
STR 600 55 5.0 – 6.0 40.4 8.72 15.2 22 (Sakai, et al., 2008)
Kluyveromyces lactis Batch, free enzyme & immobilized enzyme
STR 159 40 6.5 22 5.83 6
(Maugard, et al.,
2003) 7.5 30 37.5 2
63
Kluyveromyces lactis Free enzyme, ultra filtration
MR 200 45 7.0 31.0 13.7 2.5 (Foda and López-Leiva, 2000)
Kluyveromyces lactis Continuous, Free enzyme
MARS ~236 40 7.5 20 30 2.5 (Czermak, et al.,
2004)
Kluyveromyces lactis Ionic interactions CMCRS 200 40 7.0 24.0 48.4 98.7 1 (Engel, et al., 2008)
Kluyveromyces lactis Free enzyme, ultra filtration
MR 250 40 7.0 - 52.1 4 (Chockchaisawasdee, et al., 2005)
MOULDS
Aspergillus candidus Covalent binding PBR 400 40 6.5 37.1 87.1 480 (Zheng, et al., 2006)
Aspergillus oryzae Batch, immobilized enzyme
STR 300 50 4.5 14 (Prenosil, et al.,
1987a)
Aspergillus oryzae Continuous, immobilized enzyme
IER 200 40 4.5 14 - - (Lopez Leiva and
Guzman, 1995)
Aspergillus oryzae Immobilization on cotton cloth
RBR 200 40 4.5 20 3.6 11 (Albayrak and Yang, 2002b; Albayrak and Yang, 2002c)
Aspergillus oryzae Fed batch, free enzyme M-ACR 200 40 7.5 33 15.3 2.5 (Czermak, et al., 2004)
Aspergillus oryzae Continuous, immobilized enzyme
PBR 200 40 4.5 ~20 ~16 ~2.5 (Albayrak and Yang, 2002b; Albayrak and Yang, 2002e)
Aspergillus oryzae Batch, immobilized enzyme
STR 200 40 3 – 4.5 17.3 17.3 2 (Gaur, et al., 2006)
Aspergillus oryzae Batch, immobilized enzyme
STR 500 40 4.0 25.7 32.1 4* (Neri, et al., 2009b; Neri, et al., 2009c)
Aspergillus oryzae Covalent binding PBR 200 40 4.5 21.7 80.0 336 (Albayrak and Yang, 2002a; Albayrak and Yang, 2002d)
Aspergillus oryzae Continuous, immobilized enzyme
PFR 400 40 4.5 23 103 58 - 60 70 (Albayrak and Yang, 2002c)
Aspergillus oryzae Covalent binding PBR 400 40 4.5 26.6 106.0 336 (Albayrak and Yang, 2002a)
Aspergillus oryzae Batch, immobilized enzyme
STR 500 40 4.5 26 130 1*
(Neri, et al., 2009a;
Neri, et al., 2009b)
Aspergillus oryzae Batch, immobilized enzyme
MSR 100 40 4.5 15 1020 (Pruksasri, 2007)
64
Penicillium
Expansum
Immobilized on yeast cell surface
- 100 25 - 43.64 0.36 120 (Li, et al., 2009b)
Talaromyces
thermophilus
Covalently immobilized on Eupergit C
200 40 6.5 40 3.3 24 (Nakkharat and
Haltrich, 2007)
* Maximum is reached after 1 hour
65
1.9 Concluding remarks
A wide variety of (micro-) organisms produce β-galactosidases. They are versatile
enzymes capable of forming galacto-oligosaccharides and a variety of galactosylated
compounds. Often these reactions are performed as part of characterization studies.
Additionally, optimization studies also take place, to find the optimal conditions for the
synthesis of GOS. The focus is mainly on the production of GOS, i.e. the quantity of
product produced in a given time. However, the performance of the enzyme itself remains
underexposed, while from an industrial point of view, this parameter is of great interest.
Further optimization of the enzyme performance may be achieved by means of
immobilization. This technique has proven itself as a versatile and powerful tool for
improving the performance of enzymes and optimization of production processes. Many
variables play a role during immobilization, and also during post-immobilization. The
rationale behind the use of post-immobilization techniques is found in the fact that single
immobilization techniques, not always lead to satisfactory results in terms of robustness
of the immobilized enzyme (Cao, 2006). Combinations of conventional techniques and
additional stabilization / modification of the catalyst can thus provide an interesting
approach for successful immobilization of enzymes to be used in industrial processes.
Although the synthesis of GOS using immobilized β-galactosidases, shows opportunities
and potential for a more efficient way of producing galacto-oligosaccharides, barriers for
the application of this technology still seem to exist. While similar processes have been
applied on an industrial scale, such as the hydrolysis of lactose and the production of high
fructose corn syrup (HFCS), the application of immobilized enzymes for the production
of GOS is still behind.
66
1.10 Outline of this thesis
This study focused on the immobilization of β-galactosidase of Bacillus circulans and, in
particular, the application of the immobilized enzyme for the production of GOS. The
composition of the oligosaccharide mixture was compared with the product that was
obtained by use of the free enzyme. The premise of this study was the hypothesis that it
would possible to produce an equivalent product using immobilized β-galactosidase from
Bacillus circulans in a more efficient process than the process with the free enzyme.
In Chapter 2, the selection of a suitable carrier for the immobilization and the proposed
application in the intended process is described. The application of the Analytical
Hierarchy Process to evaluate three different carriers is dealt with.
In Chapter 3, the application of immobilized B. circulans β-galactosidase in a batch
process is described. Particularly, the fact that the conversion of lactose to GOS started
from slurry instead of the more commonly described lactose solution was given attention.
The enzymatic and the volumetric productivity of the system were compared with those
of a system with free enzyme. In addition, the compositions of the final products were
compared with the composition of the free enzyme GOS mixture.
Chapter 4 describes the synthesis of GOS by means of immobilized enzyme in a Packed
Bed Reactor (PBR). In contrast to the process described in Chapter 3, this involved a
continuous system. The inactivation constants at different temperatures and lactose
concentrations were determined. The workable limits of such a continuous process were
studied during this work and evaluated.
67
The formation of allo-lactose during the synthesis of GOS is the central topic in Chapter
5. The influence of process parameters on the formation of allo-lactose was studied. The
possible mechanisms for the formation of this lactose isomer are also discussed.
The work described in this thesis is discussed in Chapter 6. Based on the experimental
results of this study, the learnings are discussed. A perspective is also given to a possible
implementation with respect to the use of immobilized enzyme on an industrial scale, and
the accompanying consequences.
Acknowledgements
This project is jointly financed by the European Union, European Regional Development
Fund and the Ministry of Economic Affairs, Agriculture and Innovation, Peaks in the
Delta, the Municipality of Groningen, the provinces of Groningen, Fryslân and Drenthe
as well as the Dutch Carbohydrate Competence Center (CCC WP9). The authors would
like to thank Dr. Linqiu Cao, Dr. Albert van der Padt and Dr. Ellen van Leusen (all
FrieslandCampina) for their critical reading of the manuscript and helpful comments.
68
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