University of Groningen Galacto-oligosaccharide synthesis ... · Souchard, 1973). Two invariant...

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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).

4

GOS differ structurally from human milk oligosaccharides (HMOs) which are, besides

glucose and galactose, generated from N-acetyl-glucosamine, fucose and syalic acid.

Tomarelli et al. (Tomarelli, et al., 1954) identified a disaccharide consisting of galactose

and N-acetylglucosamine, derived from porcine mucin, as a Bifidus factor and this

structure is found also as the building block for HMOs. This Bifidus factor was

discovered by György et al. (György, et al., 1954) in 1954 and is a metabolic substrate

for desired bacteria in the composition of the intestinal microbiota with health benefits for

breast-fed infants (Bode, 2012). To date, more than 200 species of oligosaccharides from

human milk have been identified differing in composition, linkage type and length (Kunz,

et al., 2000).

GOS molecules contain β1→4, β1→6, β1→3 linkages in various combinations, but also

the occurrence of β1→2 and even 1↔1 linkages has been described. The linkage types

and length of the GOS molecules that are formed largely depend on the source of the β-

galactosidase used for the synthesis of GOS (Coulier et al., 2009; Torres et al., 2010 ;

Fransen, et al., 1998).

This introduction provides an overview of historical and current literature on the use of β-

galactosidases for the synthesis of (galacto-)oligosaccharides. A brief description and

background of the enzyme (β-galactosidase) and the mechanism for synthesis are

provided, as well as an overview of the current literature in the field of GOS synthesis by

β-galactosidases. Subsequently, the industrial applications of β-galactosidases are

discussed. Because of the importance of stable enzymes and the need to reduce the

production cost, immobilization of β-galactosidases will be further addressed. In addition,

5

the possibilities and potential of this technology for oligosaccharide synthesis are

discussed.

1.2 Enzyme

Enzymes that are used for research or industrial purposes are in general, derived from

bacteria, yeasts or moulds. An overview of some commercially available β-galactosidase

preparations is shown in Table 1. Despite the differences in source organism, pH and

optimum temperature, all enzymes listed in Table 1 catalyze the hydrolysis of lactose into

glucose and galactose. The preponderance of the Kluyveromyces genus can be attributed

to the fact that they are safe and highly productive (Fonseca, et al., 2008).

Table 1. Commercially available sources of β-galactosidase

Brand name Manufacturer Organism pHopt Topt [°C]

Maxilact DSM, The Netherlands Kluyveromyces lactis 6.5 40 Tolerase DSM, The Netherlands Aspergillus oryzae 4.0 40 β-galactosidase Megazyme International Ireland Ltd. Aspergillus niger 4.5 60 β-galactosidase Megazyme International Ireland Ltd. Kluyveromyces lactis 6.5 45 Lactozym Novozymes, Denmark Kluyveromyces lactis 6.0 48 Lactoles L3 Amano Enzyme Inc., Japan Bacillus circulans 6.0 65 Lactase F Amano Enzyme Inc., Japan Aspergillus oryzae 5.0 55 β-galactosidase Worthington Biochemicals Inc., UK Escherichia coli 6.0-8.0 37 β-galactosidase Sigma-Aldrich, USA Escherichia coli 6.0-8.0 37 β-galactosidase Sigma-Aldrich, USA Bos taurus 4.3 37 β-galactosidase Sigma-Aldrich, USA Aspergillus oryzae 4.5-5.5 50 L017P Biocatalysts, UK Aspergillus oryzae 4.5-5.5 55 Ha-lactase Chr. Hansen, Denmark Kluyveromyces lactis 6.5 40 Lactase NL Enzeco, USA Kluyveromyces lactis 6.5 40 Fungal lactase Enzeco, USA Aspergillus oryzae 4.0 -5.5 55 GODO-YNL2 GODO SHUSEI Co., Ltd., Japan Klyuveromyces lactis 6.5 40 Biolactase F Kerry Ingredients and Flavours, Ireland Aspergillus oryzae 4.5 55 Biolactasa NTL Biocon, Spain Bacillus circulans 6.0 65 Biolactase L Kerry Ingredients and Flavours, Ireland Kluyveromyces lactis 6.0 40 GODO-YNL2 DuPont Danisco, Denmark Kluyveromyces lactis 6.5 40 Lactase 100 Specialty Enzymes & Biotechnologies

Co., USA Aspergillus oryzae 4.5-5.5 55

6

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.

7

1.3.1 Mechanism of hydrolysis of lactose

In humans the lactase activity is at its maximum immediately after birth (Shukla, 1975)

where the β-galactosidase enzyme is present in the brush border of the small intestine

facilitating the absorption of the monosaccharides into the bloodstream. Besides its

presence in humans, this enzyme can be found in a wide variety of organisms (Chang, et

al., 2009; Shukla, 1975). In the CAZy database glycoside hydrolases are described as a

widespread group of enzymes which hydrolyze the glycosidic bond between two or more

carbohydrates or a carbohydrate and a non-carbohydrate moiety, indicating the versatility

of these enzymes. Two models for the catalytic mechanism of the hydrolysis of the

glycosidic linkage are described in the literature (Henrissat, et al., 1995; Sinnott and

Souchard, 1973). Two invariant glutamic acid residues in the enzyme active center are

directly involved in the catalytic mechanism, acting as a proton donor and a nucleophile /

base (Gebler, et al., 1992; Vasella, et al., 2002; Wallenfels and Malhotra, 1961; White

and Rose, 1997). Figure 1 schematically displays the hydrolysis of lactose by the action

of a retaining β-galactosidase.

8

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.

9

Figure 2. Glycosydic linkage hydrolysis via inverting mechanism

The attack of the nucleophilic water molecule in the inverting mechanism and cleavage of

the glycosidic linkage take place simultaneously, resulting in inversion of the

configuration around the anomeric carbon of galactose. In contrast, the retaining

mechanism operates through multiple steps, including an enzyme-galactose complex

intermediate. This intermediate forces nucleophiles to attack from the side opposite to the

bond between the base group in the enzyme’s catalytic center and the galactose moiety.

Figure 1 schematically shows that retention of the anomeric configuration is actually

achieved by double anomeric inversion; firstly in the formation of the covalent enzyme-

glycosyl intermediate, followed by another inversion after nucleophilic attack of water.

10

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).

11

O

H

HO

H

O

H

H

OHH

OH

HO

O

HO

H

H

HO

H

H

OHH

HO

O

HO

H

H

HO

H

H

OHH

HO

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

12

1.3.3 Combinations of lactose and various acceptors

In the previous paragraphs, the activity of β-galactosidases in lactose as a preferred

substrate is described, but many β-galactosidases have the ability to transfer the sugar

moieties to another sugar or alcohol, resulting in the formation of oligosaccharides

(Adamczak, et al., 2009; Albayrak and Yang, 2002d; Berger, et al., 1995b; Li, et al.,

2009a; Li, et al., 2010; Mozaffar, et al., 1989; Takayama, et al., 1996), glycoconjugates

and alkylglycosides (Bankova, et al., 2006; Bridiau, et al., 2006; Menzler, et al., 1997;

Vic, et al., 1997). The efficiency of this transglycosylating activity strongly depends on

the source of the β-galactosidases and the conditions applied during the reaction (Boon, et

al., 2000; Gekas and Lopez-Leiva, 1985; Mahoney, 1998; Prenosil, et al., 1987a;

Prenosil, et al., 1987b). The types of oligosaccharides formed or the nature of the formed

glycosidic linkages also strongly depend on the source of enzyme. For instance, A. oryzae

β-galactosidase was shown to synthesizes many oligosaccharides with β-D-(1→6)

glycosidic bonds (Toba, et al., 1985), while the β-galactosidase from B. circulans

produces mainly β-D-(1→3) and β-D-(1→4) bonds (Coulier, et al., 2009). However,

Vetere and Paoletti have shown that the preference for the linkage position of the latter

enzyme is dependent on pH and temperature during oligosaccharide formation (Vetere

and Paoletti, 1996a; Vetere and Paoletti, 1996b). Zeng et al. additionally reported that the

regioselectivity of B. circulans β-galactosidase was greatly dependent on the nature of the

acceptor. Replacement of p-nitrophenyl-β-D-galactoside by p-nitrophenyl-β-D-

galactosaminide changed the regioselectivity from predominantly β-D-(1→3) linkages to

β-D-(1→6) linked disaccharides, caused by a more favorable orientation of the acceptor

in the hydrophobic binding locus in the active site (Zeng, et al., 2010; Zeng, et al., 2000).

13

Several glycoconjugates can be synthesized using the transglycosylating properties of β-

galactosidases. In the following section, a number of examples from the literature are

discussed. Lactose can be combined with various receptor compounds to obtain specific

(galacto-) oligosaccharides like N-acetyllactosamine (Vetere and Paoletti, 1996a; Vetere

and Paoletti, 1996b) and N-acetylglucosamine containing oligosaccharides (Takayama, et

al., 1996). Benzyl-D-xylopyranoside was shown to be a suitable acceptor for the synthesis

of galactosyl-xylopyranoside-O-Benzyl (Guisán, et al., 1993), as well as 2-hydroxybenzyl

alcohol and related compounds. Compounds that were structurally related to 2-

hydroxybenzyl alcohol were also shown to be suitable acceptors. Remarkably, the

adsorption of 3-aminobenzyl alcohol on silica yielded 96% acceptor conversion. Both O-

and N-galactosylated products were obtained.

The modification of drugs by means of glucosylation is one approach that can be taken to

prolong pharmacological activity and reduce adverse effects. Bridiau et al. chose this

approach in their examination of the acceptor properties of the drugs guaifenesin and

chlorphenesin with K. lactis β-galactosidase (Bridiau, et al., 2006). Galactosylation of the

latter compound was likewise carried out by Scheckermann et al., who also carried out

the galactosylation of chloramphenicol by using A. oryzae β-galactosidase

(Scheckermann, et al., 1997). Both studies showed that chlorphenesin was a moderate

acceptor for the galactose moiety. Acceptor conversion was approximately 15% in the

case of (Bridiau, et al., 2006), who adsorbed the acceptor molecules to various solid

supports. Scheckermann et al. used cosolvents to anticipate to the hydrophobic properties

of these compounds and achieve higher yields. Although higher yields (approx. 12.5%)

14

were obtained using acetonitrile as a cosolvent, the enzyme stability was better with

dioxane.

β-D-Galactopyranosyl-(1→6)-β-D-galactopyranosyl-(1→4)-β-D-fructopyranose, together

with the α- and β- fructofuranosidic variants were derived from lactulose using a β-

galactosidase from K. lactis (Martinez-Villaluenga, et al., 2008). The synthesis of

lactulose from mixed solutions of lactose and fructose using β-galactosidases from

different sources was reported by several authors (Adamczak, et al., 2009; Kim, et al.,

2006; Mayer, et al., 2010). Cryo-protective galactosyl-trehalose trisaccharides were

produced using a β-galactosidase (Kim, et al., 2008). Although glycosyltransferases are

in general more regioselective (Berger and Rohrer, 2003; Zigova, et al., 1999), the use of

expensive activated sugars can be a major drawback for their application. The

transglycosylation reaction catalyzed by β-galactosidases does not require activated

sugars and is thus cheaper, but less selective, yielding a variation of reaction products. If

high purity of the desired compound is required, additional downstream processing is

needed.

1.3.4 Non-aqueous reaction media

A combination of water-miscible organic solvents and water can also be used as the

reaction medium. Bankova et al. demonstrated transglycosylation activity of A. oryzae β-

galactosidase in the presence of DMSO or DMF. The activity, however, was

demonstrated to be supressed. The most plausible reason for this was lowering of the

dielectric constant in the presence of miscible organic solvents, which increases the

electrostatic interactions between polar and charged residues. Due to this the flexibility of

the protein is affected and the accessibility of substrates to the active site is reduced. The

15

presence of the water immiscible solvents iso-propanol and iso-butanol yielded

oligosaccharides as well as alkylglycosides. Increasing the iso-butanol concentration to

50%, yielded 6.7% trisaccharides and 14.5% isobutylglycosides. Due to their bipolar

character, the synthesized products could possibly be applied in pharmaceutical, chemical

or cosmetic industries as emulsifiers and / or surfactants (Bankova, et al., 2006; Carretti,

et al., 2007). Sauerbrei and Thiem conducted transglycosylation reactions with A. oryzae

and E. coli β-galactosidase in aqueous solutions containing up to 50% acetonitrile.

Besides o- and p-nitrophenyl glycosides, they also synthesized galactosylated L-serine

(β-Gal-L-Ser) (Sauerbrei and Thiem, 1992). Pérez-Sánchez et al. demonstrated a change

in regioselectivity for B. circulans β-galactosidase from β-D-(1→4) linkages to β-D-

(1→6) linkages when a 2M concentration of glycerol derived solvents was used during

the synthesis of disaccharides using p-nitrophenyl-β-D-galactopyranoside and N-acetyl-

glucosamine as substrates. This phenomenon was explained by a molecular modeling

study and it was found that the three-dimensional arrangement between GlcNAc and the

water-solvent mixture in the active site of the enzyme, favors the β-D-(1→6) linkage

(Pérez-Sánchez, et al., 2011). These investigations show the usability and wide

applicability of β-galactosidases as catalysts for numerous reactions. Moreover, above

mentioned research reports show that β-galactosidases can be used for the synthesis of a

large amount of compounds besides galacto-oligosaccharides. Obviously, when lactose is

used in combination with other substrates, also regular galacto-oligosaccharides are

formed as part of the reaction mixture.

The solubility of lactose is another factor that needs to be considered. Being already

poorly soluble in water, the solubility of lactose in organic solvents is even lower. On the

16

other hand, water miscible solvents help lower the water activity and may therefore

contribute to a change in kinetic properties, thereby shifting more towards synthetic

activity.

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

17

substrate and inhibiting monosaccharides. Another elegant solution for the latter case was

cell surface engineering of yeast, for which the authors reported the immobilization of a

β-galactosidase on the outer cell membrane of yeast cells. By doing so, the β-

galactosidases could synthesize galacto-oligosaccharides. Simultaneously, the resulting

glucose was utilized by the yeast as a carbon source preventing the inhibition of the

enzyme (Li, et al., 2009b).

Enzyme membrane systems, just like the PBR systems, can be susceptible to issues like

blocking when undissolved substrate is present.

1.3.6 High substrate conditions

In order to favour the synthesis of galacto- oligosaccharides formation, the synthesis is

generally performed at higher substrate concentrations (Boon, et al., 1999; Boon, et al.,

2000; Nakkharat and Haltrich, 2007; Neri, et al., 2009a; Neri, et al., 2009b; Neri, et al.,

2009c; Park, et al., 2008). High substrate concentrations lower the water activity and

facilitate the saturation of the enzyme with nucleophilic molecules other than water.

Lactose, the natural substrate for β-galactosidases and a regenerable raw material, readily

available in large quantities, however has poor dissolving properties at low temperatures

(Machadoa, et al., 2000; McSweeney and Fox, 2009; Walstra, et al., 2006). For 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).

18

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.

19

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)

21

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 C2z 250 50 6.5 27.9 4 17.4 (Padilla, et al., 2012)



Kluyveromyces lactis BP1z 250 50 6.5 30.2 4 18.9 (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)


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 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 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)


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)


PBR 200 40 4.5 ~20 ~16 ~2.5 (Albayrak and Yang, 2002b; Albayrak and Yang, 2002e)


STR 200 40 3 – 4.5 17.3 17.3 2 (Gaur, et al., 2006)


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)


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)


STR 500 40 4.5 26 130 1*

(Neri, et al., 2009a;

Neri, et al., 2009b)


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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University of Groningen Galacto-oligosaccharide synthesis ... · Souchard, 1973). Two invariant...

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Transcript of University of Groningen Galacto-oligosaccharide synthesis ... · Souchard, 1973). Two invariant...