β-Galactosidases: A great tool for synthesizing galactose-containing carbohydrates

Journal Pre-proof β-Galactosidases: A great tool for synthesizing galactosecontaining carbohydrates

Lili Lu, Longcheng Guo, Ke Wang, Yan Liu, Min Xiao PII:

S0734-9750(19)30165-X

DOI:

https://doi.org/10.1016/j.biotechadv.2019.107465

Reference:

JBA 107465

To appear in:

Biotechnology Advances

Received date:

16 July 2019

Revised date:

26 October 2019

Accepted date:

31 October 2019

Please cite this article as: L. Lu, L. Guo, K. Wang, et al., β-Galactosidases: A great tool for synthesizing galactose-containing carbohydrates, Biotechnology Advances (2018), https://doi.org/10.1016/j.biotechadv.2019.107465

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© 2018 Published by Elsevier.

Journal Pre-proof

β-Galactosidases: a great tool for synthesizing galactose-containing carbohydrates

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Lili Lua,* , Longcheng Guob, Ke Wang a, Yan Liu a, Min Xiaob a

School of Pharmacy, Tongji Medical College, Huazhong University of Science and

Technology, 430030 Wuhan, China. b

National Glycoengineering Research Center, Shandong Provincial Key Laboratory of

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Carbohydrate Chemistry and Glycobiology, State Key Laboratory of Microbial Technology, Shandong University, 266237 Qingdao, China.

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⁎ Corresponding author.

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E-mail address: [email protected] (L. Lu)

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Abbreviations: CMP-Neu5Ac, cytidine-5′- monophospho-N-acetylneuraminic acid; DNP; dinitrophenyl; Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; GDP-Fuc, guanosine 5′-diphospho-fucose; GOS, galacto-oligosaccharides; NMR, nuclear magnetic resonance; Man, mannose; 4MU, 4-methylumbelliferyl; oNP, o-nitrophenyl; oNP-β-Gal; o-nitrophenyl β-galactopyranoside; pNP, p-nitrophenyl. 1

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Abstract

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β-Galactosidases, an important class of glycosidases, naturally catalyze the hydrolysis of β-galactosidic bonds in oligosaccharides and polysaccharides. Traditionally, these enzymes have been used to degrade lactose in dairy products, which are beneficial for lactose- intolerant people. Attractively, β-galactosidases exhibit glycosyl transfer activity under certain conditions in vitro. They are capable of synthesizing carbohydrates from cheap starting substrates in a facile, efficient, and environment- friendly way. The condensation of lactose into the well-known prebiotic galacto-oligosaccharides by β-galactosidases has become a key aspect of the industrial interest in the synthetic activity in recent years. At present, the transglycosylation activity of these enzymes has been greatly extended. It can be used not only in building glycan blocks of crucial glycoconjugates to elucidate their biological functions, but also in glycosylation of vital molecules, which have been applied in food, medicine and cosmetic industries to improve solubility, stability and bioactivity. Further molecular engineering of β- galactosidases has significantly improved their synthetic activity, expanded the substrate spectrum and made them more powerful in carbohydrate synthesis. This review covers the classification, structure and mechanism of β-galactosidases, galactosylation reactions catalyzed by these enzymes, and various strategies of enzyme engineering, with an emphasis on recent advances. Keywords: β-Galactosidases; enzyme engineering.

galactosides;

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galacto-oligosaccharides;

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1. Introduction

galactosylation;

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Enzyme catalysis has the advantages of high efficiency, specificity, sustainability and environmental friendliness. The carbohydrate-active enzymes (CAZymes) contain a large number of enzymes involved in carbohydrate synthesis and degradation (Lombard et al., 2014). In recent years, glycosidases (EC3.2.1), which play an intrinsic role in the hydrolysis of glycosides, have attracted special attention for stereo- and regio-selective synthesis of oligosaccharides, polysaccharides, and glycoconjugates with simple, low-cost substrates in one-step reations (Bojarová and Kren, 2011; Faijes et al., 2019; Hayes et al., 2017; Palcic, 1999). Because a sugar moiety has multiple reactive hydroxyl groups, obtaining the desired glycoside by conventional chemical method requires tedious procedures of group protection and deprotection to control the stereo- and regio-specificities (Sears and Wong, 2001). Therefore, glycosidase-mediated glycosylation provides a particularly useful alternative to the chemical method, allowing the defined synthesis of target glycosides (Bojarová and Kren, 2009, 2011; Scigelova et al., 1999; Yamamoto, 2013; Wang et al., 2009). β-Galactosidases (EC3.2.1.23) are among the most promising glycosidases with both hydrolytic and transglycosylation activities (Husain, 2010; Lu et al., 2017; Oliveira et al., 2011; Silvério et al., 2018; Torres et al., 2010). In the normal hydrolytic reaction, β-galactosidases hydrolyze lactose and release galactose and glucose. This characteristic has been used by many food industries to degrade lactose and improve the digestibility, sweetness, 2

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solubility and flavor of dairy products. Reduction of lactose in dairy products helps alleviate the symptoms of lactose intolerance, which is common in more than half of the world's population (Husain, 2010; Saqib et al., 2017). Some β- galactosidases have also been applied in biosensor assembly to accurately quantify lactose in dairy products (Sharma and Leblanc, 2017). In the basic research field, β-galactosidase is often used as a reporter gene because of the release of detectable chromogenic or fluorogenic groups by hydrolysis of artificial galactoside substrates. In addition to hydrolysis activity, β-galactosidases also catalyze the transfer of sugar residues from glycosyl donor substrates to acceptors, thus forming new glycosidic bond. The transglycosylation activity has been applied in industrial production of galacto-oligosaccharides (GOS), which structurally mimic human milk oligosaccharides, using lactose as self-condensation substrates. As typical prebiotics, GOS selectively stimulate the growth of beneficial intestinal bacteria, such as Lactobacilli or Bifidobacteria, and adjust the intestinal micro-ecological balance (Lu et al., 2017; Torres et al., 2010). Additionally, GOS can directly prevent the binding of pathogens to oligosaccharides on the surface of host cells. They act as decoy receptors to bind pathogens and help flush them out of the gastrointestinal tract (Searle et al., 2010; Sinclair et al., 2009). Moreover, GOS exhibit superior properties when compared with other prebiotics including lactulose, fructo- xylo-, isomalto-, and soybean oligosaccharides, owing to the large reduction of harmful bacteria growth, the high- level production of short-chain fatty acid, and the low gas generation (Rycroft et al., 2001). Notably, commercial GOS product is one of the most popular prebiotics that are expected to make about U$10.55 billion in profits in the global market by 2025 (Mano et al., 2018). As a result, the synthesis of GOS by β-galactosidases has been studied extensively. More importantly, β- galactosidases have a wide range of substrate specificity, enabling them to synthesize a number of oligosaccharides or glycosides other than GOS (Oliveira et al., 2011). Since galactose is an important component of the glycan chains of glycoconjugate that are involved in many biological events, such as cell recognition, communication, and microbial infection, this synthetic ability has drawn considerable interest (Lu et al., 2007, 2010b). β-Galactosidases facilitate the synthesis of glycoconjugate building blocks, such as ABO blood group determinants, cancer-related carbohydrate antigens and the like (Murata et al., 1996; Yoon and Ajisaka, 1996). In addition, β-galactosidases have proved to be an effective tool for glycosylation of antibiotics and natural products, resulting in attractive glycosides with optimized properties and expanded applications (Scheckermann et al., 1997; Shimizu et al., 2006; Zhang et al., 2016). Combining β- galactosidases with other enzymes can produce more complex saccharides and conjugates, even novel biomaterials with large molecular weight (Jia and Wang, 2007; Vie et al., 1997; Zeng and Uzawa, 2005). The molecular engineering of β- galactosidases further improves glycosylation activity. Rational design and directed evolution strategies have been used to increase the yield of glycosides, the ability to utilize non-natural substrates, and the access to tailor- made product structures with desired molecular weights or special glycosidic bonds. A significant example among the engineering strategies is the catalytic mechanism-based mutagenesis of 3

Journal Pre-proof nucleophilic residues of the enzymes, which completely removes the hydrolytic activity and makes them truly act as synthetases (Drone et al., 2005; Henze et al., 2014 ; Jakeman et al., 2002; Mackenzie et al., 1998). Excitingly, the nucleophilic mutants of β-galactosidases can produce galacotosylated peptides and proteins that are difficult to obtain chemically (Tolborg et al., 2002; Müllegger et al., 2006). As a whole, various enzymatic engineering strategies play an important role in promoting the application of β-galactosidase in carbohydrate synthesis.

2. Classification, mechanism and structure of β-galactosidases

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β-Galactosidases are widely distributed in nature, including microorganisms, plants and animals. Based on the sequence similarity, β-galactosidases are classified into glycosyl hydrolase (GH) family 1, 2, 35, 42, 59 and 147 in CAZy database (http://www.cazy.org/). According to the number of enzyme sequences deposited in the database, these enzymes mainly exist in GH2, GH35 and GH 42 families. Fig. 1 shows the evolutionary relationship between some enzymes and their families. In the phylogenetic tree, GH1 and GH2 β-galactosidases are distributed in the same evolutionary branch, while GH35, GH42, GH59 and GH147 enzymes belong to another branch of which the GH35 and GH42 enzymes are located in the same sub-branch, suggesting that they might have evolved from a common ancestor. β-Galactosidases from all families belong to superfamily Clan-A and share (α/β)8 barrel structure. They work through a retaining mechanism, in which the product maintains the same anomeric configuration as the starting substrate (Brás et al., 2010; Juers et al., 2001). Two glutamic or aspartic acid residues play essential roles in the catalysis : one as an acid/base catalyst and the other as a nucleophile. The catalytic process is carried out by a two-step double-displacement mechanism (Fig. 2). In the initial galactosylation step, the nucleophile residue attacks the substrate at the anomeric center, while the acid-base residue provides acid assistance to facilitate the departure of the leaving group. When lactose is used as a substrate, a covalent galactosyl-enzyme intermediate is formed and combined with the release of glucose from lactose. In the next step of degalactosylation, the acid-base residue serves as a general base and activates the acceptor molecule, which attacks the galactosyl-enzyme intermediate at the anomeric center to yield the product that retains the anomeric configuration of the substrate. If the acceptor is water, hydrolysis takes place and the galactose is released from the enzyme intermediate. However, if sugars act as the acceptor, transglycosylation occurs and galactosyl residues are transferred from the enzyme intermediate to the sugars to form galactosides. This explains why β-galactosidases can be used for synthetic purposes (Lu et al., 2017; Perugino et al., 2004). Generally, β- galactosidases follow the catalytic retaining mechanism described above, but their structure and substrate specificity vary with the enzyme source. In recent years, a series of three-dimensional structures of β-galactosidases have been obtained by X-ray crystallography and cryoelectronic microscopy, which are helpful to understand the relationship between the structure and function of the enzymes (Lu et al., 2017). 4

Journal Pre-proof 2.1. GH2 β-galactosidases

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GH2 family contains the largest number of β-galactosidases, mainly produced from bacteria and yeast. Among them, LacZ from Escherichia coli is the most clearly elucidated with a number of crystal structures of native and mutant enzymes (Jacobson et al., 1994; Lu et al., 2017). The LacZ enzyme is a 464.4-kDa homologous tetramer, in which each subunit comprises five domains and functions independently (Fig. 3A). The complete catalytic efficiency of the enzyme depends on the participation of divalent and monovalent cations. According to the substrate used, the presence of Mg2+ or Mn2+ leads to 5~100-fold activation (Brás et al., 2010; Juers et al., 2001). The active center of the enzyme is a deep pocket within a central (α/β)8 barrel, with all the catalytically important residues positioned in or near this pocket. The residues Glu461 and Glu537, approximately 5.5 Å apart, are identified as acid/base and nucleophile catalysts, respectively. The binding pose of lactose is determined by the complex network of hydrogen interactions with Asn102, Asp201, His391, Asn460, Glu461, and Glu537, accompanying by the hydrophobic interactions between the sugars rings and the side chains of Trp568 and Trp999 (Fig. 3A). Initial binding of lactose to the enzyme occurs in a shallow mode, with a stacking by Trp999, followed by a shift into a deep mode and entering the active site (Bartesaghi et al., 2015; Brás et al., 2010). Two subsites in the active sites are responsible for substrate binding. One of them has strict specificity for galactose binding while the other has less specificity for aglycon binding (Juers et al., 2001). Such relaxed specificity enables the enzyme to utilize various galactose-containing substrates with different aglycons, including natural lactose and synthesized galactosides, such as chromogenic 5-bromo-4-chloro-3-indoyl- D-galactopyranoside (X-Gal), o-nitrophenyl galactopyranoside, and fluorogenic 4- methylumbelliferyl β- D-galactopyranoside. This ability not only contributes to the application of the enzyme in basic research, especially in the field of molecular biology, but also facilitates the transglycosylation reaction due to the possibility of selecting efficient substrates as glycosyl donor. The LacZ enzyme mainly generates β-1,6 linked product from lactose, along with minor β-1,3/4 products. Due to less steric hindrance, it might be easier for the 6-hydroxyl group of glucose to attack the enzyme-bound intermediate as compared with other positions. Also the formation of β-1,6 product is thermodynamically more favorable than others, due to the most negative value of the Gibbs energy (Brás et al., 2010; Juers et al., 2001). The latest developments in dynamic nuclear polarization NMR provide direct information on reaction mechanisms in real-time assays and help to further explain LacZ's synthetic preferences. By use of the labeled substrate o-nitrophenyl β- D-[1- 13 C, 1-2 H] galactopyranoside treated by polarization and dissolution, multiple low-populated short-lived intermediates of LacZ have been detected by NMR analysis. The relative production rate of the 1,6-linked product is faster than those of 1,3, 1,4, and 1,1 linkages, whereas its relative hydrolysis rate is slower than others. Therefore it seems that the LacZ enzyme prefers making 1,6-transglycosylation. In contrast, the formation rate of β-1,1 galactoside is slower but the 5

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2.2. GH35 β-galactosidases

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hydrolysis is faster, which explains why it has not been discovered before. Hydrolysis and transglycosylation may be more complex than previously described (Kjeldsen et al., 2018). The essential amino acid residues involved in substrate binding and product specificity were described in detail in the β-galactosidase (BgaD) of Bacillus circulans ATCC 31382 (189 kDa; PDB ID: 4YPJ), which is an important enzyme for industrial GOS production. Glu532 and Glu447 residues of BgaD act as nucleophile and acid/base catalysts, respectively. Arg185 and Glu601 at −1 subsite assist substrate binding and positioning, while Tyr511 near the catalytic residues donates proton to Glu532 before attacking the anomeric center of the substrate. Residues Trp570, Trp593 and Phe616 form an aromatic pocket that shapes the active site, which determine the linkage preference and product size. In addition, Asp481, Asp484, and Lys487 at or near the +1 subsite may influence the acceptor orientation and exert an indirect effect on the linkage specificity of glycosylation (Bultema et al., 2014; Yin et al., 2017a, 2017b). GH2 family also includes cold active β- galactosidase, exemplified by the hexameric enzyme C221-β-Gal from an Antarctic bacterium Arthrobacter sp. C2-2 (660 kDa; PDB ID: 1YQ2). Despite the similarity to LacZ, significant differences exist between these two enzymes influencing the polymerization state and activity. Hexameric packing of C221-β-Gal increases the number of channels and cavities that are filled with localized water molecules, making the enzyme flexible to adapt to cold. Besides, cysteine in C221-β-Gal at 999 site replaces the Trp999 in LacZ that participates in the stacking interaction with substrate in shallow mode, which may affect ligand binding mode and enzyme flexibility (Skálová et al., 2005).

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GH35 β-galactosidases come from bacteria, fungi, plants and animals. The Tr-β-gal from Trichoderma reesei is an important GH 35 enzyme (113.6 kDa; PDB ID: 3OGS). It not only hydrolyzes disaccharides and monogalactosides, such as lactose, lactulose, galactobiose, aryl- and alkyl-β- D-galactosides, but also cracks β-1,3- and β-1,4-galactan polymers (Maksimainen et al., 2011). Unlike prokaryotic enzymes, the eukaryotic Tr-β-gal contains N-linked glycosylation sites (Fig. 3B), which may be critical for protein stability. The glycosylation of Asn930 produces the longest oligosaccharides, which contain high- mannose-type sugar units, together with two additional glucoses located near the active site that may affect the catalytic activity. The glycan at Asn930 forms a network of hydrogen bond with the enzyme protein, which helps mask the residues Phe264, Phe304, Phe307, Trp310, Phe775 and Ile955 and protects them from protease hydrolysis. Similarly, the glycan at Asn627 provides protection for the residues Tyr576, Tyr608, Arg367 and Arg527 from proteolysis (Maksimainen et al., 2011). In the natural structure of Tr-β-gal, two different conformations were observed on the side chains of Leu262-Cys266 and Phe304, which made the active site open or close slightly. This property suggests the possibility of conformational adaption for the molecular 6

Journal Pre-proof recognition. The ability to open the active site can explain the tolerance of the enzyme to various substrates, including galactan polymers. 2.3. GH42 β-galactosidases

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GH42 β-galactosidases are mainly derived from bacteria, especially thermophiles. These enzymes are particularly important for the growth of microorganisms using galactose-related polysaccharides in plant biomass as carbon sources. An example is that Gan42B, a β-galactosidase from Geobacillus stearothermophilus T-6, is a key enzyme for the degradation of galactan. It shows significant hydrolytic activity towards galactobiose and larger galacto-oligomers, but has no detectable activity for lactose (Solomon et al., 2015). The crystal structure of Gan42B (242.8 kDa; PDB ID: 4OIF) demonstrates a homotrimer shaping like a flowerpot (Fig. 3C). Catalytic residues (Glu323 and Glu159) are located at the interface between monomers. Interaction between adjacent monomers facilitates the formation of complete active sites. This special assembly may contribute to the high thermal stability and catalytic activity of the enzyme. The three symmetrical Glu435 and Arg539 residues in the tunnel lining play a key role in the selection of suitable substrates, which may penetrate the wider opening of the central tunnel and further enter the protein active site. The presence of multiple active-site pockets may increase the encounter chances between substrate and catalytic sites, thus increasing the catalytic activity. The Gan42B monomer consists of three main domains: the largest domain A of (α/β)8 TIM barrel at N-terminus, the mixed α/β domain B, and the smallest C-terminal domain C. Domain A contains two catalytic residues at the pocket-like active site. There are also four highly conserved cysteine residues (Cys124, Cys164, Cys166 and Cys169), which are responsible for binding zinc cations and may indirectly enhance protein oligomerization. Domain B appears to participate in the assembly of oligomers rather than catalysis, while the function of domain C is not yet clear (Solomon et al., 2015). 2.4. GH1, GH59 and GH147 β-galactosidases GH 1 family contains very few β-galactosidases derived from archaea. Unlike GH2, GH35 and GH42 enzymes, GH 1 β-galactosidases can utilize glycosides bearing glycosyl moieties other than galactose, thus displaying extremely broad substrate specificity. This property is particularly useful for synthetic purposes, because only one enzyme is sufficient for glycorandomization by transferring various glycosyl groups from different donor substrates to an acceptor (Gloster et al., 2004). Ssβ-Glc1 (LacS) is a GH1 enzyme derived from Saccharolobus solfataricus (originally named Sulfolobus solfataricus), an extremely thermoacidophilic archaea that optimally grows in hot springs at around 80°C and pH 3. This enzyme has drawn attractive attention because of its stability and high tolerance to organic solvent, acid, and high temperatures. It exhibits almost equal action on gluco- and galacto-configured substrates. In the crystal structure (PDB ID: 1UWU and 1UWT), the binding differences between the two substrates only reside in the 7

Journal Pre-proof interaction of residues Gln18, Glu432, Trp433 with sugar hydroxyl groups at the O3 and O4 position (Gloster et al., 2004; Moracci et al., 2001). GH 59 and GH147 each contain only one β- galactosidase, without crystal structure analysis. GH147 is a newly assigned family, involving the β- galactosidase from Bacteroides ovatus, which is particularly active against galactohexaose and galactan, and contributes to the galactan degradation during pectin utilization (Luis et al., 2018).

3. Glycosylation reactions catalyzed by β-galactosidases 3.1. Synthesis of prebiotic galacto-oligosaccharides

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Using lactose as a substrate, β-galactosidases catalyze sequential galactosyl transfer from lactose to lactose itself or to hydrolysate, galactose or glucose, forming a GOS mixture containing linear and branched oligosaccharides with polymerization degree of 2~8. It has been reported that β-galactosidases from a variety of microorganisms, including bacteria, archaea, yeast, and fungi, produce GOS in general yields of 20%~50% (Lu et al., 2017; Saqib et al., 2017). The enzymes from various sources exhibit different regioselectivity of transglycosylation from lactose. As a result, the famous commercial GOS products from different sources, like Oigomate®55 (Japan) from Aspergillus oryzae and Streptococcus thermophilus, and Vivinal® GOS (The Netherlands) from B. circulans, contain distinct oligosaccharide patterns varying in glycosidic linkages (Torres et al., 2010). β-Galactosidases have been used to synthesize GOS in free or immobilized forms. Immobilization of enzymes is particularly attractive for industrial-scale synthesis because of the reuse of enzymes, improved stability of enzymes and easy separation of products (Panesar et al., 2018). Conventional strategies for immobilization include the adsorption, entrapment, covalent binding, and cross- linking of β-galactosidase proteins with various carrier materials, such as agarose, anion exchange resin, cellulose, chitosan, cotton cloth, glass beads, grapheme, magnetic particles, membranes, polystyrene nanofiber, and silica nanoparticles (Lu et al., 2017; Misson et al., 2016; Panesar et al., 2018; Satar et al., 2016). In addition, there is an alternative immobilization strategy that does not use carriers, that is, enzyme proteins are precipitated by salt- or solvents and then cross- linked by glutaraldehyde to form insoluble biocatalysts designated as cross- linked enzyme aggregates (CLEAs) (Sheldon et al., 2011). An instance is that recombinant β-galactosidase derived from a marine metagenomic library has been prepared in the form of CLEAs, which can produce GOS in 59.4% yield from 360 g/L of lactose and retain about 82.1% activity after ten batches of reactions (Li et al., 2015). Recent advances in molecular biotechnology make it easy to fuse affinity tags with enzymes or display enzymes on the surface of yeast or spore cells, which promotes the emergence of oriented immobilization strategies. Immobilization of enzymes at specific sites can reduce the damage of active sites and maintain higher enzyme activity (An et al., 2016; Li et al., 2009; Lu et al., 2012b; Kwon et al., 2007). A notable example is the fusion of β-galactosidase from Lactobacillus bulgaricus L3 with cellulose binding domain (CBD), an industrial attractive tag for spontaneous and specific binding of low-cost cellulose. The recombinant CBD- fusion enzyme was purified and immobilized on microcrystalline cellulose 8

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by absorption in one simple step. The immobilized enzyme can produce 49% GOS from 400 g/L lactose. At least 20 batches of reactions have been recycled, and the enzyme activity remains above 85% (Lu et al., 2012b). Another example is the successful anchorage of the β-galactosidase from Penicillium expansum F3 on the cell surface of Saccharomyces cerevisiae EBY-100 as an immobilized catalyst for GOS synthesis (Fig. 4A). In this system, monosaccharide byproducts (glucose and galactose), which may inhibit the synthesis reaction, can be utilized by the recombinant yeast for cell growth and enzyme induction, thus greatly facilitating GOS synthesis. Enzyme expression and GOS production remained at a high level during 35 days of batch culture for 7 times (Li et al., 2009). GOS produced by β- galactosidases are a mixture of transgalactosylated oligosaccharides, unreacted lactose, glucose and galactose. Fermentation with GOS mixtures by yeast cells can selectively consume monosaccharide and lactose, producing high purity oligosaccharides for use by diabetic patients or for the treatment of intestinal diseases (Aburto et al. 2018; Li et al., 2008). Recently, an elaborate system has emerged to produce high-purity GOS by using only one organism in the synthesis and purification process (Fig. 4B). The cells of Kluyveromyces lactis first permeated through ethanol treatment to facilitate substrate entry and product release. Then, they were incubated with lactose to synthesize GOS. Afterwards, the product mixture was fermented with active cells of K. lactis without ethanol treatment to deplete the undesired glucose, galactose, and lactose. Ethanol is produced simultaneously in this process and can be reused for initial cell treatment. This system is recyclable and efficient to produce GOS with high purity above 95% (Sun et al., 2016).

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3.2. Synthesis of galactose-containing glycans and glycosides

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In addition to the synthesis of GOS, β-galactosidases also exhibit robust and powerful transglycosylation ability for the synthesis of a wide range of important galactose-containing chemicals. Using lactose, nitrophenyl or methylumbelliferonyl galactoside as glycosyl donors, they are able to glycosylate various compounds, including aliphatic alcohols, amino acids, nucleosides, antibiotics, sugars, sugar alcohols, natural glycoside products, and the like (Table 1, Fig. 5). Simple aliphatic alcohols are good acceptors for glycosylation by β-galactosidases to synthesize alkyl glycosides that are useful in the food and pharmaceutical industries (van Rantwijk, et al., 1999). The Antarctic cold-adapted β- galactosidase of Pseudoalteromonas sp. 22b seems to be more effective in glycosylation of C3–C6 aliphatic alcohols, including 2-propanol, 1-butanol, 1-pentanol, and 1- hexanol than the mesophilic enzyme from E. coli (Makowski et al., 2009). It was found that reverse micelle system could improve the yield of long chain alkyl glycosides. Using aerosol-OT reverse micelles, the β-galactosidase from Penicillium canescencs can synthesize octyl β-galactoside with a yield of up to 45% (Kuptsova et al., 2001). Attractively, the β- galactosidase from A. oryzae can glycosylate complex PEG (polyethylene glycol) brushes tethered on a gold surface (Fig. 5, 6), forming a glycocalyx- like structure which is effective for probing carbohydrate–protein interactions (Fang et al., 2012). 9

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It is reported that a series of anti- microbial and anti-cancer drugs bearing alcohol groups have been glycosylated by β-galactosidases in convenient way. The enzyme from A. oryzae has been shown to catalyze the enantioselective transgalactosylation of antibiotics such as chlorphenisin and chloramphenicol using lactose as the glycosyl donor (Scheckermann et al., 1997). The β-galactosidase from Aplysia fasciata can efficiently glycosylate the anticancer drug 5- fluorouridin, resulting in 5'-O-β-galactosyl-5-fluorouridin (Fig. 5, 7), which is a prodrug that is more than 100 times less toxic to bone marrow cells in Balb/c mice than the drug. A similar result was obtained by galactosylation of anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) using the same enzyme (Fig. 5, 8). All the reactions were extremely stereo- and regioselective, with only 5'-O-β-galactosyl derivatives accumulated in high yields of 43% to 60% (Giuseppina et al., 2007). β-Galactosidases also contribute to the synthesis of essential oligosaccharide units bearing galactose moiety, which constitute the glycan chains of glycoproteins and glycolipids, rich in information and involved in various biological processes (Murata and Usui, 2006; Oliveira et al., 2011). Similar to the synthesis of GOS, the glycosylation regioselectivity of β-galactosidases to acceptors depends on the enzyme source. Porcine testicular β-galactosidase prefers to form β-1,3 linkage, which has been used to synthesize Gal-β-1,3-GalNAc and Gal-β-1,3-GlcNAc whose structures are related to tumor-associated carbohydrate antigens and ABO blood group determinants, respectively. In contrast, the enzyme from Bifidobacterium bifidum is β-1,4 regioselective and has been used to synthesize Gal-β-1,4-GlcNAc, the terminal sequence of N-linked glycans (Murata et al., 1996; Yoon and Ajisaka, 1996). It seems that the desired glycosyl linkages can be obtained by choosing suitable enzymes with certain regioselectivity. The combination of various glycosidases or glycosidase and glycosyltransferase has been proved to be helpful in the construction of full- length oligosaccharide units. The sequential incubation of β- galactosidase and α- galactosidase with nitrophenyl β-galactoside, thioethyl GlcNAc, and nitrophenyl α- galactoside has produced an important Gal-α-1,3 trisaccharide, an antigen involved in the hyperacute rejection of xenotransplantation from pigs to human (Vie et al., 1997). Likewise, the successive use of β-galactosidase with α-1,3 fucosyltransferase and α-2,3 sialyltransferase, starting with lactose and GlcNAc derivatives, followed by addition of GDP-Fuc and CMP-Neu5Ac as donors for glycosyltransferases, facilitates the assembly of sialyl Lewisx , which plays a crucial role in cell-to-cell recognition (Zeng and Uzawa, 2005). Interestingly, the combination of β-galactosidase and a lipase unrelated to sugar processing has been reported to produce a promising biomaterial. Firstly, β- galactosidase formed β- galactosyl- L- lactate ethyl ester from lactose and ethyl- L- lactate. Then lipase-catalyzed polymerization was carried out to obtain poly (β- D-galactoside-co- L-lactic acid) with molecular weight between 800 and 2000. This kind of sugar-lactate copolymer has good application prospects because it incorporates sugar units into poly (lactic acid), which is highly valuable for biomedical and food industries. The introduction of the sugar moiety can endow the copolymer with the desired advantages, such as controllable degradation rates, convenience for attaching reagents through the hydroxyl groups, and easy gelation by 10

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crosslinking (Jia and Wang, 2007). Besides simple alcohols and straight-chain oligosaccharides, β-galactosidases can also accommodate complex structures as acceptors for transglycosylations (Fig. 5, 14, 15). For example, a cyclic glucotetrasaccharide, which has potential use as low-calorie sweetener, drug-delivery carrier, and affinity-chromatography material, has been modified by β-galactosidases from A. oryzae and B. circulans to yield 6' and 3' galactosyl cyclic tetrasaccharides, respectively. The branched cyclic saccharides will acquire new functions depending on the position of the branched chain or the sugar units added (Higashiyama et al., 2004). Another example is the β- galactosidase-catalyzed glycosylation of myricitrin, a highly anti-oxidative flavonol glycoside with potential value as an ingredient of functional foods, cosmetics and medicines. The resultant galactosylated derivatives of myricitrins are 480 times more soluble in water than myricitrin, breaking through the limitation of water insolubility (Shimizu et al., 2006). β-Galactosidases are not only flexible to acceptor substrates but also tolerant to engineered donor substrates. An instance is the successfully enzymatic introduction of sulfated groups into oligosaccharides by use of a sulfated donor substrate (Murata et al., 2001). Employing 4- methylumbelliferyl 6-sulfo β- D-galactopyranoside as donor, the β-galactosidase from B. circulans can transfer 6-sulfo galactosyl to GlcNAc or Glc acceptors and produce sulfated disaccharides (Fig. 5, 12, 13). Sulfation of oligosaccharides often occurs in glycoconjugates, such as glycoproteins, glycolipids, and proteoglycans, which play numerous roles in biological events including selectin-binding, laminin-binding, neural cell migration, bacteria binding, and macrophage activation (Murata and Usui, 2006). Enzymatic access to sulfated oligosaccharides will facilitate the assembly of complex glycoconjugates in the future.

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4. β-Galactosidases engineering Nowadays, numerous rational and non-rational engineering strategies have been successfully applied to optimize β-galactosidases, including site-directed mutagenesis, truncation, site-saturation mutagenesis, random mutagenesis, DNA shuffling and monobody modifications of enzyme proteins (Fig. 6). Changes in β- galactosidases at DNA or protein levels have brought promising superior properties, such as high transglycosylation efficiency, broadened substrate specificity, tailor- made product structures, increased thermal stability, and reduced product inhibition (Table 2). This has greatly promoted the development of β-galactosidase, especially in the field of carbohydrates synthesis. 4.1. Improve transglycosylation efficiency In the process of glycosidase catalyzed synthesis, the product can also be used as a substrate for hydrolysis, thus usually resulting in a moderate yield. A great breakthrough of glycosidases came from the nucleophile mutants, namely glycosynthases that lost hydrolytic activity but remained transglycosylation activity. The pioneered glycosynthase was derived from GH1 glycosidase (Abg) of Agrobacterium sp., which possess both β-glucosidase and 11

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β-galactosidase activities. Replacing the catalytic nucleophile glutamate at the 358 site with an alanine residue completely altered the enzyme mechanism. The resulting mutant enzyme E358A cannot act on normal substrate to form glycosyl-enzyme intermediate, although it still exists in correctly folded form. However, in the presence of an activated donor sugar bearing small easy- leaving group, which could mimic glycosyl-enzyme intermediate (e.g. α- glycosyl fluoride), the mutant enzyme was able to glycosylate acceptor sugars with high yields above 90% (Mackenzie et al., 1998). Owing to the lack of catalytic nucleophile, o ligosaccharide products cannot be hydrolyzed, thus accumulating to a virtually high yield. Later, it was found that mutations of nucleophilic sites to serine and glycine promoted synthesis more than alanine (Mayer et al., 2000; Tolborg et al., 2002). Further engineering of glycosynthase E358G generated the most efficient Abg enzyme, named 2F6, which carries four mutated residues: A19T, E358G, Q248R, and M407V (Kim et al., 2004). Site-directed mutagenesis outside the catalytic residue can also improve the properties of transgalactosylation, although the hydrolysis activity of the enzyme may not be completely eliminated. Three mutants (Y296F, F417S and F417Y) related to -1 and +1 subsites were obtained by rational design of GH1 β- glycosidase from Halothermothrix orenii, which increased GOS production from 39.3% to more than 50% (Hassan et al., 2016). Likewise, two mutants (F359Q and F441Y) obtained by site-directed mutagenesis of β- galactosidase from S. solfataricus P2 produced 58.3% and 61.7% GOS compared with 50.9% from natural enzyme (Wu et al., 2013). Semi- rational site-saturation mutagenesis is effective in changing the properties of an enzyme that is not conducive to synthesis. The thermophilic GH 42 β-galactosidase (BgaB) from G. stearothermophilus KVE39 can only synthesize very small amounts of GOS in 2% yield. However, the production of GOS was increased to 11.5% by replacing arginine with lysine at 109 site. Subsequent saturated mutations resulted in better mutants R109V and R109W, which could further elevate the transglycosylation performance by more than 10% (Placier et al., 2009). It has been reported that truncated mutation can transform the normal, hydrolytic β-galactosidase of B. bifidum DSM20215 into a highly efficient transgalactosylase. The C-terminal 580 amino-acid residues involved in galactose binding motif were deleted, resulting in the use of approximately 90% reacted lactose by the truncated enzyme for GOS synthesis (Jorgensen et al., 2001). 4.2. Change glycosyl donor specificity Generally, β-galactosidases have strict specificity on the galactose moiety of the glycosyl donor except for the enzymes from GH1 family. Nevertheless, this specificity can be modulated by enzyme engineering. The resulting engineered β-galactosidases can be converted into other glycosidases, such as β- fucosidase and β- glucuronidase, which act on fucose and glucuronic acid containing substrates, respectively. The lacZ from E. coli was shifted to an efficient β- fucosidase by DNA shuffling and screening through chromogenic fucose substrates. In each round of shuffling, only colonies 12

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that exhibited enhanced fucosidase activity were selected as templates for the next round of shuffling to accumulate beneficial mutations. An evolved enzyme purified from the seventh-round mutant cells became fucosidase, which displayed 1,000- fold increased specificity for oNP-fuc versus oNP-Gal and 300- fold increased specificity for pNP-fuc versus pNP-Gal (Zhang et al., 1997). Later, semi-rational design of lacZ produced more efficient β- fucosidases through the analysis of the crystal structures of enzyme-galactose complex (Parikh and Matsumura, 2005). Considering the slight difference in the structure of C6 substituent of pNP- fuc and pNP-Gal (methyl versus hydroxymethyl), the site saturation mutagenesis of His540, Asn604 and Asp201 residues that may interact directly or indirectly with C6 hydroxyl groups was carried out. The result showed that the catalytic efficiency (k cat /Km) of a double mutant (H540V, N604T) toward pNP- fuc was 180 times higher than that of the wild-type enzyme, and the specificity of pNP-gal versus pNP-fuc was reversed 700,000 times as compared with the best variant obtained by DNA shuffling (Zhang et al., 1997). A β-galactosidase can also develop into a β-glucuronidase by DNA shuffling. Using the substrate 5-bromo-4-chloro-3-indolyl-β- D-glucuronic acid (X-GlcA), more than 200,000 mutant colonies were screened out from each round shuffling of the enzyme gene from Pyrococcus woesei. After four rounds, it was observed that the activity of a mutant toward pNP-β- D-glucuronide was 7.5 and 4.9 times higher than that of wild-type enzyme at 25 and 37°C, respectively. The mutant contained seven mutated residues associated with high β-glucuronidase activity (T29A, V213I, L217M, N277H, I387V, R491C, and N496D), of which the residue site 277 play the most important role (Xiong et al., 2007). Molecular evolution of β- glactosidases also enables the enzyme to efficiently handle engineered donor substrates, such as p-nitrophenyl 3-O- methyl-β- D-galactopyranoside (3-MeOGalpNP) modified by methylation at C-3 of the galactose ring (Fig 5, 16). Based on the structurally analysis of the Abg from Agrobacterium sp., Gln24, His125, Trp126, Trp404, Glu411 and Trp412 residues that constrain binding around the 3-OH group were subjected to site-directed saturation mutagenesis. It was found that two mutants (Q24S-W404L and Q24N-W404N) showed enhanced hydrolytic activity toward 3-MeOGalpNP. Further integration of Q24S and W404L into the engineered glycosynthase 2F6 significantly increased the synthesis rate of 3-O-methylated trisaccharide by 40 times, when 3-O-methyl glucopyranosyl fluoride was used as a glycosyl donor and pNP-cellobioside as an acceptor. This enzyme is valuable for the preparation of naturally methylated glycoconjugates or artificially methyl-capped glycans (Shim et al., 2012). 4.3. Broaden glycosyl acceptor specificity β-Glactosidases have different preferences for various acceptors despite the flexibility toward acceptor structures. They generally catalyze the formation of O-glycosidic bonds between galactose and alcoholic hydroxyl groups of the acceptors, whereas the glycosylation of phenolic hydroxyl groups or the synthesis of glycosides other than O- linkage was rarely reported (Kim et al., 2010; Lu et al., 2015b). 13

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Enzymatic glycosylation of phenolic hydroxyl seem to be more difficult than that of alcoholic hydroxyl due to its low nucleophilicity. Recently, the rational design of L. bulgaricus L3 β- galactosidase has enhanced its preference for phenolic hydroxyl groups. It is speculated that the residue Trp980 at the entrance of the active center plays a key role in the initial substrate selection of the enzyme. The subsequent site saturation mutagenesis generated a mutant W980F that produced 7.6% to 53.1% higher yields of glycosides by glycosylation of catechol, hydroquinone, and phenol. Moreover, the W980F enzyme was found to glycosylate pyrogallol and caffeic acid which cannot be modified by the wild-type enzyme (Lu et al., 2015a, 2015b). This is a breakthrough in enzymatic galactosylation of challenging phenols. Many valuable phenolic compounds exhibit attractive bioactivities but their use is limited due to poor water solubility and instability under light irradiation. It is reported that glycosylation of phenols can improve their property and expand their applications (Xu et al., 2016). Strategies to develop glycosynthase can also broaden the range of acceptors. The mutant Abg E358G displayed extended application in solid-phase synthesis of oligosaccharide with excellent material recovery (Tolborg et al., 2002). Amazingly, the best mutant Abg 2F6 was able to glycosylate a modified endo-xylanase (Bcx) from B. circulan, which was introduced with a cysteine mutation at S22 and then chemically bound to cellobiosyl derivatives at the site (Fig. 5, 19). Using α-galactosyl fluoride as donor, Abg 2F6 converted above 80% of modified Bcx into glycoproteins containing three sugar units (Müllegger et al., 2006). It should be noted that natural glycosidases and glycosynthases usually glycosylate acceptors at hydroxyl groups to produce O-glycosidic linkages. Contrastly, they are hard to modify the thiosugars despite of the importance of S- glycosides. This may be related to the electrostatic repulsion between acid/base residues and thiolate, or to the steric hindrance caused by the larger sulfur atoms (Jahn and Withers, 2003). Interestingly, the site directed mutagenesis of catalytic acid/base has yielded a new class of catalysts, termed thioglycoligases, which can synthesize S-glycosidic linkages. A pioneered example of thioglycoligase was also derived from the Abg enzyme described above. The serine mutation at the acid/base site (E171) slowed down glycosylation and deglycosylation steps. Nevertheless, the use of donors with good leaving groups, such as dinitrophenyl glycoside without acid catalysis, led to rapid glycosylation. In the presence of the acceptors with strong- nucleophilic thiol groups that do not require general base catalysis, the deglycosylation step was accelerated, resulting in the formation of S-linked oligosaccharides. Interestingly, the regioselectivity of this synthetic reaction is entirely controlled by the position of thiol in acceptors (Jahn et al., 2003). Subsequent site-saturation mutagenesis at the 171 site revealed that glutamine or glycine mutants displayed higher thioglycoligase activities than the serine mutant (Müllegger et al., 2005). More importantly, Abg E171G can galactosylate the modified Bcx protein, which also introduces cysteine at 22 position by mutation and binds with 4-deoxy-4-thiocellobiosyl moiety, thus producing thioglycosylated glycoprotein (Fig. 5, 21). The method is promising to generate stable therapeutic glycoproteins in which the terminal sugars are S- linked and resistant to glycosidase- mediated hydrolysis (Müllegger et al., 2006) 14

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4.4. Modulate product specificity

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Molecular evolution of β- galactosidases also affects the specificity of products, including glycosidic linkages and polymerization state. Natural BgaD-D from B. circulans tends to synthesize β-1,4 linkages, while the mutants R484S and R484H produced by site saturation mutation of R484 near +1 subsite form new GOS structures containing alternating β-1,3 and β-1,4 linkages (Yin et al., 2017a). Later, the structure- function relationship of BgaD-D was studied in detail, and a variety of essential residues related to GOS linkage specificity were predicted. Alterations of these residues produced the mutants W593Y, W593F, F616L and D481N, which preferentially synthesized β-1,2/3 or β-1,3/6 linkages (Yin et al., 2017b). It is noteworthy that modifying BgaD-D protein with artificial monobodies (synthetic binding proteins) can change GOS polymerization. Candidate monobodies were incubated with the enzyme protein, and one of them (L23) showed little effect on the production of mono-, di- and trisaccharides, but diminished the production of tetrasaccharides and larger species. L23 monobody may occupy the subsite responsible for the binding of large oligosaccharides, thus preventing their further elongations. This new strategy expands enzyme-engineering technologies and can be extended to modify other enzymes ( Tanaka et al., 2015). 4.5. Improve thermal stability

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Enzyme reactions also require higher operational stability, especially in industrial applications at higher temperatures. The site-directed mutagenesis of BgaD-D produced a triple mutant (K166P, G307P, A833P) with better thermal stability at 60°C (Ishikawa et al., 2015). In addition, the DNA family shuffling of two enzymes sharing 56% identity at the DNA level, of which one is the stable CelB from P. furiosus and the other is the efficient LacS from S. solfataricus, produced stable hybrids with improved catalytic properties for lactose (Kaper et al., 2002) Another interesting example is the rational mutation of β-galactosidase from K. lactis by introducing cysteine at subunit interfaces, which resulted in a mutant (R116C, T270C, G818C) with a half- life increase of 6.8 times at 45 °C and a higher catalytic efficiency. The increase in thermostability and activity may be related to the formation of disulfide bonds between subunits, which enhances the interface contact and strengthens the quaternary structure. Under the same conditions, compared with natural enzymes, the mutant enzyme can synthesize higher yield of GOS with fewer enzyme amount, thus greatly reducing the processing cost (Rico-Díaz et al., 2017). 4.6. Reduce product inhibition Most beta-galactosidases are inhibited by the galactose released from lactose hydrolysis, because the binding of the enzyme with galactose may prevent the lactose from entering the 15

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active site. Rational design of β-galactosidases can reduce competitive galactose inhibition. The motif Asp258-Ser-Tyr-Pro-Leu-Gly-Phe264 of Aspergillus niger β- galactosidase, which may be involved in galactose binding, was mutated randomly to produce a galactose-insensitive mutant containing the altered motif Asp258-Phe-Tyr-Thr-Ser-Ser-Phe264. The Ki value of the mutant enzyme was 6.46 mM, while that of wild type enzyme was 0.76 mM (Hu et al., 2010). Site directed mutagenesis of β-galactosidase from Aspergillus candidus has also significantly decreased galactose inhibition. Tyr96, Asn140, Glu142, and Tyr364 residues, which form hydrogen bonds with galactose, may play an essential role in galactose binding. These residues were individually subjected to site saturation mutagenesis, and the mutations in Tyr96, Asn140, and Tyr364 caused less galactose inhibition. The mutant Y364F seemed to be the least sensitive to galactose because its Ki value (282 mM) is the highest as compared with the reported Ki values of other β-galactosidases (Zhang et al., 2018).

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5. Conclusions

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β-Glactosidases have been proved to be excellent catalysts for synthesizing oligosaccharide and glycoside. These enzymes are readily available, inexpensive, easy to handle, and flexible to simple substrates without requirement of cofactors (Scigelova et al., 1999; Bojarová and Kren, 2009). It is worth noting that the synthesis capability of β-galactosidases is continuously expanding nowadays. Numerous current studies are focusing on β-galactoidases with high transglycosylation activity, which help to increase oligosaccharide production and reduce large-scale production costs. Screening enzymes from natural sources is a long-standing method for establishing efficient glycosidase libraries (Scigelova et al., 1999; Palcic, 1999). Gene mining in online database or metagenomic library provides an alternative method for finding new enzymes with good properties (Li et al., 2015; Liu et al., 2019; Silvério et al., 2018). Today, molecular engineering of β- galactosidases greatly improves transglycosylation activity and/or substrate tolerance, and extends the application to synthesize unusual and challenging structures. The preparation of engineered β-galactosidases can be accelerated by discovering three-dimensional structures of enzymes and developing useful high-throughput screening methods to detect transglycosylation activity. New natural or engineered β-galactosidases and their promising transglycosylation products will greatly promote the basic research of glycobiology and their practical application in food, medicine, personal care, cosmetics, and other industries in the future.

Acknowledgments This work was supported by National Natural Science Foundation of China (No.21877044) and Fundamental Research Funds for the Central Universities (No. 2018KFYYXJJ020).

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101. Wang, L.X., Huang, W., 2009. Enzymatic transglycosylation for glycoconjugate synthesis. Curr. Opin. Chem. Biol. 13, 592-600. https://doi.org/10.1016/j.cbpa.2009.08.014. 102. Wojciechowska, A., Klewicki, R., Sójka, M ., Grzelak-Błaszczyk, K., 2018. Application of transgalactosylation activity of β-galactosidase from Kluyveromyces lactis for the synthesis of ascorbic acid galactoside. Appl. Biochem. Biotechnol. 184, 386-400. https://doi.org/10.1007/s12010-017-2551-z. 103. Wojciechowska, A., Klewicki, R., Sójka, M ., Klewicka, E., 2017. Synthesis of the galactosyl derivative of gluconic acid with the transglycosylation activity of β-galactosidase. Food Technol. Biotechnol. 55, 258-265. https://doi.org/10.17113/ftb.55.02.17.4732. 104. Wu, Y., Yuan, S., Chen, S., Wu, D., Chen, J., Wu, J., 2013. Enhancing the production of galacto-oligosaccharides by mutagenesis of Sulfolobus solfataricus beta-galactosidase. Food Chem. 138, 1588-1595. https://doi.org/10.1016/j.foodchem.2012.11.052. 105. Xiong, A.S., Peng, R.H., Zhuang, J., Li, X., Xue, Y., Liu, J.G., Gao, F., Cai, B., Chen, J.M ., Yao, Q.H., 2007. Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity. Appl. M icrobiol. Biotechnol. 77, 569-578. https://doi.org/10.1007/s00253-007-1182-7.. 106. Xu, L.J., Qi, T., Li, X., Lu, L., Xiao, M ., 2016. Recent progress in the enzymatic glycosylation of phenolic compounds. J. Carbohyd. Chem. 35, 1-23. https://doi.org/10.1080/07328303.2015.1137580. 107. Yamamoto, K., 2013. Recent advances in glycotechnology for glycoconjugate synthesis using microbial endoglycosidases. Biotechnol. Lett. 35, 1733-1743. https://doi.org/10.1007/s10529-013-1272-9. 108. Yin, H., Pijning, T., M eng, X., Dijkhuizen, L., van Leeuwen, S.S., 2017a. Engineering of the Bacillus circulans β-galactosidase product specificity. Biochemistry. 56, 704-711. https://doi.org/10.1021/acs.biochem.7b00032. 109. Yin, H., Pijning, T., M eng, X., Dijkhuizen, L., van Leeuwen, S.S., 2017b. Biochemical characterization of the functional roles of residues in the active site of the β-galactosidase from Bacillus circulans ATCC 31382. Biochemistry. 56, 3109-3118. https://doi.org/10.1021/acs.biochem.7b00207. 110. Yoon, J.H., Ajisaka, K. 1996. The synthesis of galactopyranosyl derivatives with beta-galactosidases of different origins. Carbohydr. Res. 292, 153-163. https://doi.org/10.1016/0008-6215(96)00187-5. 111. Zeng, Q.M ., Li, N., Zong, M .H., 2010. Highly regioselective galactosylation of floxuridine catalyzed by beta-galactosidase from bovine liver. Biotechnol. Lett. 32, 1251-1254. https://doi.org/10.1007/s10529-010-0302-0. 112. Zeng, X., Uzawa, H., 2005. Convenient enzymatic synthesis of a p-nitrophenyl oligosaccharide series of sialyl N-acetyllactosamine, sialyl Lex and relevant compounds. Carbohydr. Res. 340, 2469-2475. https://doi.org/10.1016/j.carres.2005.08.019. 113. Zeng, X., Yoshino, R., M urata, T., Ajisaka, K., Usui, T., 2000. Regioselective synthesis of p-nitrophenyl glycosides of beta-D-galactopyranosyl-disaccharides by transglycosylation with beta-D-galactosidases. Carbohydr. Res. 325, 120-131. https://doi.org/10.1002/chin.200027193. 114. Zhang, J., Lu, L., Lu, L., Zhao, Y., Kang, L., Pang, X., Liu, J., Jiang, T., Xiao, M ., M a, B., 2016. Galactosylation of steroidal saponins by β-galactosidase from Lactobacillus bulgaricus L3. Glycoconj. J. 33, 53-62. https://doi.org/10.1007/s10719-015-9632-4. 115. Zhang, J.H., Dawes, G., Stemmer, W.P., 1997. Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening. Proc. Natl. Acad. Sci. U S A. 94, 4504-4509. https://doi.org/10.1073/pnas.94.9.4504. 116. Zhang, Z., Zhang, F., Song, L., Sun, N., Guan, W., Liu, B., Tian, J., Zhang, Y., Zhang, W., 2018. Site-directed mutation of β-galactosidase from Aspergillus candidus to reduce galactose inhibition in lactose hydrolysis. 3 Biotech. 8, 452. https://doi.org/10.1007/s13205-018-1418-5. 117. Zhou, Y., Liu, K., Zhang, J., Chu, J., He, B., 2017. M g2+ -induced stabilization of β-galactosidase from Bacillus megaterium and its application in the galactosylation of natural products. Biotechnol. Lett. 39, 1175-1181. https://doi.org/10.1007/s10529-017-2344-z.

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Journal Pre-proof Figure captions Fig. 1. Phylogenetic analysis of GH2 (red), GH1 (orange), GH59 (purple), GH147 (black), GH42 (green) and GH35 (blue) β- galactosidases. The sequences of enzymes were firstly subjected to the multiple alignment by the ClustalX program and then converted to a phylogenetic tree by the MEGA7 program.

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Fig. 2. The retaining mechanism of β-galactosidase using lactose as a substrate. The β-galactosidase follows a double displacement mechanism in which a glycosyl-enzyme intermediate forms, followed by hydrolysis or transglycosylation when water or sugars are used as acceptors, respectively. Possible glycoside products produced by glycosylation of the sugars such as glucose, galactose and lactose are represented in β-1, 6 galactosyl forms. The actual composition is complex, depending on enzyme source.

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Fig. 3. Three-dimensional structures of GH2 (A), GH35 (B) and GH42 (C) β-galactosidases. (A) Lac Z from E. coli with lactose (PDB ID: 1JYN). A1, surface presentation of tetramer with monomers in different colors; A2, surface and cartoon presentations of a monomer; A3, enlarged active center. Hydrogen network and hydrophobic interactions exist between essential residues (dark blue) and lactose (red). Green spheres are water molecules. (B) Tr-β-gal monomer from T. reesei with IPTG (PDB ID: 3OGS). B1 and B2, surface and cartoon presentations, with domains in different colors. In B2, the rose red residues are glycosylated. (C) Gan42B from G. stearothermophilus (PDB ID: 4OIF). The central cavity of the trimeric enzyme is in funnel shape. Catalytic residues (red) situate at the interface between monomers. Glu435 and Arg539 (blue) participate in substrate selectivity.

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Fig. 4. GOS synthesis by recombinant (A) or native (B) yeast cells. (A) GOS synthesis by recycling β-galactosidase anchored on the cell surface of S. cerevisiae. The byproduct glucose can be used as carbon source for cell growth while the galactose is used as an inducer for enzyme production. (B) Synthesis of high-purity GOS by sequential use of permeated and active cells of K. lactis. GOS are synthesized by the ethanol-permeated cells, followed by purification with the intact active cells. Fig. 5. Examples of carbohydrate synthesis catalyzed by natural (red) and engineered (blue) β-galactosidase from various donor and acceptor substrates. The listed products are formed by glycosylation of aliphatic alcohols (1), sugar alcohol mannitol (2), antibiotics chlorphenisin (3) and chlorphenicol (4), serine derivative (5), PEG 1000 brushes on gold (6), nucleoside drugs 5-fluorouridine (7) and 3'-azido-3'-deoxythymidine (8), N-acetylglucosamine and its derivative (9-12), glucose and its derivative (13, 20), cellulose derivative (16), cyclic glucotetrasaccharide (14), natural product myricitrin (15), resin bond glucose (17), phenolic compound caffeic acid (18), modified protein Bcx S22C (19, 21). The green represents the mutation site S22C of Bcx. Fig. 6. Engineering strategies for optimizing β-galactosidases. Ep PCR, error prone PCR.

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Journal Pre-proof Table 1 Examples of natural β-galactosidases-catalyzed transglycosylation Organism

Donor

Acceptor

Major product

Reference

Aplysia fasciata

oNP-β-Gal

Uridine, Cytidine, Thymidine, Adenosine, Fluorouridine, 3 -Azido-3 -deoxythymidine, 5-Chlorocytosine arabinoside

β-Galactosides

Giuseppina et al., 200

Aspergillus oryzae

pNP-β-Gal Lactose Lactose Lactose

GalNAc Chlorphenisin, Chlorphenicol Cyclic glucotetrasaccharide PEG brushes on gold

Gal-β-1,6/4-GalNAc β-Galactosides Gal-β-1,6/3-cyclic saccharides Galactosyl PEG brushes

Yoon and Ajisaka, 199 Scheckermann et al., 1 Higashiyama et al., 20 Fang et al., 2012

Bacillus circulans

pNP-β-Gal Lactose Lactose

GlcNAc-β-1,6-Man UDP-Gal GlcNAc-β-cyclodextrin (CD)

Fujimoto et al., 1997 Nieder et al., 2003 Tanimoto et al., 2005

Gal-β-1,4/6-GalNAc Gal-β-1,4-GlcNAc

Yoon and Ajisaka, 199

Lactose Lactose Lactose

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Gal-β-1,4-GlcNAc-β-1,6-Man β-1,4-di/trisaccharides Gal-β-1,4-GlcNAc-β-CD Gal 2-β-1,4-GlcNAc-β-CD GlcNAc-β-pNP β-1,4-disaccharide Myricitrin β-Galactosides Astragalin Gal-β-1,6-astragalin Gal-β-1,4-Gal-β-1,6-astragalin Fucose Gal-β-1,3/4/2-fucose Naringin, Polydatin, Aesculin, β-Galactosides Bergenin

Zeng and Uzawa, 2005 Shimizu et al., 2006 Han et al., 2017

Lactose pNP-β-Gal

Bifidobacterium bifidum

pNP-β-Gal

GalNAc GlcNAc

Bovine testes

Lactose

Sucrose, Lactose, Isomalt, Isomaltulose, Isomelezitose, Raffinose, 1-Kestose

β-1,3-saccharides

Schroder et al., 2004

Diplococcus pneumoniaeand

pNP-β-Gal

GlcNAc-β-1,2-Man

Gal-β-1,4-GlcNAc-β-1,2-Man

Fujimoto et al., 1997

Enterobacter cloacae

oNP-β-Gal

β-Galactosides Gal-β-1,6-salicin Gal-β-tyrosol Gal-β-1,6/3-Gal-β-tyrosol

Lu et al., 2010b

Gal-β-1,6/4-Glc-α-1,1-Glc β-Galactosides

Kim et al., 2007 Seo et al., 2015

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Bacillus megaterium

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Mannitol, Sorbose Salicin Tyrosol

Escherichia coli

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Lactose

Usvalampi et al., 2018 Zhou et al., 2017

Qi et al., 2017

Lactose oNP-β-Gal

Trehalose N-carboxy benzyl benzyl ester

Lactose Lactose Lactose Lactose

Aromatic compounds Hydroquinone Sodium ascorbate Gluconic acid

β-Galactosides

Bridiau et al., 2006 Kim et al., 2010 Wojciechowska et al., Wojciechowska et al.,

Lactobacillus bulgaricus

Lactose Lactose Lactose

Lactose Sucralose Timosaponin BII Polianthoside D Terrestrinin D Typaspidoside H1

Gal-β-1,6/3-lactose Gal-β-1,6-sucralose Gal-β-1,6-steroidal saponins

Lu et al., 2010a Lu et al., 2012a Zhang et al., 2016

Penicillium canescencs

Lactose

Octyl alcohol

Octyl galactoside

Kuptsova et al., 2001

Porcine liver

oNP-β-Gal

GalNAc-pNP GlcNAc-pNP Floxuridine

β-1,6-disaccharides

Zeng et al., 2000

5-O-β-Galactosyl-floxuridine

Zeng et al., 2010

Kluyveromyces Lactis

oNP-β-Gal

L-serine

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Journal Pre-proof Porcine testes

Lactose

GalNAc-pNP

β-1,3/6-disaccharides

Murata et al., 1996

Pseudoalteromonas sp.

Lactose

C3-C6 alcohols

β-Galactosides

Makowski et al., 2009

Thermoanaerobacter sp.

pNP-β-Gal

Inositols, Pinitol

β-Galactosides

Hart et al., 2004

Table 2 Examples of engineered β-galactosidases Strategy

Mutation site

Result

Reference

Agrobacterium s p. (Abg)

Nucl eophile mutagenesis Sa turation mutagenesis Ra ndom mutagenesis

E358A E358S/E358G 2F6 (A19T, E358G, Q248R, M407V)

Gl ycosynthase with i mproved effi ciency i n glycoside synthesis (nea r 100% yi eld) and expanded a cceptor repertoire

Ma ckenzie et al., 1998 Tol borg et al., 2002 Ki m el al., 2004

Si te-saturation muta genesis

Q24S, W404L

Expa nd donor substrate s pecificity to use 3-MeOGal-pNP

Shi m et al., 2012

Aci d-base mutagenesis Sa turation mutagenesis

E170A E170G/E170Q

Thi oglycoligases that synthesize thi ogalactosides i n high yi elds

Ja hn et al., 2003 Mül l egger et al., 2005

Aspergillus candidus

Si te-saturation muta genesis

Y364F

Reduce galactose inhibition by 15.7-fol d; Ki = 282mM

Zha ng et a l., 2018

Aspergillus niger DSM 22593

Mul ti ple site-saturation muta genesis

D258-F-Y-T-S-S-F264

Reduce galactose inhibition by ~7 fol d; Ki = 6.46 mM

Hu et a l ., 2010

Aspergillus oryzae

Si te-directed muta genesis

N140C, W806F

Improve GOS yi eld from 35.7% to 59.8%

Ga o et a l., 2019

Bacillus circulans (Bga C)

Nucl eophile mutagenesis

E233G

Gl ycosynthase that efficiently gl ycosylates N-acetylglucosamine

Henze et al., 2014

Bacillus circulans ATCC 31382 (Bga D-D)

Si te mutagenesis

K166P, G307P, A833P

Increase thermostability

Is hikawa et al., 2015

Occupa tion of s ubstrate s ubsite

Sel ectively s ynthesize di- and tri s accharides

Ta na ka et a l., 2015

R484S/R484H

Preferably form β-1,3/4 l inkages

Yi n et a l., 2017a

W593Y/W593F

Preferably form β-1,2/3 l inkages

Yi n et a l., 2017b

F616L

Preferably form β-1,2/3 l inkages

D481N C-termi nal 580 a mi no-acid deletion

Preferably form β-1,3/6 l inkages Enha nced transgalactosylation a cti vi ty

Si te-directed muta genesis

F349S

Reduce galactose inhibition by 12-fol d; Ki=160 mM

Ki m et a l., 2011

Nucl eophile mutagenesis

E537S

Gl ycosynthase

Ja keman et a l., 2002

Si te-saturation Muta genesis DNA s huffling

H540V, N604T

180-fol d increase i n kcat /Km towa rd pNP-Fuc 1,000-fol d increased s ubstrate s pecificity for oNP-Fuc versus oNP-Ga l

Pa ri kh et a l., 2005

Geobacillus stearothermophilus

Si te-directed muta genesis

R109V/R109W

Increased synthesis of from 2% to a bove 20%

Pl a ci er et a l., 2009

Halothermothrix orenii,

Si te-directed muta genesis

Y296F/F417S/F417Y

Improve GOS yi elds by more than 10%

Ha s san et a l., 2016

Kluyveromyces lactis (Kl -β-Gal)

Si te-directed muta genesis

R116C, T270C, G818C

6.8 fol d increased half-lives at 45°C

Ri co-Díaz et al., 2017

Lactobacillus bulgaricus L3

Si te saturation muta genesis

W980F

Effi ciently gl ycosylate phenolic hydroxyl groups

Lu et a l ., 2015a , 2015b

Pyrococcus furiosus (Cel B) and Sulfolobus solfataricus (La cS)

DNA fa mily s huffling

Chi meras bearing a LacS N-termi nus a nd a main

1.5~3.5-fol d increased l actose hydrol ysis ra tes

Ka per et al., 2002

Si te saturation muta genesis

Caldicellulosiruptor saccharolyticus DSM 8903 Escherichia coli (La cZ)

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Bifidobacterium bifidum DSM20215

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Si te saturation muta genesis

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Monobody modification

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Enzyme source

V9I, Q135R, P511S, Q573R, D604S, D908N

Jorgensen et al., 2001

Zha ng et a l., 1997

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Journal Pre-proof Cel B s equence Pyrococcus woesei

DNA s huffling

T29A, V213I, L217M, N277H, I387V, R491C, N496D

7.5-ti me higher a ctivi ty toward pNP-β-D -gl ucuronide than the wi l d-type enzyme a t 25°C

Xi ong et al., 2007

Sulfolobus solfataricus Thermus thermophilus

Si te-directed muta genesis Nucl eophile mutagenesis

F441Y

Improve GOS yi eld from 50.9% to 61.7% Gl ycosynthases that efficiently s ynthesize β-1,3-glycosides

Wu et a l ., 2013

Xanthomonas manihotis (Bga X)

Aci d-base mutagenesis

E184A

Thi oglycoligases that efficiently s ynthesize thiogalactosides

Ki m et a l., 2006

Drone et al., 2005

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E338A/E338G/E338S

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Journal Pre-proof

Highlights 

β-Galactosidases from various families follow general retaining mechanism but differ in structure and substrate specificity



β-Galactosidases have galacto-oligosaccharides



β-Galactosidases exhibit powerful transglycosylation ability to produce important glycans and glycosides



β-Galactosidases engineering results in superior synthetic properties, like high efficiency, broadened substrate range, and tailor-made product structures

applied

in

industrial

production

of

prebiotic

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