Comprehensive study on transglycosylation of CGTase from various sources.

Transglycosylation is the in-vivo or in-vitro process of transferring glycosyl groups from a donor to an acceptor, which is usually performed by enzymatic reactions because of their simplicity, low steric hindrance, high region-specificity, low production cost, and mild processing conditions. One of the enzymes commonly used in the transglycosylation reaction is cyclodextrin glucanotransferase (CGTase). The transglycosylated products, catalyzed by CGTase, are widely used in food additives, supplements, and personal care and cosmetic products. This is due to improvements in the solubility, stability, bioactivity and length of the synthesized products. This paper's focus is on the importance of enzymes used in the transglycosylation reaction, their characteristics and mechanism of action, sources and production yield, and donor and acceptor specificities. Moreover, the influence of intrinsic and extrinsic factors on the enzymatic reaction, catalysis of glycosidic linkages, and advantages of CGTase transglycosylation reactions are discussed in detail.

Introduction

Cyclodextrin glucanotransferase also known as cyclodextrin glucosyltransferase (CGTase) is a biological catalyst that is categorized under glycoside hydrolase family 13 or α-amylase family with enzyme commission number (EC numbers) of EC 2.4.1.19 [51,63], classified under transferase (EC 2), a subclass of transglycosylase (EC 2.4) and sub-subclass of hexosyltransferase (EC 2.4.1).

In previous studies, researchers usually focused on the cyclization reaction of CGTase and its product, cyclodextrin, and its function. However, coupling and disproportionation reactions catalyzed by CGTase also play a vital role in enzymatic synthesis of compounds that are widely used in food additives, dietary supplement, detergents, and medical and cosmetic products. The coupling reaction of CGTase opens the cyclodextrin ring and transfers glycosyl groups to acceptors, while the disproportionation reaction by CGTase shifts the linear oligosaccharides to another oligosaccharides. This transition of the glycosyl group improves water solubility [85], bioavailability [94], absorption [169], and bioactivity [116] of acceptors. This paper summarizes the information on the compounds that have been transglycosylated by CGTase in the past 30 years and highlights the advantages of enzymatic transglycosylation using coupling and disproportionation reactions catalyzed by this enzyme.

Importance of enzymatic transglycosylation

Transglycosylation reaction is a process of transferring glycosyl group from a donor, carbohydrates, to an acceptor that contains hydroxyl or other functional groups and form glycoconjugates. In vivo glycoconjugates such as glycoproteins, glycopeptides, glycolipids and glycosides are involved in cellular recognition, cellular adhesion, and detoxification process. On the other hand, in vitro transglycosylation is an important method of improving functional properties, including their hydrophilicity, chemical stability, and bioavailability, of natural compounds and products [172].

The advantages of using enzymatic transglycosylation include simple reaction steps, low steric hindrance, high regiospecificity, low production cost, mild reaction conditions and being eco-friendly, while chemical transglycosylation comprises complicated reaction steps, such as protection and deprotection processes, to achieve regioselectivity and stereoselectivity. The transglycosylation provides defined anomeric configuration in a single step, without any necessity for protecting groups [171]. Chemical transglycosylation using acid catalysts requires higher reaction temperature compared with enzymatic transglycosylation and thus requires higher energy demand. Besides, harsh chemical transglycosylation decomposes an aglycone, produce lower yields and unwanted anomers, containing a mixture of α- and β-anomers, which causes purification process to be difficult and complicated. Furthermore, the catalysts used in chemical transglycosylation are heavy metals, which have negative effects such as toxicity and higher production cost [76]. The purification complication is mainly due to the interruptions by other water-soluble compounds that are extremely difficult to separate from the desired glycoside compounds. Therefore, the enzymatic transglycosylation is the most preferred method to synthesis glycoside [18].

Type of enzymes used in enzymatic transglycosylation

Enzymatic transglycosylation can be catalyzed by glycosyltransferases (EC 2.4) and glycoside hydrolases also known as glycosidases (EC 3.2.1). Glycosyltransferases catalyze the transglycosylation reaction, which transfers sugar moieties or glycosyl groups from donor to acceptor, and form glycosidic bonds between sugar moieties and acceptor [78]. There are two types of glycosyltransferase, namely Leloir glycosyltransferase and non-Leloir glycosyltransferase [178].

The Leloir glycosyltransferases use sugar nucleotides as activated donors when catalyze the transglycosylation reaction. For example, UDP-glycosyltransferase is a Leloir glycosyltransferase that catalyzes transglycosylation reaction by transferring glycosyl groups from activated donors, such as uridine diphospho-D-glucose, to acceptors [25]. On the other hand, non-Leloir glycosyltransferases use either sugar phosphates or non-activated sugars as donors when catalyze transglycosylation reaction. Glycoside phosphorylase is an example of non-Leloir glycosyltransferase that uses sugar phosphates as donor, while CGTase is a non-Leloir glycosyltransferase that uses non-activated sugars, such as starch, as donors [119, 129]. Enzymatic characteristic of glycoside hydrolases (glycosidases) is their ability to hydrolyze glycosidic bonds but some glycoside hydrolases can also synthesis glycoside under certain reaction conditions. Example of glycoside hydrolases are α-amylase, β-galactosidase and α-rhamnosidase [29, 132, 179].

Importance of CGTase for enzymatic transglycosylation

CGTase is a non-Leloir glycosyltransferase. CGTase uses non-activated sugar, which has free cleavage energy of maltooligosaccharides, as donors [123]. Normally, Leloir glycosyltransferases are rare enzymes that are more costly, more vulnerable to environmental conditions, require exclusive donors, and often require special buffers or detergents for solubilization. In contrast, non-Leloir glycosyltransferase, such as CGTase, are commonly available enzymes, which are less expensive, require commercially available donors, and can be used with a wider range of acceptors. Moreover, CGTase extracted from microorganisms with rapid growth rate, is preferred as it is readily available [146].

Although glycosidases have similar properties as CGTase but they tend to partially hydrolyze glycoside compounds and produce undistinguishable or low yields of glycosylated products [178]. Others disadvantages of glycosidases are poor regioselectivity and production of undesired compounds in various proportions, when there are more available –OH groups [76]. On the other hand, CGTase has low hydrolytic activity, good conversion yield to convert compounds into glycosylated product, effective transglycosylation of certain acceptors, and excellent regioselectivity, which particularly catalyze α (1→4)-glycosyl transfer reactions [99, 149]. For instance, synthesizing the stevioside, a sweetener, was best achieved by using CGTase from Bacillus macerans when compared with other enzymes such as pullanase, dextrin dextranase, β-galactosidase, and isomaltase [3].

Another example would be conversion of benzo[h]quinazolines to transglycosylated benzo[h]quinazolines that could be catalyzed efficiently by CGTase while other enzymes such as β-fructofuranosidase, β-amylase, pullulanase and β-galactosidase could not yield any transglycosylated benzo[h]quinazolines [95]. Furthermore, the CGTase extracted from Bacillus circulans 251 could couple the artificial rhamnoside acceptors with maltodextrin donors to synthesize α-D-glucosylated products [161]. In addition, CGTase can tolerate high percentage of organic solvent in a reaction mixture [173]. Therefore, CGTase is considered as a useful biocatalyst for enzymatic transglycosylation for its board acceptor specificity, availability of low cost donors, and effectiveness in transglycosylation.

Characteristics of CGTase

CGTase is a monomer with different number of amino acids and molecular weight depending on its source (Table 1). Molecular weights in the range of 33–110 kDa have been reported for CGTase from various organisms, such as Pyrococcus furiosus DSM3638 (65kDa) [80], Thermoactinomyces vulgaris Tac-5354 (66 kDa) [4], Thermoanaerobacterium thermosulfurigenes EM1 (68 kDa) [175], Paenibacillus pabuli US132 (70kDa) [57], Haloferax mediterranei (77 kDa) [13], Bacillus agaradhaerens WN-I (85 kDa) [52], Amphibacillus sp. NPST-10 (92 kDa) [53], and Bacillus agaradhaerens LC-3C (110 kDa) [98]. However, there are contrasting views on the molecular weight of CGTase extracted from alkaliphilic strains [2, 4, 126]. Moreover, the number of amino acids in the CGTase produced by various microorganisms, including Haloferax mediterranei (713 amino acids) [13], Anaerobranca gottschalkii and Bacillus agaradhaerens (679 amino acids) [98, 156], Thermoanaerobacterium thermosulfurigenes EM1 (567 amino acids) [176], Bacillus stearothermophilus ET1 (711 amino acids) [24], Bacillus sp. Strain 251 (686 amino acids) [122] and Bacillus sp. Strain 1011 (713 amino acids) [67], has been identified. Therefore, the properties of CGTase depend on the microorganism that they are extracted from [15].

Table 1

CGTase producing microorganisms.

Genus	Organisms	Yield (%)	Molecular mass (kDa)	Ref
Bacteria
Bacillus	Bacillus megaterium strain 5	-	-	[69]
	Bacillus sp. ATCC 21783	35	88	[112]
	Bacillus sp. HA 3-2-2	7	68	[117]
	Bacillus stearothermophilus TC-91	15.3	75.5	[77]
	Bacillus coagulans	37.8	65	[7]
	Bacillus autolyticus 11149	31.1	70	[157]
	Bacillus strain A2-5a	51	80	[74]
	Bacillus sp. PS304	58	76	[124]
	Bacillus stearothermophilus ET1	31.6	66.8	[24]
	Bacillus firmus NCIM 5119	64	78	[37]
	Bacillus macerans strain 15	66.3	54	[2]
	Bacillus agaradhaerens LS-3C	50	110	[98]
	Bacillus sp. G1	4.18	75	[144]
	Bacillus sp. 7-12	18.9	69	[19]
	Bacillus circulans ATCC 21783	75.5	97.4	[167]
	Bacillus firmus strain 7B	91.6	56.23	[106]
	Bacillus sp. BL-31	21.7	92	[38]
	Bacillus cereus N1	3. 63	75	[34]
	Bacillus sphaericus strain 41	31	59	[107]
	Bacillus pseudalcaliphilus 20RF	63	70	[11]
	Bacillus agaradhaerens strain WN-I	26.4	85	[52]
	Bacillus pseudalcaliphilus 8SB	62	71	[70]
	Bacillus halodurans	49.44	33	[104]
	B. halophilus BIO-12HB. halophilus BIO-13H	-	7070	[4]
Paenibacillus (anaerobic thermophilic)	Paenibacillus campinasensis strain 324	26.6	75	[180]
	Paenibacillus sp. KJ-12	5	82	[60]
	Paenibacillus illinoisensis ST-12 K	27	70	[31]
	Paenibacillus campinasensis H69-3	13.3	70	[9]
	Paenibacillus pabuli US132	20	70	[57]
	Paenibacillus sp. RB01	38	-	[22]
	Paenibacillus illinoisensis ZY-08	26.6	70	[82]
Klebsiella (anaerobic thermophilic)	Klebsiella pneumoniae AS-22	68	75	[37]
Thermoanaerobacter (anaerobic thermophilic)	Thermoanaerobacterium thermosulfurigenes EM1	79	68	[175]
	Thermoanaerobacter sp. P4	17.8	68.7	[12]
Anaerobranca (anaerobic thermo alkaliphilic)	Anaerobranca gottschalkii	13.5	78	[156]
Amphibacillus (anaerobic alkaliphilic)	Amphibacillus sp NPST-10	44.7	92	[53]
Brevibacterium (aerobic mesophilic)	Brevibacterium sp. No. 9605	16	75	[105]
Brevibacillus (aerobic mesophilic)	Brevibacillus brevis CD162	-	75	[64]
Microbacterium (alkaliphilic)	Microbacterium terrae KNR 9	33	27.72	[126]
Thermoactinomyces (thermophilic aerobic)	Thermoactinomyces vulgaris Tac-5354	-	66	[4]
Archaea
Thermococcus (thermophilic anaerobic)	Thermococcus sp. strain B1001	16	83	[153]
Pyrococcus	Pyrococcus kodakaraensis	-	79	[128]
	Pyrococcus furiosus DSM3638	-	65	[80]
Haloferax (halophilic archeon)	Haloferax mediterranei	3	77	[13]
Fungi
Aspergillus	Aspergillus niger CCRC 31494	-	-	[81]
Trichoderma	Trichoderma viride	-	-	[115]

The structure of CGTase extracted from Bacillus circulans strains 8, Bacillus circulans strains 251, alkalophilic Bacillus sp. 1011, and Thermoanaerobacterium thermosulfurigenes strain EM1 has been determined by X-ray structure analysis [49, 72, 73, 79]. Generally, the structure of CGTase consists of five protein domains (A to E) and its active site is situated in the A domain [79, 83]. Domain A is the catalytic (α/β)-8 domain and catalytic residues are situated at the C-terminal ends of the β-strands. Domain B contributes to substrate binding. Substrates bind with a groove formed by domains A and B containing ten sugar-binding sub sites labeled −7 to +3 [148]. Sugar binding sub sites are labeled from -n to + n whereby the ‘n’ is an integer. The -n sugar binding sub site constitutes the non-reducing end while + n constitutes the reducing end [27]. According to Leemhuis et al. [83], sugar-binding sub sites labeled from -7 to -1 are donor, and from 1 to 3 are acceptor sub sites. The domain C shows an antiparallel β-sandwich fold and its function is to bind substrate [122] while domain E used to bind starch and maltose [20]. However, the function of domain D is still unexplored [83]. CGTase from different microorganisms has different numbers of residues in 5 domains (A-E). CGTase from Bacillus circulans strains 8 has 80–108 residues in domain A, 185–192 residues in domain B, 407–494 residues in domain C, 495–580 residues in domain D and 581–684 residues in domain E [72]. CGTase from alkalophilic Bacillus sp. 1011.14 has 1–406 residues in domain A, 139–203 residues in domain B, 407–496 residues in domain C, 497–584 residues in domain D and 585–686 residues in domain E [49]. CGTase from Bacillus circulans strains 251 has 261–268 residues in domain A, 183–186 residues in domain B and 599–602 residues in domain E. However, the number of residues in domain C and D of CGTase from Bacillus circulans strain 251 have not been identified [79]. CGTase from Thermoanaerobacterium thermosulfurigenes strain EM1 -88-95 residues at domain A, 335–339 residues in domain B, 495–499 residues in domain C, 534–540 residues in domain D and 654–657 residues in domain E [73].

Catalytic mechanism of CGTase

Catalytic reactions of the CGTase are based on the α-retaining, double displacement, and random ternary complex mechanisms [165, 174]. The α-retaining mechanism maintains the substrate's α-anomeric configuration during transglycosylation [164]; however, the α-retaining mechanism of glycosyltransferase enzymes is not clearly understood [178]. Generally, catalytic carboxylate residues of the CGTase are Asp 229, Asp 328 and Glu 257, the same as other members of α-amylase family [72]. The function of three carboxylic amino acids were explained when an inhibitor, such as Acarbose, bound at active site of the CGTase. Glu257 catalyzes proton donor, Asp229 acts as the nucleophile, stabilizing the intermediate, while Asp328 acts as substrate binding [71, 110, 111].

Double displacement, also known as Ping-Pong mechanism, initiates by SN2 nucleophilic reactions. The carboxylate residues of the CGTase (Glu or Asp) interact with donors and form β-covalently linked enzyme-glycosyl intermediates. Then, another carboxylate residue activates the glycosyl acceptors to attack the enzyme-glycosyl intermediates, which results in reversing the β-covalent bonds and forming α-linkage products with retained α-anomeric configuration [135, 155, 174]. In contrast, the random ternary complex mechanism involves simultaneous and random binding of glycosyl donors and acceptors to active sites of the CGTase. The glycosyl donor covalently bonds with acceptor to form a product and subsequently the product is released.

The CGTase has four enzymatic reactions, namely cyclization, coupling, disproportionation and hydrolysis [164]. Figure 1 shows the four enzymatic reactions of the CGTase. The cyclization activity of CGTase is an intramolecular transglycosylation reaction, where linear and long maltooligosaccharides chains, such as starch, are broken down and form oligosaccharide cyclic ring structures, called cyclodextrin. Cyclodextrins can be divided into α, β and Ƴ types. The coupling and disproportionation activities of CGTase are intermolecular transglycosylation. The coupling activity is a reverse reaction to cyclization, which is opening the cyclic oligosaccharides (cyclodextrin) and converting them to linear oligosaccharides before transferring linearized oligosaccharides to an acceptor through random ternary complex mechanism [165]. Moreover, disproportionation activity, the main transferase reaction of CGTase, is the process of cleaving linear maltooligosaccharides and then transferring glycosyl groups to an acceptor molecule via double displacement mechanism [165]. Furthermore, CGTase also has a weak hydrolysis reaction.

Figure 1

Schematic representation of four enzymatic reactions by CGTase. A) Cyclization B) Coupling C) Disproportionation D) Hydrolysis. glucose residue reducing end glucose.

Generally, CGTase cleaves α (1–4) glycosidic bond of donor between sub sites -1 and +1 and yields a stable covalent glycosyl-intermediate complex covalently linked to catalytic residues, Asp 229, at sub site -1. The covalent glycosyl-intermediate complex which binds at the sub sites (sugar-binding sub sites labeled from -7 to -1) is called donor. The leaving group is expelled from acceptor sites (sugar-binding sub sites labeled 1 to 3) before being replaced by acceptor molecule. Finally, a new α (1–4) glycosidic bond is formed between the donor and acceptor to form a product and subsequently the product is being released [99, 160]. The following sections of this paper focus on the coupling and disproportionation activities of CGTase, which transfer glucosyl groups to the acceptors.

Synthesis of glycosidic linkage catalyzed by CGTase

Generally, CGTase has been used to catalyze α (1→4) glycosyl transfer reactions. However, production of other glycosidic linkages such as α (1→3) and α (1→6) glycosidic linkage are also reported. For example, α (1→3) glycosidic linkage is produced by transglycosylation of rhamnopyranoside using CGTase [161]. This is because the formation of a glycosidic linkage can be influenced by time and anomeric configuration of the acceptor [149]. The glycosidic linkages produced by the CGTase have been investigated using α and β anomers of p-nitrophenyl-D-glucopyranoside as acceptors. The α anomers of p-nitrophenyl-D-glucopyranoside only form α (1→4)-glycosyl linkages with their donors, while α (1→4), α (1→3) and α (1→6) glycosylation products are produced when β anomers of p-nitrophenyl-D-glucopyranoside used as acceptors. Moreover, during longer reaction times, α (1→4)-glycosylation products slowly change to α (1→3) and α (1→6) glycosylation product, by using β anomers of p-nitrophenyl-D-glucopyranoside as acceptors.

Sources and production yield of CGTase

Most CGTase enzymes are produced extracellularly by various genera of bacteria. Bacillus [11, 69], Paenibacillus [9], Klebsiella [38], Brevibacterium [105], Brevibacillus [64], Thermoanaerobacter [175], Thermococcus [153], Thermoactinomyces [4], Haloferax [13], Pyrococcus [80], Anaerobranca [156], Microbacterium [126] and Amphibacillus [55] are some of the bacteria that produce CGTase. The archaea and fungi can also produce CGTase. The genera of archaea that are commonly studied for production of CGTase are Thermococcus [128, 153], Haloferax [13] and Pyrococcus [80]. Aspergillus [81] and Trihoderma viride [115] are the fungi that are used to produce CGTase. However, the CGTase is mostly produced by strains of Bacillus, such as B. circulans [163], B. macerans [2], B. firmus [36], B. cereus [54], and B. pseudalcaliphilus [11].

Other known CGTase producing bacteria include Paenibacillus illinoisensis [81], Klebsiella pneumoniae [37], Thermoanaerobacter sp. P4 [12], Amphibacillus sp NPST-10 [53], Brevibacterium sp. 9605 [105], Brevibacillus brevis [64], Microbacterium terrae KNR 9 [127] and Thermoactinomyces vulgaris Tac-5354 [4]. The CGTase producing microbes are mainly found in soil, lake water, hot springs, wastewater from flour industry and rotten potatoes [22, 28, 53]. Table 1 shows a summary of the CGTase producing microorganism.

Donor specificity

Generally, since CGTase is a common starch-degrading enzyme, donors of CGTase are carbohydrate sources. Common carbohydrates used by CGTase are dextrin, maltodextrin, maltose, maltoheptaose, maltotriose, starch and cyclodextrin, as summarized in Table 2. The usage of cyclodexrins as donors has the advantage of enhancing the solubility of acceptor through complex inclusion [179]. However, the substrate may become inert to enzymatic catalysis and cyclodexrins are more costly compared with other donors.

Table 2

Factors influencing the CGTase enzymatic reaction and its conversion yield.

Acceptor	Donor	Strain/Type of CGTase	Product	pH	Temp (˚C)	Yield (%)	Ref.
Sugar
lactose (4-O-β-D-galactosyl-D-glucose)	Soluble Starch	Bacillus stearothermophilus (CGTase)	α-D-glucosyl O-β-D-galactosyl-(1–4)-β-D-glucoside	5.6	50 °C	64	[68]
1,5-anhydro-D-fructose	β-CD	Bacillus stearothermophilus (CGTase)	Glucosyl 1,5-anhydro-D-fructose (1,5-anhydro-3-O-α-glucopyranosyl-D-fructose)	5.0	35 °C	-	[183]
Acarbose	α-CD	Bacillus macerans (CGTase)	D-glucopyranosyl-acarbose	6.0	37 °C	-	[181]
Sucrose	β-CD	Thermoanaerobacter sp. (CGTase)	Polyglucosyl-fructosides	5.5–6.5	60 °C	-	[102]
Sucrose	Soluble Starch	Thermoanaerobacter sp. (CGTase)	Maltooligosyl fructofuranosides	5.5	60 °C	≥80	[96]
Sugar glycosides
i-menthyl α-D-glucopyranoside	Starch	Bacillus macerans (CGTase)	i-Menthyl α-D-glucopyranosyl-(1→4)-α-D-glucopyranoside	6.0	70 °C	74.2	[30]
Sugar alcohols
Myo-inositol	β-CD	Bacillus ohbensis (CGTase)	Oligoglucosyl-inositols	6.0	50 °C	58.8	[134]
Inositol	β-CD	Thermoanaerobacter sp. (CGTase)	Monoglucated product of L-chiro-, D-chiro-, muco-, and allo-inositol.	6.0	50 °C	-	[101]
Sugar Fatty Acid Esters
Sucrose monolauroyl esters	Dextrin	CGTase	Additional 1–3 glucose residues on the pyranose ring of sucrose moiety in the ester	6.5	50 °C	58.8	[120]
Polyols
Pentaerythritol	Starch	Bacillus stearothermophilus (CGTase)	Glucosyi pentaerythritol	5.5	40 °C	-	[114]
Trimethylolethane			Glucosyl trimethylolethane			-
Trimethylolpropane			Glucosyl trimethylolpropane			-
Glycerol	Soluble Starch	Thermoanaerobacter sp. (CGTase)	Glycosyl Glycerol-O-α-D-glucosyl-(1-1)-glycerol-O-α-D-glucosyl-(1–2)-glycerol-O-α-D-Glucosyl-(1–4)-O-α-D-glucosyl-(1-1)-glycerol	6.0	80 °C	-	[113]
		G.stearothermophilus (CGTase)			60 °C
Phenolic compound
(+)catechin(flavan-3-ol)	Soluble Starch	Bacillus macerans (CGTase)	(+)catechin3′-O-α-D-glucopyranoside	6.5	40 °C	18.3	[34]
Hesperidin (Flavan-on glycoside)	Soluble Starch	Alkalophilic Bacillus sp. (CGTase)	Hesperidin monoglucoside	9.0	40 °C	75	[75]
Hesperetin 3′- O-α-glucosidesHesperetin 5- O-α-glucosidesHesperetin 7-O-α-glucosides	Starch	CGTase	Hesperetin 3′-O-α-maltosideHesperetin 5-O-α-maltosideHesperetin 7-O-α-maltoside	7.0	40 °C	496250	[139]
Hesperidin	Soluble Starch	Thermoanaerobacter sp. (CGTase)	Hesperetin 7-O-α-D-glucopyranoside	5.0	60 °C	4.1	[40]
4-hydroxyphenyl α-glucopyranoside (α-arbutin)	Soluble Starch	Bacillus macerans (CGTase)	Arbutin-α-glycosides-4-hydroxyphenyl β-maltoside (β-Ab-α-G1)-4-hydroxyphenyl β-maltotrioside (β-Ab-α-G2)	7.0	40 °C	70	[149]
Genistin	Soluble Starch	alkalophilic Bacillus sp. I-5 (CGTase)	Glucosyl-α(1–4)-genistinMaltosyl-α(1–4)-genistin	6.0	60 °C	-	[87]
Genistin	α-CD	Paenibacillus macerans (CGTase)	Genistin diglucoside	6.5	30 °C		[42]
Quercetin 7-O-β-D-glucoside	Starch	CGTase	Quercetin 7-O-β-maltoside	7.0	40 °C	36	[141]
			Quercetin 7-O-β-maltotrioside			10
Quercetin 3-O-β-D-glucoside			Quercetin 3-O-β-maltoside			40
			Quercetin 3-O-β-maltotrioside			14
Genistein 4-O-β-D-glucoside			Genistein 4-O-β-maltoside			32
			Genistein 4-O-β-maltotrioside			12
Genistein 7-O-β-D-glucoside			Genistein 7-O-β-maltoside			30
			Genistein 7-O-β-maltotrioside			7
Naringin	Malto dextrin	alkalophilic Bacillus sp. BL-31 (CGTase)	Monoglycosyl naringin	8.5	40 °C	92.1	[39]
α-tocopheryl β-glucoside	Starch	CGTase	α-tocopheryl β-maltoside	7.0	40 °C	51	[137]
			α-tocopheryl β-maltotrioside			35
δ-tocopheryl β-glucoside			δ-tocopheryl β-maltoside			45
			δ-tocopheryl β-maltotrioside			29
Hesperetin α-glucoside (Vitamin P)	Starch	Bacillus macerans (CGTase)	Hesperetin 3′-O-α-maltoside	7.0	40 °C	49	[92]
			Hesperetin 5-O-α-maltoside			62
			Hesperetin 7-O-α-maltoside			50
Curcumin 4′-O-β-D-glucopyranoside	Starch	Bacillus macerans (CGTase)	Curcumin 4′-O-β-glucooligosaccharidesCurcumin 4′-O-β-maltosideCurcumin 4′-O-β-maltotrioside	7.0	40 °C	5121	[139]
Glycitein	Soluble Starch	Bacillus macerans (CGTase)	Glycitein 4′-O-β-D-maltoside	7.0	40 °C	45	[140]
			Glycitein 4′-O-β-maltotrioside			39
			Glycitein 7-O-β-maltotrioside			33
Daidzein	Soluble Starch		Daidzein 4′-O-β-maltoside			46
			Daidzein 4′-O-β-maltotrioside			35
			Daidzein 7-O-β-maltoside			39
			Daidzein 7-O-β-maltotrioside			30
Resveratrol	Starch	Thermoanaerobacter sp.(CGTase)	α-glucosyl derivatives of resveratrol (3,5,4′-trihydroxy stilbene)	5.6	60 °C	50	[159]
Epicatechin	Soluble Starch	Paenibacillus sp. RB01 (CGTase)	Epicatechin glucosides	6.0	40 °C	22.6	[10]
	β-CD					22.4
	Malto heptaose					21.4
Piceid (stilbenoid glucoside)	Maltose	Bacillus macerans (CGTase)	Piceid glycosides	6.0	40 °C	17.8	[99]
	Malto triose					56.4
	Malto dextrin					72.1
	α-CD					78.9
Pterostilbene (trans-3,5-dimethoxy-4′-hydroxystilbene)	Partial hydrolysed starch	Thermoanaerobacter sp.(CGTase)	Pterostilbene α-Glucoside	-	60 °C	-	[39]
Hydroquinone	α-CD	Thermoanaerobacter sp.(CGTase)	Hydroquinone-1-O-α-D-glucopyranoside (α-arbutin)	5.5	40 °C	31.8	[100]
	Malto dextrin					29.2
Kaempferol (flavonol)	Ƴ-CD	CGTase (Toruzyme)	Kaempferol-3-O-β-D-glucopyranosyl-(1→4)-O-α-D- glucopyranoside	6.0	60 °C	8.6	[23]
Capsaicinoids
Capsaicin	Starch	CGTase	Capsaicin β-maltoside	7.0	40 °C	41	[143]
			Capsaicin β-maltotrioside			35
Saponins
α-glucosyl ginsenoside, Rb1	Dextrin	Bacillus macerans (CGTase)	α-maltosyl ginsenoside	6.0	50 °C	10.3	[66]
α-glucosyl ginsenoside, Rc			α-maltosyl ginsenoside			10.5
α-glucosyl ginsenoside, Re			α-maltosyl ginsenoside			16.9
α-glucosyl ginsenoside, Rg1			α-maltosyl ginsenoside			18.6
Ginsenoside F1	Dextrin	CGTase	α-Glucosyl Ginsenoside F1	7.0	50 °C	-	[105]
Vitamins
L-ascorbic acid (Vitamin C)	α-CD	Bacillus stearothermophilus (CGTase)	2-O-α-D-Glucopyranosyl-L-ascorbic acid	5.5	60 °C	97	[5]
D-Erythorbic acid	Ƴ-CD	Thermoanaerobacter sp. (CGTase)	2-O-α-D-glucopyranosyl-d-erythorbic acid	4.0	40 °C	49.1	[154]
L-ascorbic acid (Vitamin C)	α-CD	Thermoanaerobacter sp. (CGTase)	2-O-α-D-Glucopyrano syl-L-ascorbic acid	4.5	50 °C	30	[41]
L-ascorbic acid (Vitamin C)	α-maltosyl fluoride (aG2F)	engineeredcyclodextrin glucanotransferase	3-O-α-maltosyl-L-ascorbate	7.5	25 °C	29	[6]
Diterpenoid
Rubusoside	Soluble starch	Bacillus circulans (CGTase)	Tri and tetra-glucosylated rubusoside	5.4	40 °C	-	[119]
Stevioside	Malto dextrin	Alkalophilic Bacillus sp. BL-12 (CGTase)	mono-, di-, and oligo-glycosyl stevioside	9.0	40 °C	76	[62]
Taxol (paclitaxel)	Soluble starch	Bacillus macerans (CGTase)	7-Glycolylpaclitaxel 2″-O-α--glucobioside	7.0	40 °C	17	[139]
			7-glycolylpaclitaxel 2′-O-α-glucotrioside			14
			7-glycolylpaclitaxel 2′-O-α-glucotetraoside			11
			7-glycolylpaclitaxel 2′-O-α-glucopentaoside			7
Stevioside	Corn starch	Paneibacillus macerans JFB05-01 (CGTase)	Monoglucosylated stevioside	7.0	60 °C	77.11	[89]
			Diglucosylated stevioside			76
Stevioside	Starch	Paenibacillus sp.CGMCC 5316 (CGTase)	mono-glycosylated stevioside	7.0	37 °C	85.6	[184]
Benzo[h]quinazolines	Ƴ-CD	Bacillus stearothermophilusST-88 (CGTase)	3-Ethyl-2-carboxy methylsulfanyl-4-oxo-3,4,5,6 tetrahydrospyro (benzo[h]quinazoline-5,1′-cyclohexane)	5.5	60 °C	19.8	[95]
			4-Carboxy-6-oxo-3,4,5,6,7,8-hexahydrospyro (thiazolidine-[2.3-b]-benzo[h]quinazoline-7,1′-cyclohexane)			18.7
Triterpene glycoside
Mogroside V	Tapioca starch	Bacillus macerans (CGTase)	Monoglycoside mogroside VDiglycoside mogroside VTriglycoside mogroside V	6.5	50 °C	≥90	[182]
Mogroside IIE	Starch	Bacillus macerans (CGTase)	Diglycoside mogroside IIE	6.0	55 °C	≥80	[170]
Alkyl glucoside
Dodecyl β-maltoside	α-CD	Bacillus macerans (CGTase)	Dodecyl β-D-maltooctaoside	5.2	60 °C	50	[152]
Dodecyl-β-D-maltoside	α-CD	Bacillus macerans (CGTase)	Dodecyl-β-D-maltooctaoside	5.2	60 °C	80	[151]
Dodecyl β-D-glucoside	α-CD	Bacillus macerans (CGTase)	Dodecyl glucooligosides	6.0	70 °C	78	[99]
Dodecyl β-D-maltoside						93
Tert-butyl alcohol	Starch	Thermoanaerobacter sp. (CGTase)	tert-butyl-α-D-glucosidetert-butyl-α-D-maltoside	6.0	60 °C	44	[35]
Non-natural
Methyl or allyl α-L-rhamno-pyranoside	Malto dextrin	Bacillus circulans 251 (CGTase)	Cis-Glucosylated L-Rhamnosides	6.0	50 °C	70	[161]

Nβ-CD = β-cyclodextrin, α-CD = α-cyclodextrin, Ƴ-CD = Ƴ-cyclodextrin.

Several cheaper carbohydrate sources have been studied for their application as donors, such as maltodextrin, maltose, maltotriose, sucrose and glucose. Although the effectiveness of donor substrates used for glycoside synthesis are in decreasing order of α-cyclodextrin > maltodextrin > maltotriose > maltose, but conversion yield of transglycosylated products can be improved by increasing the donor concentrations [100]. Thus, it has been found that, if used in high concentrations, maltodextrin can be used as an efficient alternative to α-cyclodextrin [100].

According to Go et al. [38], starch and maltodextrin can act as a better donor than low molecular weight carbohydrates, such as cyclodextrin, maltotriose, maltose, and dextrin. Moreover, donor substrates for CGTase should have at least two glucose units. Therefore, glucose cannot be used as a donor for the CGTase [100]. Furthermore, donor's chain length and concentration will also influence the glycosides' chain length. For instance, short chain donors such as maltose would form shorter glycoside, while long chain donors, such as starch, or high concentrations of donor would result in glycosides with longer chain length [99, 100].

Acceptor specificity

Generally, good acceptors for CGTase contain available hydroxyl group(s) (-OH group) located at carbon position(s) 2, 3 and 4. There are two groups of acceptors which are sugar and non-sugar compounds. The sugar compounds acting as an acceptor for CGTase include lactose, sucrose, 1, 5-anhydro-D-fructose and methyl α-D-glucopyranoside. Meanwhile, non-sugar compounds used as acceptors for CGTase are sugar alcohols, sugar fatty acid esters, polyols, phenolic compounds, capsaicinoids, saponins, vitamins, diterpenoid, triterpene glycoside, alkyl glucoside and artificial rhamnoside. Table 2 provides a list of acceptors previously used in transglycosylation.

Effects of pH and temperature on transglycosylation reaction

The pH range of buffer is vital for enzymatic activity since extreme acidity or alkalinity will denature the enzyme. The optimum pH for CGTase isolated from alkaliphilic, mesophilic, halophilic and thermophilic bacilli is in the range of 6.0–7.5 [3]. For example, optimum pH for transglycosylation of the piceid by the CGTase from Bacillus macerans is in the rage of 5.5–7.0. The conversion yield of piceid was reduced when enzymatic reactions carried out at lower pH such as pH 4.5 and pH 5.0 [94].

The reaction temperature plays an important role in the enzymatic activity. The CGTase extracted from different microorganisms have different optimum reaction temperatures. For instance, the optimum temperature for the CGTase isolated from halophilic and mesophilic strains is in the range of 45–50 °C, while for the CGTase isolated from alkalophilic and thermophilic strains is 50–55 °C [3]. The temperature used in most studies on CGTase has been in the range of 40–60 °C [23, 129]. Table 2 provides a summary of temperatures used in transglycosylation of different compounds.

Achieving the optimum temperature can be assisted by using the microwave and ultrasound techniques. Microwave and ultrasound produce heat and can be used to maintain the optimum temperature for enzymatic reaction, using different frequencies and heat transfer mechanism [13, 92, 108, 131, 133, 166]. The ultrasound produces sound with frequencies between 2 to 15 MHz, while microwave produces electromagnetic radiation with frequencies between 1 to 1000 GHz. The ultrasound is a transmitter to produce sound, which vibrating molecules transfer energy from one location to another with back and forth motion. The energy produced by ultrasound can be absorbed by the reaction mixture and converted into heat. On the other hand, a microwave transmits thermal radiation that induces polar molecules in the reaction mixture to rotate and produce thermal energy, providing optimum temperature for enzymatic reaction [5, 118].

According to Jaitak et al. [56], transglycosylation by using microwave-assisted techniques was faster than traditional and ultrasound-assisted techniques. The reaction by microwave-assisted techniques yielded 66% 4′-O-alpha-D-glycosyl stevioside and 24% 4′-O-alpha-D-maltosyl stevioside in 1 min, while the ultrasound assisted techniques were able to yield 50% and 25% for 4′-O-alpha-D-glycosyl stevioside and 25% 4′-O-alpha-D-maltosyl stevioside, respectively, but the process required 10 min to achieve those results. In comparison, the production yield for traditional technique (without any assistance) was 46% for 4′-O-alpha-D-glycosyl stevioside and 24% for 4′-O-alpha-D-maltosyl stevioside but only after 12h.

Effects of organic reagents and metal ions on transglycosylation reaction

In order to achieve good solubility for a hydrophobic acceptor, like polyphenols, in an aqueous system, it is first dissolved in water-miscible organic solvents or co-solvents, such as dimethyl sulfoxide (DMSO), before being added into the buffer solution. The examples of suitable co-solvents include DMSO, ethanol, dioxane, sulfolane, dimethylformamide, isopropanol, n-propanol, glycerol and ethyl-lactate. The ratio of buffer to organic solvent must be adjusted in such a way that ensures it does not pose denaturing or inhibiting effects on the enzyme while ensuring a good level of solubility for the hydrophobic acceptor. DMSO mixed with buffers exhibits the best performance in terms of solubility of the hydrophobic acceptors and product's yield as compared with other co-solvents [159]. According to Blackwood and Bucke [16], the addition of small quantities of organic solvents increases the production yield of transglycosylation reaction carried out by CGTase.

Addition of metal ions also shows similar effects on the production yield of transglycosylation reaction by CGTase. This is because some metal ions such as Ca2+ can enhance the enzyme stability. Most CGTase enzymes contain two Ca2+ binding sites which located at the N-terminal end and the active site region, which influence their activity and stability [87]. Calcium also acts as a protective agent for CGTase against heat [87]. It has been shown that the β-CGTase activity can be stimulated by presence of Ca2+ which resulted in an increase, up to 123%, of its transglycosylation yield [62]. Besides, Mn2+ metal ions also exhibit positive effect on transglycosylation by CGTase. Enzyme activity of CGTase from Alkalophilic Bacillus sp. BL-31 was stimulated by presence of Mn2+ which resulted in increased yield of glycosylated naringin from 80.2% to 92.1% [38]. However, some other metal ions such as Zn2+, Cu2+ and Fe2+ are inhibitors for most CGTase [158].

Effects of concentration and source of CGTase on transglycosylation reaction

The transglycosylation activity of CGTase is dependent on the amount of CGTase in reaction mixture and enzyme source. Although, high amount of CGTase added into a reaction mixture would increase the initial reaction rate of transglycosylation significantly, but excess amount of CGTase used in catalyzing reaction would produce unwanted side reactions such as excess formation of cyclodextrins [38, 89].

Furthermore, conversion yield can be affected by the source, microorganism, of CGTase isolation [3]. In their study, Abelyan et al. [3] used CGTase extracted from mesophilic (B. macerans and B. circulans), thermophilic (B. stearothermophilus), alkaloiphilic (B. alcalophilus), and halophilic (B. halophilus) bacilli to synthesize stevioside glycoside. The effectiveness of enzymes in stevioside glycoside formation was in the decreasing order with CGTase from B. halophilus and B. alcalophilus > B. circulans > B. stearothermophilus and B. macerans. The enzyme activity of CGTase from B. halophilus and B. alcalophilus was 15–16 U/g followed by B. circulans, and lastly B. macerans and B. stearothermophilus, producing 11–11.5 U/g and 8–9 U/g respectively. According to Jaitak et al. [56], the enzyme activity of CGTase extracted from B. firmus was 2 U/g; although they used the same acceptor and stevioside as the experiment conducted by Abelyan and his colleagues [3]. Other studies have shown that CGTase from Thermoanaerobacter sp. produces higher yield of resveratrol glucoside when compared with B. macerans [159]. This might be due to the higher thermo-stable properties of CGTase isolated from Thermoanaerobacter sp. (thermophilic anaerobic bacterium) which displays a high disproportionation activity [96]. Moreover, studies on the elongation of carbohydrate groups in alkyl glycosides found that CGTase from Paenibacillus macerans yield the highest β-dodecyl maltooctaoside concentration compared with the CGTase from Paenibacillus Thermoanaerobacter sp., Carboxydocella sp. and B. stearothermophilus [129].

Strategies to improve the transglycosylation activity of CGTase

Several protein modification techniques have been used in order to improve the transglycosylation activity of CGTase, including chemical modification, immobilization and recombinant DNA techniques. For example, succinylation, a chemical modification, has been applied on CGTase from Thermoanaerobacter sp. 501 to improve its disproportionation [8]. Succinylation is the reaction of transferring a succinyl group to lysine (K) residue of protein molecule [50].

Immobilization is another method of improving the transglycosylation activity of CGTase. Advantages of enzyme immobilization include reusability of enzymes, shortening purification process as enzymes are not mixed with the final product, and simplicity of steps for enzyme immobilization on supports (insoluble matrix) [26]. In addition, immobilization can improve stability of the enzyme that useful for application in industrial scale. The covalent binding, adsorption and entrapment are different methods applied for the immobilization of CGTase. The CGTase has been covalently immobilized on different supports, including porous glass [147], polyvinyl chloride (PVC) [1], polyethylene film [21], Eupergit® C [97], alumina [125], and chitosan [128]. The synthetic adsorption resin (DIAION® HP-20) [61] and purified sand [55] are examples of supports used for immobilization of CGTase through absorption method. Meanwhile, alginate, polyacrylamide [80], curdlan and vegetable sponge natural supports [33] have been used in entrapment immobilization technique.

The immobilized CGTase has been used to synthesize xylitol [65], maltooligosyl fructofuranosides [96], 2-O-α-glucopyranosyl L-ascorbic acid [125, 185] and alkyl glycosides [150]. The α-CGTase immobilized on sodium alginate has shown higher optimal temperature (45 °C) and 2-fold 2-O-α-glucopyranosyl L-ascorbic acid production yield compared with free α-CGTase [185]. Furthermore, the immobilized CGTase from Paenibacillus sp. A11, used for transglycosylation of L-ascorbic acid, exhibits 20% higher thermal stability than free CGTase and can be stored for a longer period of up to two months [125]. In addition, the CGTase from Thermoanaerobacter sp. immobilized on Amberlite® IRA-900, which has been used for transglycosylation of xylitol, have longer half-life and higher optimal temperature (90 °C) compared with it free form (70 °C) [65].

Protein engineering is an alternative method of improving the transglycosylation activity of CGTase. Different approaches have been studied for protein engineering of CGTase, including site-directed mutagenesis, directed evolution [84], and combination of x-ray crystallography with site-directed mutagenesis [176]. The site-directed mutagenesis has been used for replacement of Tyr 188 in CGTase from Bacillus ohbensis [145]; Tyr 195 in CGTase from Bacillus circulans Strain 251 [121]; Lys-232, Phe-183, Phe-259 and Glu-264 in CGTase from Bacillus circulans strain 251 [162]; Ala 223 and Gly 255 in CGTase from Bacillus clarkia 7364 [109]; lysine 47 in CGTase from Paenibacillus macerans strain JFB05-01 [90]; and Asp372 and Tyr89 in CGTase [91]. The protein engineering of CGTase is mainly used to improve the specificity in the cyclodextrin production, such as enhanced production of α-cyclodextrin.

Moreover, protein engineering is used to synthesize other products such as ascorbic acid and genistein diglucoside [42]. The 2-O-D-glucopyranosyl-L-ascorbic acid can be synthesized by chimeric modification and/or site-saturation mutagenesis of the α-CGTase from Paenibacillus macerans. Previous studies have found that maltodextrin specificity is enhanced by site-saturation engineering of lysine 47 [46], tyrosine 195, tyrosine 260, and glutamine 265 [48], or the iterative saturation mutagenesis of -6 sub site residues [47] in α-CGTase extracted from Paenibacillus macerans when used to synthesize 2-O-D-glucopyranosyl-L-ascorbic acid. Moreover, starch specificity can be enhanced by chimeric modification, including fusion of the carbohydrate-binding module to the carboxyl terminus of CGTase, fusion of self-assembling amphipathic oligopeptides with α-CGTase [45], or replacing the E domain of α-CGTase [43], to synthesize 2-O-D-glucopyranosyl-L-ascorbic acid. The donor's specificity improvement makes the glycosyl donors more soluble and the synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid cheaper [44]. In addition, fusion of carbohydrate-binding modules with CGTase enhances hydrolysis and disproportionation activities of CGTase, resulting in an increased 2-O-D-glucopyranosyl-L-ascorbic acid production yield. The CGTase hydrolysis and disproportionation activities play an important role in enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid. The hydrolysis activity leads to conversion of starch to oligosaccharides while disproportionation activity transfers the glycosyl group from the oligosaccharides to the acceptor. The changes in hydrolysis and disproportionation activities of fusion enzymes can lead to an increase in 2-O-D-glucopyranosyl-L-ascorbic acid production yield.

The mutation of Glu 284 in CGTase to alanine residue catalyzes regioselective transglycosylation at 3-OH group of L-ascorbic acid resulting in direct production of 3-O-α-maltosyl-L-ascorbate, without requiring any hydrolysis process by glucoamylase [6, 86]. 3-O-α-maltosyl-L-ascorbate has higher anti-antioxidant activity than ascorbic acid. Furthermore, protein engineering not only improves the transglycosylation of CGTase, but it also can increase the yield of enzymatic products by employing systematic codon optimization strategy. For instance, the systematic codon optimization strategy improves the heterologous expression of Paenibacillus macerans cgt gene in Escherichia coli [58], and it has been used to increase production of the genistein diglucoside by α-CGTase, which resulted in 1.5-fold increase in production compared with the native α-CGTase [42].

Improved conversion yield of transglycosylated products

The conversion yield of transglycosylated products is the most important factor in the industry. Li et al. [89] had used starch as glycosyl donor and the CGTase extracted from B. macerans to synthesize diglucosylated stevioside with 76% conversion yield. Similarly, Mathew et al. [100] reported that use of the CGTase from B. macerans to transfer the glycosyl group from a donor, α-cyclodextrin, to acceptor lead to a very good conversion yield of 93%. According to Svensson et al. [146], the conversion yield of dodecyl-β-D-maltoside to dodecyl-β-D-maltooctaoside using CGTase and α-cyclodextrin, as a donor, was 80%. Table 2 provides information on the conversion yield of different transglycosylated products.

Carbohydrate elongation of alkyl glycosides

Alkyl glucosides are non-ionic surface-active agents and widely used in food, personal care and detergent productions, and protein extractions [17]. Surface-active agents are amphipathic molecules with hydrophobic and hydrophilic moieties. The reasons for wide application of alkyl glucosides are their antimicrobial activity, biodegradability and low toxicity [99]. CGTase not only can synthesize alkyl glycosides, using sugars and fatty alcohols with simple reaction steps compare with chemical synthesis [129], it can also elongate the carbohydrate group of alkyl glycosides to a desired length, such as elongation of dodecyl-β-maltoside to dodecyl-β-maltooctaoside. In fact, commercial alkyl glycosides are only available with relatively short carbohydrate parts. Elongation of the sugar moieties of alkyl glycosides makes the surfactant milder on cells and tissues [32]. The dodecyl-β-maltooctaoside and tert-butyl α-D-glucopyranosides are examples of alkyl glycosides synthesized by CGTase.

Improved water-solubility of transglycosylated products

Transglycosylation reaction influences both physicochemical and biological properties of molecules. The most obvious advantage of this reaction is to render hydrophobic compounds to have a better solubility in aqueous systems. Improvement of the solubility of hydrophobic compounds can increase their bioavailability in our body and their biological and pharmacological functions. For instance, polyphenols are hydrophobic compounds, which exhibit deficient absorption, causing entry of a low concentration of polyphenols into circulatory streams [14]. Adding a sugar moiety to improve the water solubility of a hydrophobic compound may improve its intestinal absorption and subsequently its bioavailability [130, 136]. Phenolic compounds such as tocopherols exhibit useful biological, including anti-cancer, anti-aging, anti-carcinogenic and anti-allergic, effects [137]. However, low water solubility of tocopherols limits their biological and pharmacological applications. The transglycosylation of α- and δ-tocopherols catalyzed by CGTase has shown to improve their anti-allergic activities [137]. In addition, transglycosylation of genistein, quercetin, curcumin, hesperetin, glycitein, and daidzein can improve their anti-allergic activities, through inhibiting the release of histamine and increasing the production of IgE antibody [137, 140, 141, 142].

The water solubility of different phenolic compounds has been improved by transglycosylation reaction using CGTase, including hesperidin [75], genistin [88] naringin [38], resveratrol [159], epicatechin [10], hydroquinone [95] and kaempferol [23]. Besides, transglycosylation reaction catalyzed by CGTase also could improve water solubility of the sugar-glycoside-menthyl α-D-glucopyranoside [30], and diterpenoid compound benzo[h]quinazolines [95]. A summary of transglycosylated products and their water solubility is provided in Table 3.

Table 3

Increase in water solubility of glycosylated products compared with their aglycon.

Acceptor	Product	Increase in Water Solubility, Fold	Ref.
Sugar glycosides
i-menthyl α-D-glucopyranoside	i-Menthyl α-D-glucopyranosyl-(1→4)-α-D-glucopyranoside	1.57 × 103	[30]
Phenolic compound
Hesperidin	Hesperidin Glycosides	3.0 × 102	[75]
Genistin	Glc(α1-4)-Glc(β1-7)-genistein	3.7 × 103	[88]
	Glc(α1-4)-Glc(α1-4)-Glc(β1-7)-genistein	4.4 × 104
Naringin	Monoglycosyl naringin	1.0 × 103	[38]
Quercetin 7-O-β-D-glucoside	Quercetin 7-O-β-maltoside	2.7 × 102	[142]
	Quercetin 7-O-β-maltotrioside	6.9 × 102
Quercetin 3-O-β-D-glucoside	Quercetin 3-O-β-maltoside	3.3 × 102
	Quercetin 3-O-β-maltotrioside	8.5 × 102
Genistein 4-O-β-D-glucoside	Genistein 4-O-β-maltoside	6.4 × 102
	Genistein 4-O-β-maltotrioside	1.7 × 103
Genistein 7-O-β-D-glucoside	Genistein 7-O-β-maltoside	4.5 × 102
	Genistein 7-O-β-maltotrioside	1.0 × 103
Resveratrol	α-glucosyl derivatives of resveratrol (3,5,4′-trihydroxy stilbene)	6.5 × 101	[159]
Epicatechin	Epicatechin glucosides	4.4 × 101	[10]
Hydroquinone	Hydroquinone-1-O-α-D-glucopyranoside	1.8	[100]
Kaempferol (flavonol)	Kaempferol-3-O-β-D-glucopyranosyl-(1→4)-O-α-D- glucopyranoside	1.21 × 102	[23]
Diterpenoid
Taxol (paclitaxel)	7-Glycolylpaclitaxel 2″-O-α-glucooligosaccharide	6.8 × 103	[138]

Improved stability of transglycosylated products

Transglycosylation can improve the stability of compounds. For instance, it has been shown that the epicatechin-3′-O-α-D-glucopyranoside has browning resistance [10] due to presence of the glucosyl moiety that helps to protect the epicatechin catechol rings from polymerization, the cause of browning [159]. Kometani et al. [75] found that transglycosylation of hesperidin stabilizes the color of pigments by absorbing ultraviolet light. The L-ascorbic acid undergoes rapid degradation when exposed to heat, neutral pH, and heavy metals [151]. However, transglycosylation of L-ascorbic acid to 2-O-α-D-glucopyranosyl-L-ascorbic acid improves its stability, and transglycosylated L-ascorbic acid is widely used in food and cosmetic products [46, 124].

Application of transglycosylated products in foods and cosmetics

Tyrosinase (EC 1.14.18.1) is a copper-containing oxidase enzyme that catalyzes melanin synthesis [148]. Melanin is a natural pigment that protects, through melanogenesis, human skin cells when exposed to UV radiation. The melanin pigment in skin cell absorbs the UV light and dissipates absorbed UV radiation. Melanogenesis is a process that produces melanin in melanosomes [168]. Excessive melanin synthesis causes hyperpigmentation in the skin cell and darkens the skin [148]. One way to control the excessive melanin synthesis is by using inhibitors to inhibit tyrosinase, the enzyme that synthesizes melanin, and thus they are considered as skin whitening agents and a vital element of skin whitening products. According to Kazuhisa et al. [62], tyrosinase inhibition effect of α-arbutin was more effective than arbutin in human malignant melanoma cells. Newly synthesized α-arbutin, catalyzed by CGTase, had shown 39.7% inhibitory effect on tyrosinase [100]. Another study showed that (+) catechin-α-glucoside exhibits 50% tyrosinase inhibitory activity [34]. Furthermore, α-glycosyl ginsenoside F1 shows higher tyrosinase inhibitory activity than ginsenoside F1, its non-transglycosylated form [103].

Transglycosylation is also able to reduce the bitter taste or aftertaste of food additives such as mogroside V. The sweetness of stevioside and mogroside V are 300–400 times higher than sucrose but they have a bitter aftertaste [170, 177]. Previous studies [93] have shown that glucosyl, sucrosyl, sophorosyl and fructosyl groups can be attached to the C13 position of stevioside, which eliminates the bitter aftertaste of stevioside. Currently, transferring glycosyl groups to stevioside through transglycosylation catalyzed by CGTase is the most common method used to remove bitterness of stevioside [59, 89].

Conclusion

Transglycosylation reaction is a process of transferring glycosyl groups from a donor to an acceptor, which contains hydroxyl or other functional groups, to form a glycoconjugate. Transglycosylation can be catalyzed by enzymatic reactions using CGTase. Catalytic mechanisms of the CGTase are retaining, double displacement, and random ternary complex mechanisms. The CGTase is mainly used to catalyze glycosyl transfer reactions. Most CGTase are produced extracellularly by bacteria and predominantly by strains of Bacillus. The CGTase can be used with a wide range of acceptors, using non-activated sugars as the donor, and tolerate high concentrations of organic solvent in the reaction mixtures. In addition, the transglycosylation reaction using CGTase improves the water solubility and stability of transglycosylated products, inhibits tyrosinase, and reduces bitterness of transglycosylated products. Therefore, it is widely used in the production of ingredients used in dietary supplements, detergents, and food, medical and cosmetic products.

Declarations

Author contribution statement

All authors listed have significantly contributed to the development and the writing of this article.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

No data was used for the research described in the article.

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The authors declare no conflict of interest.

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