**2. Production of NDOs through glycosyl transfer reaction**

#### **2.1. Galactosidases**

pathogenic microorganisms, and an increase of the bifidobacteria population [2]. The bioactive properties of NDOs can be influenced by monosaccharide composition, type of glycosidic

Nondigestible oligosaccharides can be produced using chemical or enzymatic processes. The synthesis using chemical methods are complicated, with numerous protection and deprotection steps required in order to achieve regioselectivity [3]. Other challenges of chemical synthesis are the low yields, expensive chemicals, and impossibility for scaleup. For those reasons with few exceptions, most of the NDOs are produced by enzymatic

The enzymatic production of NDOs can be achieved by two different approaches, the use of glycosyltransferases or glycosidases. Glycosyltransferases catalyze the stereospecific and regiospecific transfer of a monosaccharide from a donor substrate (glycosyl nucleotide) to an acceptor substrate. Some of the difficulties associated with the application of glycosyltransferases are availability of enzymes and sugar nucleotide donors, product inhibition, and reagent costs. These factors decrease the applications of these enzymes in the production of NDOs [4]. The glycosidases offer a good alternative for enzymatic production of NDOs, where they can be synthetized from monosaccharides using transglycosylation reactions, or formed by controlled enzymatic hydrolysis of polysaccharides. Some advantages of the glycosidases in relation to glycosyltransferases are availability, good stability, and the fact that they act on easily found substrates and do not need

The transglycosylation route can be performed by the use of a good glycosyl donor that can be a disaccharide, in high concentrations. This donor will form an intermediate glycosyl-enzyme that can be intercepted by an acceptor to give a new glycoside or oligosaccharide [3]. When the substrate is a monosaccharide, it will be acting as a donor and acceptor. Some glycosidases used to produce NDOs using this approach are α-galactosidases, β-fructofuranosidase, cyclo-

The production of NDOs by controlled hydrolysis of polysaccharides involves the break of glycosidic bonds, the reaction is acid base catalyzed by an oxocarbenium ion-like transition state and involves two carboxylic groups at the active site [5]. The glycosidases can be divided into inverting or retaining depending on the configuration of the glycosidic linkage after the hydrolysis. Inverting glycosidases operate through direct displacement of the leaving group by water. The two carboxylic groups are responsible for the reaction, one provides base catalytic assistance to the attack of water and the other provides acid catalytic assistance to cleavage of the glycosidic bond. Retaining glycosidases use a double displacement mechanism involving the formation of a covalent glycosyl enzyme intermediate, where one carboxylic group acts as acid catalyst for the glycosylation step and base catalyst for the deglycosylation step [3]. The second carboxylic group acts as a nucleophile and a leaving group. The enzymes inulinase, pullulanase, amylase, xylanase, endogalactanase, rhamnogalacturonase, endogalacturonase, and chitosanase are used for NDOs production using the controlled hydrolysis

maltodextrin glucanotransferase, and α-glucosidase [4].

linkage, and degree of polymerization [2].

processes.

182 Enzyme Inhibitors and Activators

cofactors [3].

approach [4].

β-Galactosidases (EC 3.2.1.23) hydrolyze the nonreducing terminal of β-D-galactose residues in β-D-galactosides. The enzyme can be used in the production of galacto-oligosaccarides (GOs) by transgalactosylation reaction in which a galactosyl is transferred into the hydroxyl group of the galactose residue of lactose [6]. Due to the strong prebiotic factor, GOs can modulate the grown of microorganisms of the gut flora, increasing the population of bifidobacteria, this enhancement is associated with beneficial effects, inhibition the grown of potentially pathogens, improvement, elimination, prevention, stimulation mineral adsorption, and decrement cholesterol and lipids [7].

When using concentrated solutions of lactose (40%), high yields of GOs can be achieved. The β-galactosidase of *Pseudozyma tsukubaensis* showed high transgalactosylation capability, yielding of 18.28% of GOs with concentration of 73.12 g/L from a 40% lactose solution [8]. The immobilization of chemically aminated β-galactosidase from *Aspergillus oryzae* onto Purolite® A-109 leads to an increase in the operational stability and transgalactosylation capacity of the enzyme, producing in the optimum conditions (400 g/L lactose, pH 4.5, 50°C) 100 g/L of GOs in a fluidized bed reactor [9]. The utilization of an ultrafiltration membrane bioreactor, allows the synthesis and separation in one system. Using high lactose concentrations (470 g/L) and β-galactosidase from *A. oryzae*, the system yielded 1.88 gGOS/mgE that is 2.44-fold higher than the conventional batch (0.77 gGOS/mgE) [10].

The milk whey, a by-product from the dairy industry, is a valuable substrate for GOs productions due to its lactose contend (45–60%). The whey is produced by the processing and manufacturing of raw milk into products such as yogurt, ice cream, butter, and cheese through processes such as pasteurization, coagulation, filtration, centrifugation, chilling, etc. [11]. Depending on the procedure used to precipitate the casein, two types of whey are formed, the acid whey (pH < 5) is obtained after fermentation or addition of organic or mineral acids, whereas the sweet whey (pH 6–7) is obtained by addition of proteolytic enzymes like chymosin [12]. The production of GOs from milk whey using a two-dimensional packed bed bioreactor yielded 97% [13], while a yield of 29.9% of GOs with a concentration of (119.8 mg/mL) was achieved using cheese whey as substrate in a 4 h process [14]. When whey permeate was used as substrate in a membrane reactor system, a mixture of GOs with 77–78% of purity was produced [15]. A high lactose conversion was achieved (70–80%), when using whey as a substrate in the production of GOs, yielding 10–20% of total sugars and producing oligomers with DP3, DP4, and DP5 [16]. The GOs production from whey permeate yielded 50% corresponding to 322 g prebiotics/kg whey permeate, presenting tagatose and lactulose in the oligosaccharides mixture [17]. Galacto-oligosaccharides were synthesized by enzymatic transgalactosylation in UF-skimmed milk permeate fortified with lactose (40% w/w). The GOs yields, expressed as a percentage of the initial lactose content, were 41, 21, 13, and 11% with β-galactosidase from *Bacillus circulans, A. oryzae, Aspergillus aculeatus*, and *Kluyveromyces lactis*, respectively, under optimal conditions [18].

#### **2.2. β-fructofuranosidases**

The β-D-fructofuranosidases catalyze the hydrolysis of β-D-fructofuranoside residues at the nonreducing end of β-D-fructofuranosides [19]. Fructooligosaccharides (FOs) can be produced by transfructosylation of sucrose by β-fructofuranosidases, which is carried out through the breaking of the β(2-1) glycosidic bond and the transfer of the fructosyl moiety onto any acceptor other than water, such as sucrose or a FO. The sucrose is used as substrate acting as the glycosyl donor and as the glycosyl acceptor in competition with water (hydrolysis) in a glycosyl transfer reaction [20]. Besides the strong prebiotic factor, many bioactivities have been associated with FOs as anti-inflammatory effect on Crohn's disease and ulcerative colitis, antimicrobial activity against gut flora pathogens, and prevention of colon cancer [21].

A β-fructofuranosidase from *Penicillium oxalicum* was able to produce neokestose from a 500 g/L sucrose solution, giving 94.2 and 224.7 g/L of neokestose and total FOs, respectively [22]. An invertase produced by *Aspergillus niger* using salt-deoiled cake as substrate was able to form kestose during enzymatic hydrolysis using glucose (50%) [23]. *Penicillium sizovae* and *Cladosporium cladosporioides* were used to produce FOs from a 600 g/L of sucrose solution with maximum yield of 184 and 339 g/L, respectively [24]. The filamentous fungus *Gliocladium virens* was able to produce 6-kestose with a yield of 3in media containing 150 g/L sucrose after 4–5 days of culture [25]. An extracellular β-fructofuranosidase from *Rhodotorula dairenensis* produced a varied type of FOs containing β(2→1)- and β(2→6)-linked fructose oligomers with a maximum concentration of 87.9 g/L (75% sucrose conversion) [26]. A fructosyltransferase from *Aureobasidium pullulans* presented maximum transfructosylation rate at 600 g/L [27].

#### **2.3. Cyclomaltodextrin glucanotransferase**

Cyclomaltodextrin glucanotranferase (CGTase, EC 2.4.1.19) catalyze the cyclization of oligosaccharides composed of D-glucose monomers joined by α(1-4) glycosidic linkages. This enzyme catalyzes mainly transglycosylation reactions leading to the formation of nonreducing cyclic oligosaccharides, named cyclodextrins. The main types are α-, β-, and γ-cyclodextrins consisting of six, seven, and eight glucose monomers in cycles, respectively. The majority of the CGTases usually produce a mixture of α-, β-, and γ-cyclodextrins, and the product ratio can vary depending on condition and reaction time [28].

The CGTase can produce cyclodextrins from starch, amylose, and other polysaccharides by catalyzing different transglycosylation steps: intermolecular coupling and disproportionation and modification of the length of noncyclic dextrins [29]. Between main microbial sources of CGTases, the *Bacillus, Geobacillus, and Paenibacillus* species are highlighted. The optimum temperature and pH for this enzyme range from 4 to 10.3°C and 10 to 85°C, respectively, whereas the molecular weight ranges from 33 to 200 kDa.

The products of the CGTases α, β, and γ-cyclodextrins are not completely digested in the gastrointestinal tract, rising to the colon where they are fermented by the intestinal microflora and for this reason are considered prebiotics. The microbial degradation results in linear malto-oligosaccharides, which are further hydrolyzed and fermented to absorbable and metabolize short-chain fatty acids. Several studies showed that CDs reduce the digestion of starch and the glycemic index of food. Other bioactivities include hypocholesterolemic and antithrombotic activity [30].

**2.2. β-fructofuranosidases**

184 Enzyme Inhibitors and Activators

**2.3. Cyclomaltodextrin glucanotransferase**

can vary depending on condition and reaction time [28].

the molecular weight ranges from 33 to 200 kDa.

The β-D-fructofuranosidases catalyze the hydrolysis of β-D-fructofuranoside residues at the nonreducing end of β-D-fructofuranosides [19]. Fructooligosaccharides (FOs) can be produced by transfructosylation of sucrose by β-fructofuranosidases, which is carried out through the breaking of the β(2-1) glycosidic bond and the transfer of the fructosyl moiety onto any acceptor other than water, such as sucrose or a FO. The sucrose is used as substrate acting as the glycosyl donor and as the glycosyl acceptor in competition with water (hydrolysis) in a glycosyl transfer reaction [20]. Besides the strong prebiotic factor, many bioactivities have been associated with FOs as anti-inflammatory effect on Crohn's disease and ulcerative colitis, antimicrobial activity against gut flora pathogens, and prevention of colon cancer [21]. A β-fructofuranosidase from *Penicillium oxalicum* was able to produce neokestose from a 500 g/L sucrose solution, giving 94.2 and 224.7 g/L of neokestose and total FOs, respectively [22]. An invertase produced by *Aspergillus niger* using salt-deoiled cake as substrate was able to form kestose during enzymatic hydrolysis using glucose (50%) [23]. *Penicillium sizovae* and *Cladosporium cladosporioides* were used to produce FOs from a 600 g/L of sucrose solution with maximum yield of 184 and 339 g/L, respectively [24]. The filamentous fungus *Gliocladium virens* was able to produce 6-kestose with a yield of 3in media containing 150 g/L sucrose after 4–5 days of culture [25]. An extracellular β-fructofuranosidase from *Rhodotorula dairenensis* produced a varied type of FOs containing β(2→1)- and β(2→6)-linked fructose oligomers with a maximum concentration of 87.9 g/L (75% sucrose conversion) [26]. A fructosyltransferase from *Aureobasidium pullulans* presented maximum transfructosylation rate at 600 g/L [27].

Cyclomaltodextrin glucanotranferase (CGTase, EC 2.4.1.19) catalyze the cyclization of oligosaccharides composed of D-glucose monomers joined by α(1-4) glycosidic linkages. This enzyme catalyzes mainly transglycosylation reactions leading to the formation of nonreducing cyclic oligosaccharides, named cyclodextrins. The main types are α-, β-, and γ-cyclodextrins consisting of six, seven, and eight glucose monomers in cycles, respectively. The majority of the CGTases usually produce a mixture of α-, β-, and γ-cyclodextrins, and the product ratio

The CGTase can produce cyclodextrins from starch, amylose, and other polysaccharides by catalyzing different transglycosylation steps: intermolecular coupling and disproportionation and modification of the length of noncyclic dextrins [29]. Between main microbial sources of CGTases, the *Bacillus, Geobacillus, and Paenibacillus* species are highlighted. The optimum temperature and pH for this enzyme range from 4 to 10.3°C and 10 to 85°C, respectively, whereas

The products of the CGTases α, β, and γ-cyclodextrins are not completely digested in the gastrointestinal tract, rising to the colon where they are fermented by the intestinal microflora and for this reason are considered prebiotics. The microbial degradation results in linear malto-oligosaccharides, which are further hydrolyzed and fermented to absorbable and metabolize short-chain fatty acids. Several studies showed that CDs reduce the digestion of The most frequently used raw material for CDs production is starch. The product inhibition effect of cyclodextrins on CGTases, make the complete conversion of starch a challenge. Strategies to decrease this effect involve the continual removal of CDs by filtration or the precipitation using agents that forms a specific insoluble complex with CDs. Filtration devices can be coupled to the production systems, hollow fiber and [31]. **Table 1** shows the yields or concentration of CDs obtained through the action of microbial CGTase on different substrates.



**Table 1.** Production of cyclodextrins by microbial CGTases.

*Bacillus* sp. species are the main microbial source of CGTase, in some cases thermophiles are used to obtain enzymes with unusual characteristics. Most of studies are focused on the β-cyclodextrin or mixture production and higher concentrations are usually obtained for β-and γ-cyclodextrins. The substrate is usually corn starch, although tapioca, cassava, wheat, and potato starches are also observed. The conditions for cyclodextrin production are usually 40–60°C, pH 6–7, and aqueous media, however, depending on the microbial source of the CGTase some unusual condition may be observed, as 25°C or pH 12. In some cases, the organic media is used to decrease the inhibition of the CD. The highest productivity is reported to the production of a mixture by a recombinant CGTase of *Thermoanaerobacter* using soluble starch that yielded 75 g/L.

#### **2.4. Alpha-glucan acting enzymes**

*Bacillus* sp. species are the main microbial source of CGTase, in some cases thermophiles are used to obtain enzymes with unusual characteristics. Most of studies are focused on the β-cyclodextrin or mixture production and higher concentrations are usually obtained for β-and γ-cyclodextrins. The substrate is usually corn starch, although tapioca, cassava,

**Enzyme source Substrate Conditions Yield (%) Concentration** 

 *B. clarkii 7364* Potato starch 10 h; 50°C; pH 7 72.5 [45]  *P. macerans* 5% soluble starch 10 h; 45°C; pH 5.5 – 1.8 [34]  *B. clarkii 7364* 15% soluble starch 55°C; pH 12 47 [46]  *T. thermosulfurigenes* 10% paselli SA2 0.1 U/mL; pH 5.9; 60°C; 8 h 13 5.0\* [35]

 *B. macerans* Glucans 24 h; 40°C 21.1 15.1 [47]  *P. macerans* 5% soluble starch 22 h 36.9 [34]  *B. circulans DF 9R* 5% cassava starch 4 h; 56°C 55.6 99.5<sup>a</sup> [48]  *Toruzyme 3.0 l* Tapioca starch 4 h; 60°C 85 23.0 [49]  *T. fusca* 15% potato starch 24 h; 30°C; pH 5.6 84 [50]  *B. cereus* 6% sago starch 8 h; 55°C – 13.7 [51] Toruzyme 3.0 l 8% tapioca starch 2 h; 70°C;pH 5 – 12.1 [52] Toruzyme 3.0 l 8% tapioca starch 3 h; 60°C 25 40.0 [49]

120 h;1000 U/g CGTase

 *B. circulans 251* 10% potato starch pH 6; 50°C; 45–50 h 40 – [55]

 *B. clausii E16* 1% soluble starch 10 U/g; pH 5.5; 55°C; 24 h 80 – [28]

25°C; 5–10 days

Mutant CGTase H43T 1% tapioca starch 1% toluene; pH 6; 60°C; 18 h 15.2 – [58]

pH 7.5; 40°C; 6 h

 *Thermoanaerobacter* sp. 5% soluble starch 60°C; pH 6 29 74.0 [60]  *B. stearothermophilus* 5% soluble starch 500U/g; 65°C; pH 6; 24 h 22 – [61]  *E. coli NV601* 5% soluble starch 60°C; pH 6 30 75.0 [60]

 *B. megaterium* 50 g/L corn starch pH 7; 45°C; 12 h; 2 U/g CGTase 30 –

24 h

12 h

 *B. macerans* 30% potato starch pH 5.5–8.5; 40–55°C;

 *B. macerans* 7.5% corn starch 48 U/g CGTase; pH 6; 60°C;

 *Bacillus* sp. 277 10% potato starch 400 U/g CGTase; pH 8; 60°C;

 *B. macerans* 10% tapioca starch 0.4 mmol cyclodecanone; pH 7;

 *K. pneumoneae* 12.5% wheat starch 20 U/g CGTase; 2% butanol;

**(g/L)**

30–35 – [53]

25 – [54]

34 – [56]

91–93 – [57]

42.5 – [59]

**Reference**

**Table 1.** Production of cyclodextrins by microbial CGTases.

Mixture (α, β, and γ)

186 Enzyme Inhibitors and Activators

Alpha-glucans are polysaccharides consisting of glucose units connected by α(1-4) or α(1-6) glycosidic linkages. Pullulan, a glucan produced by the fungus *A. pullulans* of α(1-4) linked maltotriose repeats connected by α(1-6) linkages, amylopectin, formed by shorter α(1-4) glucan chains connected by α(1-6) branch points, and dextran are some examples of alphaglucans [62].

Enzymes that act as hydrolyzing or debranching alpha-glucans are suitable for nondigestible oligosaccharides production. Pullulanase, dextransucrase, and starch acting enzymes can be used in the preparation of maltooligosaccharides and isomalto-oligosaccharides. Maltooligosaccharides contain α-D-glucose residues linked by α(1-4) glycosidic linkages, while isomaltooligosaccharides (IMOs) contain two to five glucose units with one or more α(1-6) linkages. While MO may exhibit immunoregulatory activity [63], the intake of IMO decreases serum cholesterol concentrations and improve bowel movement, stool output, and microbial fermentation in the colon [64]. IMOs also upregulate the Th1 response that play a triggering role in allergic diseases, such as rhinitis, asthma, and eczema [65].

Dextransucrases (EC 2.4.1.5) catalyze the synthesis of high molecular weight D-glucose polymers from sucrose to form a glucan called dextran. The synthesis of dextran occurs by successive transfer of glucosyl units to the polymer, while the presence of acceptor molecules in the reaction medium, the transfer of glucosyl units is made onto these molecules, leading to oligosaccharide synthesis. They can also transfer glucosyl units onto water molecules and simply hydrolyze sucrose [66]. *Leuconostoc citreum* KACC 91035 produced panose (8.63 mM), isomaltosyl maltose (6.56 mM), and isomaltotriosyl maltose (1.74 mM) after 12 days (10°C), using glucose (29 mM) as donor and maltose (28 mM) as acceptor through the transglycosylation activity of the dextransucrase [67]. An endodextranase D8144 from *Penicillium* sp. immobilized on epoxy produced IMOs (DPs 8–10) from dextran T40 in an enzymatic reactor [68]. A productivity of 42.95 mmol/L.h was obtained using 100 mmol/L of sucrose and 200 mmol/L of maltose, using dextransucrase (1 U/mL) from *Leuconostoc mesenteroides* NRRL B-512F [69]. A productivity of 7.26 mmol/L.h of IMOs was obtained a using an immobilized mixture of dextransucrase and dextranase [70], while a purified dextransucrase yielded 35 mmol/L.h of panose [71]. A productivity of 55.6 mmol/L of oligosaccharides was obtained by fermentation with *L. mesenteroides* B-742 [72]. Higher yields (70–90%) of IMOs were obtained from maltose/sucrose solutions using dextransucrase of *L. mesenteroides* B-512F [73]. Isomaltooligosaccharides of controlled molecular weight were produced using an *L. mesenteroides* NRRL B-512F dextransucrase with a yield of 58% by the acceptor reaction with glucose, and reached a degree of polymerization of at least 27 glucosyl units [74]. The use of dextransucrase associated with dextranase in the production of IMOs lead to oligosaccharide mixtures containing mainly sugars (up to 36%) with DP varying between 10 and 60 together lower and higher molecular weight sugars [75].

Alpha-amylase (EC 3.2.1.1) also can be used to obtain maltooligosaccharides. This enzyme hydrolyses the internal α(1,4) linkages in starch in a random fashion, leading to the formation of soluble maltooligosaccharides, maltose, and glucose. A protein engineering approach of the amylase from *Bacillus lehensis* G1-produced mutated proteins with an increase in the transglycosylation to hydrolysis ratio of up to 4.0-fold and reduction in the concentration of maltotriose required for use as a donor/acceptor for transglycosylation. A reduction of steric interference and hydrolysis suppression introduced a synergistic effect to produce MOs with a higher degree of polymerization [76]. Amylases from *Streptomyces sp*. were able to produce mainly maltotriose (55–75%) from soluble starch at 20–30°C pH 6.5 [77]. The *Bacillus subtilis* strain SDP1 amylase hydrolyses starch to produce maltotriose and maltotetraose along with maltose after prolonged reactions of 5 h [78]. A recombinant alpha-amylase (145 mg/mL) from *Streptomyces avermitilis* was able to yield maltose (4.49) and maltotriose (1.77 g/L) from 10.0 g/L of soluble starch [79]. An amylase from Bacillus megaterium produced a maltooligomer mixture with high proportion of maltopentaose (G5) and maltotriose (G3) during hydrolysis of starch, amylopectin, and amylose [80]. Malto-oligosaccharide production by commercial α-amylase (liquefying amylase 6 T) using freeze-thaw infusion resulted in a maximum production of 6.5 g/L after 60 min at 1.0% (w/v) enzyme [81]. A productivity of 8.9 g/L of maltopentaose was achieved using a *Bacillus* sp. AIR-5 amylase and a 40 g/L solution of soluble starch [82]. A *S. solfataricus* KM1 amylase was able to give an 80% yield of trealose from a 10% amylose solution [83].

Pullulanase (EC 3.2.1.41), a debranching enzyme, hydrolyses the α(1-6) linkage in pullulan and branched polysaccharides, producing maltotriose. An amylopullulanase from the hyperthermophilic archaeon *Caldivirga maquilingensis* was able to act on a wide range of substrates. Assays with the enzyme produced linear MOs (≤G8–G1) from cyclodextrins, amylodextrins (DP6-96) from amylose, and amylodextrins (DP1-76) from amylopectin and potato starch [84]. A one-step method using neopullulanase and α-amylase for the bioconversion of purified rice starch slurry (30% w/w) resulted in a syrup containing 59.2% of IMO (dry basis) after 72 h of bioconversion (Lin et al. 2011).

Alpha-glucosidase (EC 3.2.1.20), an exo-acting hydrolase, attacks the substrates from the nonreducing end producing α-D-glucose and presents some transglycosylation activity that can be used in the production of oligosaccharides [85]. Liquefied banana slurries were used for IMO synthesis by Transglucosidase L, producing after 12 h of transglucosylation, a yield of 76.6% with a concentration of 70.74 g/L. The IMOs mixture was composed of 53 isomaltotriose, 21 isomaltotetraose, and 26% maltooligoheptaose and larger oligomers [86]. A yield of 58.1% with a concentration of 93 g/L was obtained for IMOs production from a immobilized glucosidase using as substrate a maltose solution (160 mg/mL) in a membrane reactor system [87]. Partially purified a-glucosidase from *Aspergillus carbonarious*, immobilized on glutaraldehyde-activated chitosan beads in a packed bed reactor, produced isomaltooligosaccharides at a yield of 60% (w/w) using 30% (w/v) maltose solution. Using intact mycelia attached with polyethyleneimine-glutaraldehyde, a yield of 46% (w/w) was obtained using 30% (w/v) maltose solution [88]. A high yield of IMOs (67%) with concentration of 2 g/L was obtained when 30% (w/v) of soluble tapioca starch was incubated with amylomaltase (120 U) for 0.5 h (pH 7.0; 40°C). While a yield of 53% and concentration of 1.63 g/L was obtained using transglucosidase (6 U) in the same condition for 1 h [89]. When amylomaltase (1.5 U) and transglucosidase (8 U) of were incubated with 20% (w/v) maltotriose for 30 min at 40°C, 9.9 mg/mL of IMOs were produced to with DP 2–7 [90].
