**3. Productions of NDOs through polysaccharide hydrolysis**

#### **3.1. Inulinase**

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

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

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

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

higher molecular weight sugars [75].

188 Enzyme Inhibitors and Activators

from a 10% amylose solution [83].

bioconversion (Lin et al. 2011).

Fructooligosaccharides can be produced by the controlled hydrolysis of fructans. Fructans are fructose-based polysaccharides, representing the major reserve carbohydrates in about 15% of flowering plant species [91]. According to differences in glycosidic linkages they can be classified in many types, being linear inulin the most studied and best-characterized fructan. Inulin consists of β(2-1)-linked fructose units terminating at the reducing end with a glucose residue attached through a sucrose-type linkage [92]. Inulinases can hydrolyze the β(2-1) linkages in inulin and can present endo- or exo-activity. Exo-acting inulinases (EC 3.2.1.80) produce fructose as the main end product, whereas endoinulinases (EC 3.2.1.7) act randomly and hydrolyze internal linkages of inulin to yield FOs and minor amounts of monosaccharides [93].

The highest yield (92%) for the conversion of chicory inulin (50 g/L) in to FOs was reported by the application of a dual system of *Xanthomonas* sp. and *Pseudomonas* sp. endoinulinases [94]. On another approach, an endoinulinase from *Xanthomonas* sp. yielded 86% of FOs from dahlia tubers inulin (10 g/L) after 10 h [95]. A production of 78% and 79% of FOs was achieved from a solution (100 g/L) of chicory inulin and chicory juice, respectively [96]. An endoinulinase produced by *Streptomyces rochei* E87 yielded 70% of FOs after 3 days of incubation with inulin producing mainly inulotriose [97]. A maximum yield of 75.6% in total of FOs was obtained by hydrolysis of a solution containing 50 g/L of inulin by *Pseudomonas* sp. inulinase, producing a mixture of oligosaccharides with DP2-7 [98]. A commercial inulinase preparation yielded 96% of FOs from dahlia tubers inulin (pH 6.0; 100 g/L). The product presented FOs with DP ranging from 1 to 6 but the major products were DP3 (23%) and DP4 (24%) [99].

The production of FOs by a inulinase from *A. niger* immobilized in montmorillonite lead to a yield of 18.32% on aqueous media and 16.03% in organic media [100], while high yields of DP3 (70.3 mM), DP4 (38.8 mM), and DP5 (3.5 mM) FOs were obtained through the enzymatic hydrolysis of inulin (150 mg/ml; 60°C; pH 6.0; 48 h) by other *A. niger* inulinase (60 U/mL) [101]. When a commercial endoinulinase preparation (Novozym®960) from *A. niger* was used in the production of FOs from inulin (60°C; pH 6.0), a productivity of F3 (70.3mM), F4 (38.8mM), and F5 (12.43 mM) was achieved [102]. Inulinases from *K. marxianus* NRRL Y 7571 produced DP2 (11.89%) and DP3 (20.83%) oligomers using inulin (20%) as substrate at 24 h at 50°C [103]. A maximum FOs production of 11.9 g/L.h and specific productivity of 72 g/g.h were observed when a mutant *X. campestris* pv. *phaseoli* grown in a 5 L fermenter containing 3%inulin and 2.5% tryptone [104]. A continuous production of FOs from inulin was carried in a bioreactor packed with immobilized cells of *Escherichia. coli* expressing a *Pseudomonas* sp. endoinulinase. Under the optimal operation conditions, continuous production of FOs was achieved by 150 g/L.h (17 days; 50°C) [105]. Continuous production of FOs from chicory juice (100 g/L) was carried out using the polystyrene-bound endoinulinase in an enzymatic reactor achieving an oligosaccharide yield of 82% [106]. *Aspergillus ficuum* endoinulinase (10 U/g) yielded 50% of FOs from Jerusalem artichoke inulin (50 g/L; 45°C; pH 6.0) after 72 h. With Jerusalem artichoke the yield reached 89% and the maximum IOS production was up to 80% after 72 h [107].

#### **3.2. Xylanases**

Xylan is also a heteropolysaccharide with a backbone formed by xylose homopolymer subunits linked through β(1-4) linkages. This polymer can be found in the hemicellulose fraction of lignocellulosic materials associated with lignin and cellulose. Through the hydrolysis of xylan with xylanases, xylooligosaccharides (XOs) can be produced. The intake of XOs is associated with many health benefits as improvement of bowel function, immunomodulatory, and anti-inflammatory activities, preventive effects on cancer and inhibitory effects on carcinogenesis, antimicrobial, antiallergic, and antioxidant activities [108].

The xylanase (β-1,4-d xylan xylanohydrolase, EC 3.2.1.8) is the main enzyme applied for xylan hydrolysis and XOs production, due its action on the main chain of xylan and release of oligosaccharides. Before the enzymatic hydrolysis of xylan, the hemicellulosic materials can be submitted to a pretreatment to enhance the xylan availability. Many types of pretreatments that can be performed, one approach uses NaOH or H<sup>2</sup> SO<sup>4</sup> solutions associate with high temperatures to disrupt the hemicellulose structure. Between the substrates used for XOs production agroresidues and food by-products are highlighted due to their high contends of hemicellulose [109].

Hydrolysis of alkali pretreated corncob powder using a commercial endoxylanase produced 81 ± 1.5% of XOs in the hydrolysate equivalent to 5.8 ± 0.14 mg/mL of XOs. Reaction parameters for the production of XOs from corncob using endoxylanase from *A. oryzae* MTCC 5154 were optimized and an XOs yield of 10.2 ± 0.14 mg/mL corresponding to 81 ± 3.9% with 73.5% xylobiose [110]. The optimization of the XOs production from corncob using the thermostable endoxylanase from *Streptomyces thermovulgaris* TISTR1948, showed that for an enzyme concentration of 129.43 U/g of substrate, 53.80°C, and pH 6.17, the yield of XOs reached 162.97 mg/g of substrate or 752.15 mg/g of hemicellulose in KOH-pretreated corncob [111]. When corncob was hydrolyzed with a xylanase from *Aspergillus foetidus* MTCC 4898 a yield of 6.73 ± 0.23 mg/mL was obtained after 8 h of reaction time using 20 U of xylanase at 45°C [112]. A commercial xylanase produced 1.208 mg/mL of xylobiose and 0.715 mg/mL of xylotriose, using 5.83 U for 16.59 h of incubation (pH 5.91; 40.8°C) [113]. Steamexploded liquor of corncobs was treated using a thermostable xylanase from *Paecilomyces themophila* J18 resulting in a XOs yield of 28.6 g/100 g xylan [114]. After a pretreatment with H2 SO<sup>4</sup> (60°C; 12 h), the corncob was hydrolyzed by xylanase, yielding 67.7% of XOs with 70% of purity [115]. Three commercial xylanase preparations (Rapidase Pomaliq, Clarex ML, and Validase) were evaluated for the enzymatic production of pentoses from the hemicellulose fraction of corn husks and corn cobs. Rapidase Pomaliq produced 104.1 g of XOs for each kg of corn husks or 133 g of XOs for each kg of corn cobs (480 min of reaction at pH 5.0 and 50°C) [116].

were observed when a mutant *X. campestris* pv. *phaseoli* grown in a 5 L fermenter containing 3%inulin and 2.5% tryptone [104]. A continuous production of FOs from inulin was carried in a bioreactor packed with immobilized cells of *Escherichia. coli* expressing a *Pseudomonas* sp. endoinulinase. Under the optimal operation conditions, continuous production of FOs was achieved by 150 g/L.h (17 days; 50°C) [105]. Continuous production of FOs from chicory juice (100 g/L) was carried out using the polystyrene-bound endoinulinase in an enzymatic reactor achieving an oligosaccharide yield of 82% [106]. *Aspergillus ficuum* endoinulinase (10 U/g) yielded 50% of FOs from Jerusalem artichoke inulin (50 g/L; 45°C; pH 6.0) after 72 h. With Jerusalem artichoke the yield reached 89% and the maximum IOS production was up to 80%

Xylan is also a heteropolysaccharide with a backbone formed by xylose homopolymer subunits linked through β(1-4) linkages. This polymer can be found in the hemicellulose fraction of lignocellulosic materials associated with lignin and cellulose. Through the hydrolysis of xylan with xylanases, xylooligosaccharides (XOs) can be produced. The intake of XOs is associated with many health benefits as improvement of bowel function, immunomodulatory, and anti-inflammatory activities, preventive effects on cancer and inhibitory effects on

The xylanase (β-1,4-d xylan xylanohydrolase, EC 3.2.1.8) is the main enzyme applied for xylan hydrolysis and XOs production, due its action on the main chain of xylan and release of oligosaccharides. Before the enzymatic hydrolysis of xylan, the hemicellulosic materials can be submitted to a pretreatment to enhance the xylan availability. Many types of pretreat-

high temperatures to disrupt the hemicellulose structure. Between the substrates used for XOs production agroresidues and food by-products are highlighted due to their high con-

Hydrolysis of alkali pretreated corncob powder using a commercial endoxylanase produced 81 ± 1.5% of XOs in the hydrolysate equivalent to 5.8 ± 0.14 mg/mL of XOs. Reaction parameters for the production of XOs from corncob using endoxylanase from *A. oryzae* MTCC 5154 were optimized and an XOs yield of 10.2 ± 0.14 mg/mL corresponding to 81 ± 3.9% with 73.5% xylobiose [110]. The optimization of the XOs production from corncob using the thermostable endoxylanase from *Streptomyces thermovulgaris* TISTR1948, showed that for an enzyme concentration of 129.43 U/g of substrate, 53.80°C, and pH 6.17, the yield of XOs reached 162.97 mg/g of substrate or 752.15 mg/g of hemicellulose in KOH-pretreated corncob [111]. When corncob was hydrolyzed with a xylanase from *Aspergillus foetidus* MTCC 4898 a yield of 6.73 ± 0.23 mg/mL was obtained after 8 h of reaction time using 20 U of xylanase at 45°C [112]. A commercial xylanase produced 1.208 mg/mL of xylobiose and 0.715 mg/mL of xylotriose, using 5.83 U for 16.59 h of incubation (pH 5.91; 40.8°C) [113]. Steamexploded liquor of corncobs was treated using a thermostable xylanase from *Paecilomyces themophila* J18 resulting in a XOs yield of 28.6 g/100 g xylan [114]. After a pretreatment with

(60°C; 12 h), the corncob was hydrolyzed by xylanase, yielding 67.7% of XOs with

SO<sup>4</sup>

solutions associate with

carcinogenesis, antimicrobial, antiallergic, and antioxidant activities [108].

ments that can be performed, one approach uses NaOH or H<sup>2</sup>

after 72 h [107].

190 Enzyme Inhibitors and Activators

**3.2. Xylanases**

tends of hemicellulose [109].

H2 SO<sup>4</sup> The application of agroresidues as a source of xylan for XOs production is a strategy that has been produced excellent results. The xylan obtained by alkali extraction from cotton stalk, was hydrolyzed using a commercial xylanase preparation produced XOs in the DP range of 2–7 (X6 ≈ X5 > X2 > X3) and also minor quantities of xylose, yielding 53% (40°C; 24 h) [117]. Tobacco stalks were hydrolyzed by xylanase producing a XOs yield of 8.2%after 8 h and 11.4% after 24 h reaction period [118]. Another process yielded 7.28 and 4.52 g/L of XOs from wheat straw and rice straw xylan, respectively, after hydrolysis with a from *A. foetidus* MTCC 4898 [119]. Using xylanases from two glycoside hydrolase families, yields of 60% and 40% were obtained for rye bran arabinoxylan hydrolysis by GH10 and GH11, respectively [120]. Wheat straw xylan was hydrolyzed using a variant of the alkali-tolerant *Bacillus halodurans* S7 endoxylanase A, resulting in 36% conversion of the xylan to predominantly xylobiose [121]. The XOs produced from garlic straw hemicelluloses hydrolyzed with xylanase secreted by *B. mojavensis* were composed of xylobiose, xylotriose, and xylotetrose, together with a small amount of xylopentaose and xylohexose yielding 29 ± 1.74% after 8 h [122]. Xylan extracted of *Mikania micrantha* was hydrolyzed by a recombinant *Paenibacillus* xylanase, yielding 68% of XOs [123]. Oil palm empty fruit bunch fiber was hydrolyzed by *Aspergillus terreus* xylanase with a maximum 262 mg of xylobiose was produced from 1.0 g of pretreated fiber [124]. Several crop by-products were subjected to an enzymatic treatment to obtain a XOs through the action of a Buzyme 2511 (R). The hydrolysis lead to a concentration of 5.3 (apple pomace), 1.3 (white poplar), 2.9 (giant cane), and 6.5 g/L (grape stalk) [125]. The enzymatic hydrolysis of hard shell almond yielded 34.0% of XOs with 70% of purity [126]. A process for producing XOs from *Sehima nervosum* grass through enzymatic hydrolysis yielded 11 g/100 g xylan of xylobiose [127]. The treatment of wheat bran with the commercial xylanase preparation enzymes, produced a yield of approximately 31.2% of XOs, with a purity of 95% (w/w) and degree of polymerization of 2–7 [128]. Viscose fiber mills were used as substrate in the production of XOs yielding 68.9% after enzymatic hydrolysis [129].

When sugarcane bagasse was hydrolyzed with a crude xylanase secreted by *Pichia stipites*, XOS accumulated with a maximum yield of 31.8% of the total xylan was achieved at 12 h, which contained 29.8% xylobiose, 47.1% xylotriose, and 18.4% xylotetraose [130]. The hydrolysis of sugarcane bagasse with a *B. subtilis* xylanase produced xylotriose (X3), xylotetraose (X4), and xylopentaose (X5) and also is less amounts xylooligomers (X11). The process yielded was 113 and 119 mg/g sugarcane bagasse for 7 and 8 h, respectively [131]. In another approach using sugarcane bagasse treated with hydrogen peroxide, the enzymatic hydrolysis by crude extracts from *Thermoascus aurantiacus* produced a maximum yield of 37.1 with 2.6% of substrate and xylanase load of 60 U/g [132]. A productivity of 2.36, 2.76, 2.03, and 2.17 mg/mL of X2, X3, X4, and X5, respectively, was obtained after hydrolysis of sugarcane bagasse by *Streptomyces rameus* L2001 xylanase [133]. A maximum yield of 5.96% was obtained for the conversion of sugarcane bagasse being xylobiose and xylotriose the main products [134]). The enzymatic hydrolysis of *Camellia oleifera* shell pretreated with NaOH produced 1.76 g/L of xylooligosaccharides (DP 2–6) [135].

#### **3.3. Pectinases**

Pectins are components of the cell walls of most higher plants, this heteropolysaccharide is characterized by a high content of galacturonic acid (GalA) monomers bonded together by α(1-4) linkages, showing acetylatilation or esterification with methyl groups. They are composed of homogalacturonans, xylogalacturonanes, rhamnogalacturonans, arabinans galactans, and arabinogalactans. Depending on how these polysaccharides are associated, pectin can be classified as homogalacturonan and rhamnogalacturonans I and II [136].

Studies using piglets showed that POs can modulate the grown of microbial communities in the ileum increasing, for example, the *Lactobacillus* counts [137, 138]. POs were also able to interfere with the toxicity of Shiga-like toxins from *E. coli* O157:H7, which play a key hole in diarrhea and hemorrhagic colitis, hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura [139].

Enzymes that act on pectins with a hydrolyzing or debranching activity have the potential to produce nondigestible oligosaccharides. The pectinolytic enzymes can be divided into: pectinesterases, pectin-methylesterases, and depolymerases being this last one more suitable for POs production. Endopolygalacturonases are depolymerases produced by various microorganisms such as bacteria, yeasts, and molds. They are also found in some plants and especially in fruits. In general, they release mono-, di-, and tri-galacturonic acid by a multiple attack mechanism single chain. Rhamnogalacturonases produce linear oligomeric compounds of alternating rhamnose and galacturonic acid (4–6 residues) with galactose residues connected to some or all the rhamnose residues. Galactanases can be divided in to endo-β-1,4 galactanases and exo-β-1,3-galactanases. The difference between these enzymes lies in their ability to hydrolyze the β(1-3), β(1-4), or β(1-6) linkages between the galactose residues [136].

Because of its high pectin content, potato, sugar beet, and apple by-products are often used as substrate for POs production. The hydrolysis of sugar beet pectin by combining endopolygalacturonase and pectinmethylesterase produced POs with a DP 1–9, with a maximum yield of trigalacturonic acid of 3.7% [140]. POs were obtained by the action of commercial enzymes on the potato rhamnogalacturonan, with a yield of 93.9 and 66.2% using Depol 670L and endoβ-1,4-galactanase, respectively. The hydrolysates yielded up to 50.6% of oligomers with DP of 13–70. Major oligosaccharides obtained with Depol 670L were DP 5 (26.3%) and DP6 (24.9%), whereas the endo-β-1,4-galactanase were DP3 (19.0%), DP5 (10.6%), and DP8 (12.6%) [141]. A high yield (93.9%) of POs was achieved using multienzymatic preparation (Depol 670 L) to hydrolyze a potato pulp by-product rich in galactan-rich rhamnogalacturonan I. Main products were oligosaccharides with DP of 2–12 (79.8–100%), whereas the oligomers with DP of 13–70 comprised smaller proportion (0.0–20.2%) [142]. A pool of pectinases was used to produce POs with degree of polymerization from 2 to 8 and six different rhamnogalacturonide structures. Total recoveries were 200 (homogalacturonides) and 67 mg/g (rhamnogalacturonides) [143]. The use of commercial pectinase preparations (Endopolygalacturonase M2, Pectinase, Viscozyme L, Pectinex Ultra SP-L, Pectinase 62 L, and Macer8 FJ) to produce POs from polygalacturonic acid. Best results were obtained with endopolygalacturonase M2 after 2 h of reaction, yielding 58, 18, and 13% of DP3 > DP2 > DP1, respectively [144].

In some cases, other food by-products were applied in the production of POs. A initial amount of 100 kg of orange peel can yield 7.5 kg of gluco-oligosaccharides, 4.5 kg of galacto-oligosaccharides, 6.3 kg of arabino-oligosaccharides, and 13 kg of oligogalacturonides [145]. Through the action commercial enzymes (EPG-M2, Viscozyme, and Pectinase) on onion skins a yield 5.6% of pectic oligosaccharides (POS) was obtained [146].

#### **3.4. Chitosanase**

conversion of sugarcane bagasse being xylobiose and xylotriose the main products [134]). The enzymatic hydrolysis of *Camellia oleifera* shell pretreated with NaOH produced 1.76 g/L

Pectins are components of the cell walls of most higher plants, this heteropolysaccharide is characterized by a high content of galacturonic acid (GalA) monomers bonded together by α(1-4) linkages, showing acetylatilation or esterification with methyl groups. They are composed of homogalacturonans, xylogalacturonanes, rhamnogalacturonans, arabinans galactans, and arabinogalactans. Depending on how these polysaccharides are associated, pectin

Studies using piglets showed that POs can modulate the grown of microbial communities in the ileum increasing, for example, the *Lactobacillus* counts [137, 138]. POs were also able to interfere with the toxicity of Shiga-like toxins from *E. coli* O157:H7, which play a key hole in diarrhea and hemorrhagic colitis, hemolytic uremic syndrome (HUS), and thrombotic throm-

Enzymes that act on pectins with a hydrolyzing or debranching activity have the potential to produce nondigestible oligosaccharides. The pectinolytic enzymes can be divided into: pectinesterases, pectin-methylesterases, and depolymerases being this last one more suitable for POs production. Endopolygalacturonases are depolymerases produced by various microorganisms such as bacteria, yeasts, and molds. They are also found in some plants and especially in fruits. In general, they release mono-, di-, and tri-galacturonic acid by a multiple attack mechanism single chain. Rhamnogalacturonases produce linear oligomeric compounds of alternating rhamnose and galacturonic acid (4–6 residues) with galactose residues connected to some or all the rhamnose residues. Galactanases can be divided in to endo-β-1,4 galactanases and exo-β-1,3-galactanases. The difference between these enzymes lies in their ability to hydrolyze the β(1-3), β(1-4), or β(1-6) linkages between the galactose residues [136]. Because of its high pectin content, potato, sugar beet, and apple by-products are often used as substrate for POs production. The hydrolysis of sugar beet pectin by combining endopolygalacturonase and pectinmethylesterase produced POs with a DP 1–9, with a maximum yield of trigalacturonic acid of 3.7% [140]. POs were obtained by the action of commercial enzymes on the potato rhamnogalacturonan, with a yield of 93.9 and 66.2% using Depol 670L and endoβ-1,4-galactanase, respectively. The hydrolysates yielded up to 50.6% of oligomers with DP of 13–70. Major oligosaccharides obtained with Depol 670L were DP 5 (26.3%) and DP6 (24.9%), whereas the endo-β-1,4-galactanase were DP3 (19.0%), DP5 (10.6%), and DP8 (12.6%) [141]. A high yield (93.9%) of POs was achieved using multienzymatic preparation (Depol 670 L) to hydrolyze a potato pulp by-product rich in galactan-rich rhamnogalacturonan I. Main products were oligosaccharides with DP of 2–12 (79.8–100%), whereas the oligomers with DP of 13–70 comprised smaller proportion (0.0–20.2%) [142]. A pool of pectinases was used to produce POs with degree of polymerization from 2 to 8 and six different rhamnogalacturonide structures. Total recoveries were 200 (homogalacturonides) and 67 mg/g (rhamnogalacturonides) [143]. The use of commercial pectinase preparations (Endopolygalacturonase M2,

can be classified as homogalacturonan and rhamnogalacturonans I and II [136].

of xylooligosaccharides (DP 2–6) [135].

**3.3. Pectinases**

192 Enzyme Inhibitors and Activators

bocytopenic purpura [139].

Chitin is a polysaccharide formed by *N*-acetyl-glucosamine monomers, joined by β(1-4) linkages and chitosan is the N-deacetylated form of chitin. Chitosanases (EC 3.2.1.132) are glycosyl hydrolases that catalyze the hydrolysis of β(1-4) glycosidic bond in chitosan to produce glucosamine oligosaccharides [147]. Studies using pigs indicated a modulating effect of chito-oligosaccharide (COs) inhibiting growth of harmful bacteria in the gut [67]. Strong antibacterial activity was also reported with complete inhibition of *E. coli* growth with a 0.5% solution [148]. They can also inhibit the growth of tumor cells by exerting immunoenhancing effect [149] and stimulate the growth of *Lactobacillus* sp. and *B. bifidium* KCTC 3440 indicating considerable bifidogenic potential [150].

A chitosanase (EC 3.2.1.132) from *Aspergillus* sp. Y2K showed preference for higher deacetylated chitosan as substrate, producing chitotriose, chitotetraose, and chitopentaose as the major products after hydrolysis with a total yield of 115% [151]. The chitosanolytic enzymes of *Metarhizium anisopliae* produced dimers (0.2 g/L), trimers (0.19 g/L), tetramers (0.06 g/L), and pentamers (0.04 g/L) from chitosan hydrolysis [147]. The enzymatic hydrolysis of chitosan by a chitosanase from *Bacillus* sp. yielded 60% of COs with 95% of purity [152], whereas *Bacillus pumilus* BN-262 chitosanase yielded above 80% in a UF membrane reactor [148]. Through the action of a *B. pumilus* BN-262 chitosanase, a COs productivity of 20 and 15 g/L was obtained in a batch and membrane reactor, respectively [153]. The hydrolysis with *B. pumilus* chitosanase yielded 52% of COs, producing mainly pentameric and hexameric chitosan oligosaccharides was steadily produced at 2.3 g/L (46% yield) for a month [154].
