Chapter 6. Extraction, characterization and anti-cancer activity of pectic oligosaccharides produced from agro-waste of Orange
1.6 Types of Pectinase
Pectinases are class of enzymes, which break down complex pectin polysaccharide of plant tissues into simple form D-galacturonic acids. Pectinases, member of the glycoside hydrolases (GH), polysaccharide lyases (PL) and carbohydrate esterases family of enzymes (Table 1.1). GH and PL family enzymes are categorized as endo or exo based on enzyme mode of action, i.e., random or terminal, respectively.
Pectinases are the growing enzymes of biotechnological sector, showing gradual increase importance in their market.
Polygalacturonases (PG, E.C.3.2.1) belong to family 28 of glycoside-hydrolases (GH28) (http://www.cazy.org/GH28.html). Endo-polygalacturonase (E.C.188.8.131.52) hydrolyses the α-(1,4)-glycosidic linkages of polygalacturonic acid randomly and resulting in oligogalacturonides. Exo-polygalacturonase (E.C. 184.108.40.206) cleaves the polygalacturonic acid substrate from one end (reducing and non-reducing) to produce mono-galacturonate (Rombouts and Pilnik, 1980).
Polymethylgalacturonases (PMG, E.C. 220.127.116.11) facilitates the hydrolytic cleavage of α-(1,4)-glycosidic bonds in highly esterified pectin, producing 6-methyl-D- galacturonate (Jayani et al., 2005). They show either endolytic or exolytic mode of cleavage mechanism towards their substrates (Blanco et al., 1999).
1.6.3 Pectin methyl esterases
Pectin methyl esterases (PME, E.C 18.104.22.168) catalyse the de-esterification of the methoxyl (methyl) group substitution of galacturonic acid moiety in pectin. Removal of the methoxyl group by PME facilitates polygalacturonases and pectate lyases to cleave the non-esterified polygalacturonan chain (Yadav et al., 2009). PME have been classified under family 8 carbohydrate esterase (CE8) in the CAZy database (http://www.cazy.org/CE8. html).
1.6.4 Pectin acetyl esterases
Pectin acetyl esterases (PAE, E.C 22.214.171.124) cleave the acetyl group present at the C-6 carbon of the galacturonic acid moiety of pectin producing pectic acid and acetate (Shevchik et al., 1997). They have been classified under family 12 and 13 carbohydrate esterase (CE12 and CE13) in the CAZy database (http://www.cazy.org/html).
1.6.5 Acetyl xylan esterase
Acetyl xylan esterase (AXE, E.C 126.96.36.199) catalyses the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, p-nitrophenyl acetate but not from triacetylglycerol. It does not act on acetylated mannan or pectin. They have been classified under family 1 to 7 carbohydrate esterase (CE1 to CE7) in the CAZy database (http://www.cazy.org/html).
1.6.6 Pectate lyases
Endo-pectate lyases (PL, E.C 188.8.131.52) randomly cleave polygalacturonic acid, while exo-pectate lyases (E.C 184.108.40.206) catalyze the cleavage of polygalacturonic acid from non-reducing end (Jayani et al., 2005). As a result of their action α-Δ-4,5- unsaturated-GalpA is produced at the non-reducing end (http://www.cazy.org/PL1.html). Pectate lyases have been classified under family 1, 2, 3, 9 and 10 polysaccharide lyase in the CAZy database (http://www.cazy.org/html).
1.6.7 Pectin lyases
Pectin lyases (PNL, E.C 220.127.116.11) catalyse the breakdown of the α-1,4- glycosidic bond in highly esterified polygalacturonic acid. They possess an endo mode of action (Yadav et al., 2009). They have been classified under family 1 polysaccharide lyase (http://www.cazy.org/PL1. html).
1.6.8 Rhamnogalacturonan I rhamnohydrolases
Rhamnogalacturonan I rhamnohydrolases (E.C 18.104.22.168) cause the hydrolytic cleavage of the α-1,4-glycosidic bonds between L-Rhap and D-GalpA at the non- reducing end releasing single L-Rhap residue (Silva et al., 2016). They have been classified glycoside hydrolase family 78 (GH78) (http://www.cazy.org/GH78.html).
1.6.9 Rhamnogalcturonan I galacturonohydrolases
Rhamnogalcturonan galacturonohydrolases (E.C 22.214.171.124) catalyse the removal of terminal non-reducing galacturonosyl residue by breaking of α-1,2- glycosidic bond between D-GalpA and L-Rhap at the non-reducing end releasing single D-GalpA residue (Silva et al., 2016). They have been classified into glycoside hydrolase family 28 (GH28) (http://www.cazy.org/GH28.html).
1.6.10 Rhamnogalacturonan I endo-hydrolases
Rhamnogalacturonan I endo-hydrolases (E.C 126.96.36.199) catalyse the cleavage of α-1,2-glycosidic bonds between D-GalpA and L-Rhap in a random fashion releasing oligogalacturonates (Silva et al., 2016). They have been classified into glycoside hydrolase family 28 (GH28) (http://www.cazy.org/GH28.html).
1.6.11 Rhamnogalacturonan lyases
Rhamnogalacturonan lyases catalyze the cleavage of α-1,4-glycosidic bond between Rhap and GalpA residues of rhamnogalacturonan I main chain through β- elimination reactin mechanism (Silva et al., 2016). The oligosaccharides products formed have α-Δ-4,5-unsaturated-GalpA residue at their non-reducing end. Endo- rhamnogalacturonan (E.C 188.8.131.52) lyases cleave the main chain randomly while exo- rhamnogalacturonan lyases (E.C 184.108.40.206) specifically cleave at the terminal glycosidic bond. Rhamnogalacturonan lyases are grouped in families 4 and 11 of polysaccharide lyases (PL4 and PL11) (http://www.cazy.org/PL4.html, http://www.cazy.org/PL11.
1.6.12 Unsaturated rhamnogalacturonyl hydrolases
Unsaturated rhamnogalacturonyl hydrolases (E.C 220.127.116.11) are active only on RG I oligomer with Δ-4,5-unsaturated-GalpA residue at the non-reducing end (Silva et al., 2016). They catalyse the cleavage of the α-1,2 glycosidic bond between the Δ-4,5- unsaturated-GalpA and L-Rhap releasing single unsaturated D-GalpA. They have been classified into glycoside hydrolase family 105 (GH105) (http://www.cazy.org/GH105.
1.6.13 Rhamnogalacturonan acetylesterases
Rhamnogalacturonan acetylesterases (E.C 18.104.22.168) hydrolyze acetyl groups from the rhamnogalacturonan I chain (Searle et al., 1992). They have been classified into carbohydrate esterase family 12 (http://www.cazy.org/CE12. html).
1.6.14 Xylogalacturonan hydrolase
Xylogalacturonan hydrolase or xylogalacturonase (E.C 3.2.1.-) hydrolytically cleaves the α-(1,4)-glycosidic linkages between two galacturonate residues in xylose containing rhamnogalacturonan chain, hence producing xylose-galacturonate dimers (Vlugt-Bergmans et al., 2000). These enzymes are classified into glycoside hydrolase family 28 (http://www.cazy.org/GH28.html).
Endo-arabinases (E.C 22.214.171.124) catalyse the hydrolysis of α-1,5-linked arabinan side chains of rhamnogalacturonan I (Silva et al., 2016). Terminal non-reducing arabinose residues are removed by a-L-arabinofuranosidases (EC 126.96.36.199). They have been classified into glycoside hydrolase family 43 (GH43) (http://www.cazy.org/
Endo-galactanases (E.C 188.8.131.52) cleave the β-1,3-, β -1,4-, or β-1,6 linked galactose residues in the side chains They have been classified into glycoside hydrolase family 53. β-D-galactosidases (EC 184.108.40.206) remove the β-D-galactose residues from the terminal non-reducing end of galactan side chains (Silva et al., 2016). β-D- galactosidases have been classified into glycoside hydrolase family 1, 2 and 42 (GH1 and GH42) (http://www.cazy.org).
Table 1.1 Types of pectin degrading enzymes in CAZy database.
Enzyme Name Enzyme
Known enzyme activity Reference Polygalacturonase GH28 Endo-polygalacturonase
(E.C.220.127.116.11) & Exo- polygalacturonase (E.C.
Rombouts and Pilnik, 1980 Polymethyl
GH28 Polymethylgalacturonases (E.C. 18.104.22.168)
Blanco et al., 1999
Pectin methyl esterases CE8 Pectin methyl esterases (E.C.
Yadav et al., 2009
Pectin acetyl esterase CE12, CE13
Pectin acetyl esterases (E.C.
Shevchik et al., 1997
Pectate lyase PL1 to 3, PL9,PL10
Endo-pectate lyases (E.C 22.214.171.124) and Exo-pectate lyases (E.C. 126.96.36.199)
Jayani et al., 2005
Pectin lyase PL1 Pectin lyases (E.C. 188.8.131.52) Yadav et al., 2009
Rhamnogalacturonan I rhamnohydrolase
GH78 Rhamnogalacturonan I rhamnohydrolase (E.C.
Silva et al., 2016 Rhamnogalcturonan I
GH28 Rhamnogalcturonan galacturonohydrolase (E.C.
Silva et al., 2016 Rhamnogalacturonan I
GH28 Rhamnogalacturonan I endo- hydrolases (E.C 184.108.40.206)
Silva et al., 2016 Rhamnogalacturonan
PL4, PL11 Endo-rhamnogalacturonan lyases (E.C 220.127.116.11) and Exo-
rhamnogalacturonan lyases (E.C.
Silva et al., 2016
GH105 Unsaturated rhamnogalacturonyl hydrolases (E.C. 18.104.22.168)
Silva et al., 2016 Rhamnogalacturonan
acetylesterase (E.C. 22.214.171.124)
Searle et al., 1992 Xylogalacturonan
GH28 Xylogalacturonan hydrolase or xylogalacturonase (E.C. 3.2.1.-)
Vlugt- Bergmans et al., 2000 Arabinase GH43 Endo-arabinases (E.C. 126.96.36.199) Silva et al.,
2016 Galactanase GH53 Endo-galactanases
Silva et al., 2016