Pectinase

Pectinases are a group of enzymes that breaks down pectin, a polysaccharide found in plant cell walls, through hydrolysis, transelimination and deesterification reactions.^[1][2] Commonly referred to as pectic enzymes, they include pectolyase, pectozyme, and polygalacturonase, one of the most studied and widely used^{[citation needed]} commercial pectinases. It is useful because pectin is the jelly-like matrix which helps cement plant cells together and in which other cell wall components, such as cellulose fibrils, are embedded. Therefore, pectinase enzymes are commonly used in processes involving the degradation of plant materials, such as speeding up the extraction of fruit juice from fruit, including apples and sapota. Pectinases have also been used in wine production since the 1960s.^[3] The function of pectinase in brewing is twofold, first it helps break down the plant (typically fruit) material and so helps the extraction of flavors from the mash. Secondly the presence of pectin in finished wine causes a haze or slight cloudiness. Pectinase is used to break this down and so clear the wine.

Endopolygalacturonase I
Identifiers
Organism	Aspergillus niger
Symbol	pgaI
UniProt	P26213
Other data
EC number	3.2.1.15
Search for Structures Swiss-model Domains InterPro

Pectinases can be extracted from fungi such as Aspergillus niger. The fungus produces these enzymes to break down the middle lamella in plants so that it can extract nutrients from the plant tissues and insert fungal hyphae. If pectinase is boiled it is denatured (unfolded) making it harder to connect with the pectin at the active site, and produce as much juice.

Characterizations

Pectinase is a generic term used for a group of enzymes that catalyse the degradation of pectin by hydrolysis, trans-elimination, as well as de-esterification reactions. The degradation of pectic polymers is mainly caused by exo- and endo-polygalacturonases (exo- and endo-PGs), pectate and pectin lyases (PLs), pectin methylesterase (PME) and acetylesterase (PAE), β-galactosidase (β-Gal), and α-L-arabinofuranosidase (α-L-Af), among others.^[4][5]

• Endo-polygalacturonase (E.C. 3.2.1.15) is known to be the most important enzyme responsible for pectic depolymerization and solubilization. This enzyme hydrolyses the α-1 → 4 glycosidic bonds of the methyl de-esterified homogalacturonan backbone. The enzyme randomly attacks its substrate and produces a number of D-GalA oligosaccharides.
• Exo-polygalacturonase (E.C. 3.2.1.67 and E.C. 3.2.1.82) attacks the substrate from the non-reducing end and is able to remove terminally (1→)–linked D-GalA residues from homogalacturonan chains. The enzyme requires a non-esterified D-GalA unit at subsites −2, −1, and + 1 and is tolerant for xylose substitution (able to remove a D-GalA-Xyl dimer), hence XGA is also an exo-polygalacturonase substrate.
• PLs (pectate lyases, E.C. 4.2.2.2, and pectin lyases, E.C. 4.2.2.10) acts through the β-elimination of methyl esterified homogalacturonan in the presence of Ca²⁺, Mn²⁺, or Ni²⁺.^[4]
• PME (E.C. 3.1.1.11) and PAE (E.C. 3.1.1.6) de-esterifies homogalacturonan chains by removing the methoxyl and acetyl residues, respectively. It decreases the degree of pectin methylation thereby providing suitable conditions for the hydrolysis of the α-1 → 4 link in the homogalacturonan backbone by polygalacturonase. The degradation of rhamnogalacturonans (RGs) involves the participation of numerous enzymes.
• RG hydrolase (RGH, E.C. 3.2.1.171) hydrolyses the α-D-1 → 4-D-GalA-α-L-1 → 2-Rha linkage in the RG-I backbone, leaving Rha at the non-reducing side. RG lyase (RGL, E.C. 4.2.2.23) hydrolyses RG-I α-L-1 → 2-Rha-α-D-1 → 4-GalA backbone leaving a 4-deoxy- β -L-threo-hex-4-enepyranosyluronic acid (unsaturated GalA) group at the non-reducing end.
• RG rhamnohydrolase (RGRH, E.C. 3.2.1.174) is an exo-acting pectinase, which possesses the specificity to release terminal rhamnosyl residues (1 → 4)-linked to α-galacturonosyl residues.
• RG galacturonohydrolase (RGGH, E.C. 3.2.1.173) is able to release a GalA moiety connected to a rha residue from the non-reducing side of RG-I chains but is unable to liberate GalA from homogalacturonan.
• β-Gal (E.C. 3.2.1.23) and α-L-Af (E.C. 3.2.1.55) are two enzymes responsible for removing galactosyl and arabinosyl residues from the RG-I backbone, respectively, while the acetyl groups are liberated by the action of RG acetylesterase (RGAE, E.C. 3.1.1.86).

The following table shows a summary of enzymes involved in pectin degradation. HG-PUL = homogalacturonan polysaccharide utilization loci; RG-I PUL = rhamnogalacturonan I polysaccharide utilization loci.

PUL	CAZyme families	EC number	Accepted name	Reaction
HG-PUL	PL1	EC4.2.2.2	Pectate lyase	Eliminative cleavage of (1 → 4)-α-D-galacturonan to give oligosaccharides with 4-deoxy-α-D-galact-4-enuronosyl groups at their non-reducing ends
	PL1	EC4.2.2.10	Pectin lyase	Eliminative cleavage of (1 → 4)-α-D-galacturonan methyl ester to give oligosaccharides with 4-deoxy-6-O-methyl-α-D-galact-4-enuronosyl groups at their non-reducing ends
	GH28	EC3.2.1.67	Galacturonan 1,4-α-galacturonidase	[(1 → 4)-α-D-galacturonide]n + H2O = [(1 → 4)-α-D-galacturonide]n-1 + D-galacturonate
	GH28	EC3.2.1.82	Exo-poly-α-digalacturonosidase	[(1 → 4)-α-D-galacturonosyl]n + H2O = α-D-galacturonosyl-(1 → 4)-D-galacturonate + [(1 → 4)-α-D-galacturonosyl]n-2
	GH28	EC3.2.1.15	Endo-polygalacturonase	(1,4-α-D-galacturonosyl)n+m + H2O = (1,4-α-D-galacturonosyl)n + (1,4-α-D-galacturonosyl)m
	CE8	EC3.1.1.11	Pectinesterase	Pectin + n H2O = n methanol + pectate
	CE4	EC3.1.1.6	Acetylesterase	An acetic ester + H2O = an alcohol + acetate
RG-I PUL	PL9	EC4.2.2.23	Rhamnogalacturonan endolyase	Endotype eliminative cleavage of L-α-rhamnopyranosyl-(1 → 4)-α-D-galactopyranosyluronic acid bonds of rhamnogalacturonan I domains in ramified hairy regions of pectin leaving L-rhamnopyranose at the reducing end and 4-deoxy-4,5-unsaturated D-galactopyranosyluronic acid at the non-reducing end
	GH28	EC3.2.1.171	Rhamnogalacturonan hydrolase	Endohydrolysis of α-D-GalA-(1 → 2)-α-L-Rha glycosidic bond in the rhamnogalacturonan I backbone with initial inversion of anomeric configuration releasing oligosaccharides with β-D-GalA at the reducing end.
	GH2	EC3.2.1.146	β-Galactofuranosidase	Hydrolysis of terminal non-reducing β-D-galactofuranosides, releasing galactose
	GH138	EC3.2.1.173	Rhamnogalacturonan galacturonohydrolase	Exohydrolysis of the α-D-GalA-(1 → 2)-α-L-Rha bond in rhamnogalacturonan oligosaccharides with initial inversion of configuration releasing D-galacturonic acid from the non-reducing end of rhamnogalacturonan oligosaccharides
	GH105	EC3.2.1.172	Unsaturated rhamnogalacturonyl hydrolase	2-O-(4-deoxy-β-L-threo-hex-4-enopyranuronosyl)-α-L-rhamnopyranose + H2O = 5-dehydro-4-deoxy-D-glucuronate + L-rhamnopyranose
	GH106	EC3.2.1.174	Rhamnogalacturonan rhamnohydrolase	Exohydrolysis of the α-L-Rha-(1 → 4)-α-D-GalA bond in rhamnogalacturonan oligosaccharides with initial inversion of configuration releasing β-L-rhamnose from the non-reducing end of rhamnogalacturonan oligosaccharides
	GH43	EC3.2.1.99	Arabinan endo-1,5-α-L-arabinanase	Endohydrolysis of (1 → 5)-α-arabinofuranosidic linkages in (1 → 5)-arabinans
	GH51	EC3.2.1.55	Non-reducing end α-L-arabinofuranosidase	Hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in α-L-arabinosides
	GH146	EC3.2.1.185	Non-reducing end β-L-arabinofuranosidase	β-L-arabinofuranosyl-(1 → 2)-β-L-arabinofuranose + H2O = 2 β-L-arabinofuranose
	GH53	EC3.2.1.89	Arabinogalactan endo-β-1,4-galactanase	The enzyme specifically hydrolyses (1 → 4)-β-D-galactosidic linkages in type I arabinogalactans
	GH43	EC3.2.1.145	Galactan 1,3-β-galactosidase	Hydrolysis of terminal, non-reducing β-D-galactose residues in (1 → 3)-β-D-galactopyranans
	GH27	EC3.2.1.88	Non-reducing end β-L-arabinopyranosidase	Removal of a terminal β-L-arabinopyranose residue from the non-reducing end of its substrate
	GH43	EC3.2.1.181	Galactan endo-β-1,3-galactanase	The enzyme specifically hydrolyses β-1,3-galactan and β-1,3-galactooligosaccharides
	CE12	EC3.1.1.86	Rhamnogalacturonan acetylesterase	Hydrolytic cleavage of 2-O-acetyl- or 3-O-acetyl groups of α-D-galacturonic acid in rhamnogalacturonan I.
RG-II PUL	GH43	EC3.2.1.55	Non-reducing end α-L-arabinofuranosidase	Hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in α-L-arabinosides
	CE19	EC3.1.1.11	Pectinesterase	Pectin + n H2O = n methanol + pectate
	GH142	EC3.2.1.185	Non-reducing end β-L-arabinofuranosidase	β-L-arabinofuranosyl-(1 → 2)-β-L-arabinofuranose + H2O = 2 β-L-arabinofuranose
	GH78	EC3.2.1.40	α-L-Rhamnosidase	Hydrolysis of terminal non-reducing α-L-rhamnose residues in α-L-rhamnosides
	GH33	EC3.2.1.124	3-deoxy-2-Octulosonidase	Endohydrolysis of the β-ketopyranosidic linkages of 3-deoxy-D-manno-2-octulosonate in capsular polysaccharides
	GH95	EC3.2.1.63	1,2-α-L-Fucosidase	Methyl-2-α-L-fucopyranosyl-β-D-galactoside + H2O = L-fucose + methyl β-D-galactoside
	GH2	EC3.2.1.31	β-Glucuronidase	a β-D-glucuronoside + H2O = D-glucuronate + an alcohol
	GH2	EC3.2.1.23	β-galactosidase	Hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides
	GH138	EC3.2.1.173	Rhamnogalacturonan galacturonohydrolase	Exohydrolysis of the α-D-GalA-(1 → 2)-α-L-Rha bond in rhamnogalacturonan oligosaccharides with initial inversion of configuration releasing D-galacturonic acid from the non-reducing end of rhamnogalacturonan oligosaccharides
	GH141	EC3.2.1.51

Properties

Crystal structures

All pectinase enzyme structures include a prism-shaped right-handed cylinder made up of seven to nine parallel β-helices. The three parallel β-helices that create the prism shape of the structure are referred to as PB1, PB2 and PB3, with PB1 and PB2 creating an antiparallel β and PB3 sitting perpendicularly to PB2. All substrate binding sites of the various esterases, hydrolases, and lyases are located on an outer cleft of the central parallel β-helix structure between protruding loops on the structure and PB1.^[6]

Optimum environment

As with all enzymes, pectinases have an optimum temperature and pH at which they are most active. For example, a commercial pectinase might typically be activated at 45 to 55 °C and work well at a pH of 3.0 to 6.5.^[3]

Reaction pathway

Pectinases depolymerise pectin through hydrolysis, trans-elimination and deesterification reaction processes, breaking down the ester bond that holds together the carboxyl and methyl groups in pectin.^[7]

Endo-polygalacturonase progresses through a reaction along the following pathway:^[8]

(1,4-alpha-D-galacturonosyl)_n+m + H₂O = (1,4-alpha-D-galacturonosyl)_n + (1,4-alpha-D-galacturonosyl)_m

Occurrence and Application

Pectinase in nature

Pectinase enzymes used today are naturally produced by fungi and yeasts (50%), insects, bacteria and microbes (35%) and various plants (15%),^[9] but cannot be synthesized by animal or human cells.^[10] In plants, pectinase enzymes hydrolyze pectin that is found in the cell wall, allowing for new growth and changes to be made. The chemical and structural properties of pectin is especially prone to changes in the fruit due to solubilization and enzymatic degradation which are considered to be the key processes responsible for the softening of fruit during ripening. Structural changes that occur in the middle lamella and primary cell wall during ripening result in cell separation and softening of the tissues. The molecular components of primary walls are modified during fruit ripening by the temporally and spatially regulated action of endogenous enzymes.^[11][12]

Similarly to their role in plants, pectinases break down pectin during the developmental stage of fungi.

Industrial uses

Pectinase enzymes play various roles in both the fruit juice and wine industries. They are used for clarification in fruit juices and also speed up fruit juice extraction through enzymatic liquefaction of fruit pulp. In addition, pectinase enzymes aid in formation of pulpy products in the fruit juice industry. Pectinase enzymes are used for extracting juice from purée. This is done when the enzyme pectinase breaks down the substrate pectin and the juice is extracted. The enzyme pectinase lowers the activation energy needed for the juice to be produced and catalyzes the reaction.

Pectinases are useful in the wine industry by extracting anthocyanin from the fruit, effectively intensifying the wine coloring.^[7] Pectinase can also be used to extract juices from cell walls of plants cells.

Pectinases are also used for retting in the textile industry.^[13] Addition of chelating agents or pretreatment of the plant material with acid enhance the effect of the enzyme.