Pectin Structure and Composition

Structure of Pectin

• Pectin is comprised of heteropolysaccharides
• Pectin composes the primary cell wall of higher plants and the middle lamella
• It is responsible for the cohesion and structural integrity of plant tissues
• Rhamnogalacturonans and galacturonans are the main chemical components in pectic materials
• D-Galacturonic residue forms most molecules
• Polymers are curved and extended with high flexibility

Smooth Region or Homogalacturonan (HG)

• About 65% of pectin involves long stretches of (1 → 4)-linked d-galactopyranosyl uronic acid residues
• Further modification occurs by Methyl esterification at C-6 or acetyl groups at C-2 and C-3 position

Hairy Region or Rhamnogalacturonan (RG I)

• Makes up 20-35% of pectin as a branched area
• Involves the side chain of the repeating disaccharide unit [α- 1, 2-rahmnopyranose residues] linked by (1 → 4) disaccharide
• Contains glucuronic acids and fucose that create the structure more complex in lateral chains
• The region is acylated and often substituted with arabinans, galactans, arabinogalactans linked to rhamnose residue; plus xylose
• RGII region is a branched pectic domain-containing HG backbone substituted with heteropolymeric side chains involving different sugars, although it is not structurally related to RGI
• Includes side chains of D-Apiose, 2-Omethyl-D-xylose, and 2-O-methyl-L-Fructose
• Galacturonic residues are acetylated at C-2 or C-3 position in rhamnogalacturonan 1

Types of Pectin Substances

• Pectin's configuration varies across sources
• Pectin properties strongly relate to methylation of galact-uronic acids residues, often around 70%
• Acidic and neutral pectin has ferulic acid on non-reduced ends of neutralarabinose and/or galactose bearing domains
• Each pectin matrix carries about one teruloyl residue
• Interacting domain structures of pectin with inorganic and organic compounds make it complex

Molecular Arrangements of Pectic Substances

• Pectic substances are grouped into four categories:
• Protopectin yields pectinic acid or pectin upon restricted hydrolysis
• It is in the middle lamella and acts as glue for cell walls
• Protopectin is water-insoluble due to its molecular weight
• Ester-bond formation occurs between carboxylic acid groups and hydroxyl groups
• Salt bonding happens between carboxyl groups and basic groups
• Pectic acid or pectate, is a polymer of galacturonan with few methoxy groups and is water-soluble
• Pectinic acid is a galacturonan polymer; it contains around or up to 75% methoxy groups that can form a gel with sugar and acid
• Pectin, or polymethyl galacturonate, is a soluble polymer with roughly 75% of galacturonate units esterified with a methyl group that can for a gel with sugar and acid

Pectinolytic Enzymes

• Pectinolytic or pectinase enzymes degrade pectin-containing substances
• These enzymes represent 10% of global industrial enzyme production
• Sources include plants, insects, Nematoda, protozoa, fungi, yeast, and bacteria
• Microbial sources (fungi, yeast, and bacteria) are the primary choice for production due to technical and commercial factors
• Pectinolytic enzymes are grouped according to the substrate like pectin, pectic acid, or oligo GalA, the cleavage type like trans elimination or hydrolysis, or the mode of action like random cleavage-depolymerizing or endo/exo-enzymes

Protopectinase

• Catalyzes solubilization of protopectin with water to release soluble pectin
• Catalyzation happens at sites with three or more non-methylated GalA molecules by hydrolyzing the glycosidic bond
• Enzymes classified into two types based on catalytic action
• Type-A: Reacts with the smooth region/inner site of insoluble protopectin, supports trans-elimination by cleaving glycosidic linkages, and include endo (random) or exoenzymes (terminal)
• Type-B: Responds at polysaccharide chains/outside of protopectin, binds polyglacturonic acid chain and cell wall components

Esterase

• Esterase removes methoxyl and acetyl esters from pectin producing polygalacturonic acid and catalyzes the de-esterification of pectins
• Enzymes from fungi eliminate methyl groups via a multi-chain mechanism
• Enzymes sourced from plants attack at/near either the free carboxyl group or non-reducing-end of pectin, progressing linearly through a a single-chain mechanism
• Some purified esterases act against the reducing end or non-reducing end of pectin
• Effective esterase function requires a pH range of 5-11 and temperature of 40-70°C
• Fungal esterases optimized product at a lower pH than bacterial esterases

Methyl-esterase

• Divides the methyl ester group of pectin in a single-chain mechanism
• Freeing methanol and converting pectin into pectic acid or pectate
• Does not reduce chain length of pectin polymer
• Includes two types of pectin methylesterase- A (PmeA) and B (PmeB)

Acetyl-esterase

• Catalyzes hydrolysis of acetyl ester residues on pectin forming acetate in pectic acid

Depolymerase

• A range of depolymerizing enzymes degrade pectic substances
• Cleaves α-(1 → 4)-glycosidic bonds in DGalA or GalA units either by trans elimination or hydrolysis
• Split the -(1,4)-glycosidic bonds are split via hydrolysis (polygalacturonase) or transelimination (lyases)
• Further groups include hydrolases or lyases

Hydrolases

• Includes polygalacturonase (PG) and polymethylgalacturonase (PMG)
• Polygalacturonases (PG) splits the glycosidic linkage using water across the oxygen bridge
• Forms D-galacturonic acid monomers
• Enzyme structure degrades upon reaction with pectin particularly with free carboxylic groups in the target molecule
• Viscosity decreases with an increase of reducing end-groups
• PG studied and applied for depolymerization via hydrolysis
• PG is categorized based on action pattern

Exo-PG

• Targets terminal groups of the pectic molecule with gradual chain length reduction
• Attacks all chain links arbitrarily for more incisive and faster effect

Rhamnopolygalacturonase

• Catalyzes cleavage within/at the nonreducing terminals of the rhamnogalacturonan core chains
• Microorganisms can produce PG with various biochemical qualities and modes of action
• PGs catalyze hydrolysis at optimum temperature (30-50°C) and pH (3.5-5.5)
• Both exo-PG and endo-PG are synthesized in acidic conditions
• Some Bacterial and fungal species produce exo-PG at high basic pH (11.0) that includes Bacillus sp. KSM-P410, Bacillus licheniformis, Fusarium oxysporum
• Rhamno-PG is more efficient/stable at pH 4.0 and temperature 50°C

Polymethylgalacturonase (PMG)

• Catalyzes hydrolytic cleavage of α-1,4- glycosidic linkage in pectin
• Divided according to action pattern:
• Exo-PMG: Targets terminal groups of the non-reducing end of pectin that releases methyl mono-galacturonate
• Endo-PMG: Attacks all chain links randomly for more incisive and faster effect that results in oligomethyl-galacturonates

Lyases

• Trans eliminases breaks down pectinate polymers/pectate using Polygalacturonate depolymerization and pectin esterification
• C-4 of glycosidic linkage follows hydrogen removal from C-5 and releasing an unsaturated product with the unsaturated bond between C-4 and C-5
• Activation requires cytoplasmic/intracellular lyases and ions like Ni2+, Co2+, and Mn2+
• Polygalacturonate lyase (PGL) requires Ca2+ ions for its activity
• Classified and used in most baby food products
• Exo-PGL: Target the non-reducing terminal of pectic acid and releasing unsaturated di-galacturonates
• Endo-PGL: Works in an unsystematic cleavage that produces unsaturated oligogalacturonates
• Oligo-D-galactosiduronate lyase: Acts on the terminal position of unsaturated di-galacturonate (released) by pectate lyase and produces produces ono-galacturonates

Polymethlygalacturonate lyase (PMGL)

• Needs no metal ions for activation
• Arginine Arg 236 residues at position of Ca2+ ion are found in pectate
• Exo-PMGL: Degrades pectin using stepwise transeliminative cleavage and releasing unsaturated methylmonogalacturonates
• Endo-PMGL: acts randomly cleaving α-1,4-glycosidic linkages and producing unsaturated methyloligogalacturonates.

Pectin Lyases

• Originate mainly from microorganisms for unique biochemical properties according to each microbe
• Work at 40-50°C and alkaline pH 7.5-10.0
• Molecular weight ranges from 22-90 kDa
• PMGL reaches 89 kDa from Aureobasidium pullulans LV-10/90 kDa from Pichia pinus
• The weight for PGL of 55/74 kDa recorded in Yersinia enterocolitica/Bacteroides thetaiotaomicron
• Isoelectric points range from 5.2-10.7.
• Many other enzymes act in adjacent chains of RGI and RGII with act as exogalactanase, endogalactanase, α-/β-galactosidase, α-L-arabinofuranosidase, exoarabinase, endoarabinase

Fungal Sources

• Fungal classes include
• Phycomycetes/Mucor
• Ascomycetes/Aspergillus
• Basidiomycetes/white-rot fungi
• Many fungal strains can produce all types of pectinase enzymes
• Aspergillus niger produced metabolites, safely used involve GRAS and broadly applied it in industries
• A. niger produces various pectinases including esterase, PGL, PMGL used in wine/fruit juice industries
• Fungi pectinases are acidic that applies to acidic conditions
• Aspergillus strains yield higher pectinase production via SSF than in SmF
• Commercial Pectinases produces by fungal sources in the industry involves Trichoderma and Aspergillus

Yeast

• 4 identified yeast species: Saccharomyces fragilis, Torulopsis kefir, Candida pseudotropicalis (later renamed Kluyveromyces marxianus), and Saccharomyces thermantitonum (reclassified as Saccharomyces cerevisiae)
• Pectolytic Yeasts can produce PG, lyase, esterase based on pH, temperature, and availability
• Candida, Saccharomyces, Kluyveromyces producing PG, Rhodotorula produces pectin esterase and PG
• S. cerevisiae (most experienced yeast) was supposed to be pectolytic enzymes-free but strains since show since reduction ability for pectin
• PG is the main activity in S. cerevisiae
• Pectolytic activities show in indigenous yeasts in different fermentation or clarification and pressing of concentrated juices

Bacteria

• Pectinolytic bacteria screened that includes Erwinia carotovora subsp. carotovora, Erwinia chrysanthemi and Bacillus sp.
• Those bacteria can produce PG, pectate lyase, and pectin lyase.
• Bacillus sp. strain and E. chrysanthemi possess the highest PG production
• Bacillus sp. comes from paper mulberry bark, E. chrysanthemi comes from onion
• Bacillus licheniformis can comes from rotten vegetables and produces pectinase at conditions.
• Aeromonas caviae, B. licheniformis, and Lactobacillus are bacteria for pectinase production