Pectin and Pectinases: Production, Characterization and Industrial Application of Microbial Pectinolytic Enzymes

Pectinases are a big group of enzymes that break down pectic polysaccharides of plant tissues into simpler molecules like galacturonic acids. It has long been used to increase yields and clarity of fruit juices. Since pectic substances are a very complex macromolecule group, various pectinolytic enzymes are required to degrade it completely. These enzymes present differences in their cleavage mode and specificity being basically classified into two main groups that act on pectin " smooth " regions or on pectin " hairy " regions. Pectinases are one of the most widely distributed enzymes in bacteria, fungi and plants. This review describes the pectinolytic enzymes and their substrates, the microbial pectinase production and characterization, and the industrial application of these enzymes. 1. INTRODUCTION The increasing energy demands have focused worldwide attention on the utilization of renewable resources, particularly agricultural and forest residues, the major components of which are cellulose, starch, lignin, xylan and pectin. These materials have attracted considerable attention as an alternative feedstock and energy source, since they are abundantly available. Several microbes are capable of using these substances as carbon and energy sources by producing a vast array of enzymes in different environmental niches [1, 2].


INTRODUCTION
The increasing energy demands have focused worldwide attention on the utilization of renewable resources, particularly agricultural and forest residues, the major components of which are cellulose, starch, lignin, xylan and pectin.These materials have attracted considerable attention as an alternative feedstock and energy source, since they are abundantly available.Several microbes are capable of using these substances as carbon and energy sources by producing a vast array of enzymes in different environmental niches [1,2].
Pectinase production occupies about 10% of the overall manufacturing of enzyme preparations.Pectinolytic enzymes are widely used in the food industry for juice and wine production [3].This review describes the pectinolytic enzymes and their substrates, the microbial pectinase production and characterization, and the industrial application of these enzymes.

PECTIC SUBSTRATES
Pectic substances are complex high molecular mass glycosidic macromolecules found in higher plants.They are present in the primary cell wall and are the major components of the middle lamellae, a thin extracellular adhesive layer formed between the walls of adjacent young cells.In short, they are largely responsible for the structural integrity and cohesion of plant tissues [4,5].
Three major pectic polyssacharides groups are recognized, all containing D-galacturonic acid to a greater or a lesser extent.

Homogalacturonan (HG)
HG is a linear polymer formed by D-galacturonic acid which can be acetylated and/or methyl esterified.It can be called smooth regions of pectin.The molecule is classified according to its esterification level: pectin has at least 75 % of the carboxyl groups methylated; pectinic acid has less than 75 % of the carboxyl groups methylated; pectic acid or polygalacturonic acid has no methyl esterified carboxyl groups.Frequently the word pectin is used as a generic name for pectic substances [6].

Rhamnogalacturonan I (RGI)
RG I is composed of the repeating disaccharide rhamnose-galacturonic acid.The galacturonic residues can be acetylated and both residues can carry side chains of neutral sugars as galactose, arabinose and xylose [7].

Rhamnogalacturonan II (RGII)
Despite its name, RGII is a homogalacturonan chain with complex side chains attached to the galacturonic residues [7].Vincken and coworkers [8] have proposed a pectin molecule structure model in which HG and RGII are long side chains of RGI backbone (Fig. 1).Both RG chains are also called hairy regions of pectin molecule.
In unripe fruit, pectin is found as a water insoluble pectic substance, the protopectin, bounded to cellulose microfibrils conferring rigidity on cell walls.During ripening the fruit enzymes alter the pectin structure by breaking the pectin backbone or side chains, resulting in a more soluble molecule [9].Pectic subsances tend to form a gel structure when portions of HG are cross-linked forming a three dimensional crystalline network in which water and solutes are trapped (Fig. 2).Various factors determine gelling properties including temperature, pectin type, esterification degree, acetylation degree, pH, sugar and other solutes, and mainly the interaction between calcium ions and pectin unesterified carboxyl groups.In high-ester pectins, the junction zones are formed by the cross-linking of HG by hydrogen bridges and hydrophobic forces between methoxyl groups, both promoted by high sugar concentration and low pH [7].
Pectic polysaccharides have been used as bioactive food ingredients and as detoxifying agents.It is an adequade infant food supplement [10].

PECTINOLYTIC ENZYMES
Pectinases are an enzyme group that catalyzes pectic substance degradation through depolymerization (hydrolases and lyases) and deesterification (esterases) reactions.
The well-known pectinolytic enzymes are homogalacturonan degrading enzymes.Fig. (3) shows the action mode of the most studied pectinases.

Protopectinases
Protopectinases solubilize protopectin forming highly polymerized soluble pectin [5,9].They are classified into two types: one reacts with the polygalacturonic acid region of protopectin, A type; the other with the polysaccharide chains that may connect the polygalacturonic acid chain and cell wall constituents, B type [12].

Pectin Methyl Esterases (PME)
Pectin methyl esterase or pectinesterase (EC 3.1.1.11)catalyzes deesterification of the methoxyl group of pectin forming pectic acid and methanol.The enzyme acts preferentially on a methyl ester group of galacturonate unit next to a non-esterified galacturonate unit.It acts before polygalacturonases and pectate lyases which need non-esterified substrates [9].It is classified into carbohydrate esterase family 8 [13].

Pectin Lyases (PL)
Pectin lyase catalyzes the random cleavage of pectin, preferentially high esterified pectin, producing unsaturated methyloligogalacturonates through transelimination of glycosidic linkages.PLs do not have an absolute requirement of Ca 2+ but they are stimulated by this and other cations [6].Up until now, all described pectin lyases are endo-PLs (EC 4.2.2.10) [28].Van Alebeek and coworkers [29] conducted a detailed study of the action mode of pectin lyase A from Aspergillus niger which produces mono-, di-, tri-and tetragalacturonates, besides unsaturated di-, tri-and tetragalacturonates from methyloligogalacturonates. Unsaturated monogalacturonates were not identified in the reaction products in any assay.
Both lyase groups are classified into polysaccharideslyase family 1.As shown in Table 2, lyases from fungi present optimum activity in acid and neutral medium, while those from bacteria are more active in alkaline medium.
Complete degradation of pectin substrate still requires enzymes that cleave the rhamnogalacturonan chain.

Rhamnogalacturonan Lyases
RG lyase (EC 4.2.2.-) catalyzes the random transelimination of the rhamnose-galcturonate linkage from rhamnoga- lacturonan chain, producing an unsaturated galacturonate at nonreducing end of one oligomer and a second oligomer containing a rhamnose as a reducing end residue [42].These enzymes are classified into polysaccharides-lyase families 4 and 11 [13].

MICROBIAL PECTINASES
Pectinolytic enzymes are naturally produced by many organisms like bacteria, fungi, yeasts, insects, nematodes, protozoan and plants.Microbial pectinases are important in the phytopathologic process, in plant-microbe symbiosis and in the decomposition of dead plant material, contributing to the natural carbon cycle.Pectinases are abundantly produced by saprophytic fungi, and decaying plant tissue represents the most common substrate for pectinase-producing microorganisms [10].Plant attack by pathogenic microorganisms usually starts by pectinolytic enzymes attack since pectic substances are more accessible than other fibers in plant tissue [4,45].
Several studies on microbial enzymes have shown the production of multiple pectinase forms which differ on molecular mass and kinetic properties [15,46,47].The production of multiple forms of enzymes improves the microorganism ability to adapt to environmental modifications [48].The cell wall is the first defense line of plants, having a crucial role in preventing infections.It consists mainly of the large biopolymers cellulose, hemicellulose, lignin and pectin; however, the composition can change according to the age and physiological conditions of the plant.In such case, the pathogenic microorganism needs to produce a pool of enzymes to successfully infect the plant tissue [49,50].
Pectic substances are rich in negatively charged or methyl-esterified galacturonic acid.The esterification level and the distribution of esterified residues along the pectin molecule change according to the plant life cycle and between different species.Thus, the ability of some phytopathogenic microorganisms to poduce a variety of pectinolytic enzymes that differ in their characteristics, mainly in their substrate specifity, can provide them with more efficacy in cell wall pectin degradation and consequently more success in the plant infection [54].
Several polygalacturonases can be expressed from only one gene or from some different genes.Caprari and coworkers [55] reported the production of four endo-polygalacturonases by the pathogenic fungus Fusarium moniliforme that differ in the extent of glycosylation of the same polypeptide arisen one gene.On the other hand, the polygalacturonases of Aspergillus niger are encoded by a family of diverged genes.The PG isozymes present significant differences in substrate specificity, kinetic parameters and optimum pH [56,57].The polygalacturonases isozymes produced by the phytopathogenic fungus Botrytis cinerea have been isolated by using different fermentation conditions [58][59][60][61][62][63][64].
Most of the plant cell wall degrading enzymes are encoded by a large multigenic family showing diverged expression pathways suggesting functional specialization [65].These genic families result from gene duplication and are linked in tandem [66].
Since pectin can not enter the cell, it has been suggested that compounds structurally related to this substrate might induce pectic enzyme productions by microorganisms.Low levels of constitutive activities may attack the polymeric substrate and release low molecular products which act as inducers [67,68].
De Vries and coworkers [69] had studied the expression of 26 pectinolytic genes from Aspergillus niger under 16 different growth conditions to obtain an expression profile for each gene.Their results provide strong indication for a general activating system for pectinolytic gene expression in A. niger responding to the presence of D-galacturonic acid or a metabolite derived from it.This system regulates both genes encoding enzymes acting on the main chain of pectin as well as genes encoding accessory enzymes.Regulation of the pectinolytic genes appears to be complex, individual genes are expressed at different times and in response to different D-galacturonic acid containing carbon sources.
Submerged fermentation is a well developed system used in industrial scale to produce a large variety of microbial metabolites.SmF is technically easier than SSF and has been strongly developed from the 1940s onwards because of the necessity to produce antibiotics in large scale [74].
On the other hand, solid state fermentation provides higher productivity due to larger enzyme yields [75].SSF processes simulate the living conditions of many higher filamentous fungi.Ascomycetes, basidiomycetes and deuteromycetes developed in terrestrial habitats on wet substrates.Higher fungi and their enzymes, as well as spores or metabolites, are well adjusted to growth on solid wet substrates.For instance, fungal spores produced by SSF show higher stability, are more resistant to drying and exhibit higher germination rates for extended periods of time after freeze-drying than do spores produced by SmF.Solid state fermentation has only found restricted applications in processes using unicellular organisms.Despite the advantages, the industrial application of SSF is, at least at present, hard to envisage.The main obstacles are the low amenability of the process to regulation, the strongly heterogeneous fermentation conditions and the ensuing frequently unsatisfactory reproducibility of the results, difficult scale-up, the often unfeasible biomass determination and complicated product purification by downstream processes resulting from the use of heterogeneous organic growth substrates [74].

PURIFICATION OF PECTINOLYTIC ENZYMES
The analysis of enzyme activity in the crude extract does not indicate either an isolated action or the presence of a multienzymic system working in synergy on the substrate degradation.The characterization of purified enzymes is an important research line since it provides discrimination between the enzymic complex components about substrate degradation mechanism, optimum activity conditions and enzyme synthesis regulation.
Contreas-Esquivel and Voget [17] purified 470 fold the PGI from a culture extract of Aspergillus kawakii with a recovery of 8.6 % of the initial activity in three steps: acetone precipitation, Sepharose Q and Sephacryl S-100 column chromatographies.The PG from Thermoascus aurantiacus was isolated with 21 fold increase in specific activity with a recovery of 24.6 % by Sephadex G-75 gel filtration followed by SP-Sepharose ion exchange chromatography [26].Celestino and coworkes [76] purified 9.37 fold one pectinase produced by Acrophialophora nainiana which has exopolygalacturonase and pectin lyase activity, 60.6 % of the enzyme was recovered after three steps: Sephacryl S-100 gel filtration, DEAE-Sepharose ion exchange and another gel filtration on Sephadex G-50.Kashyap and coworkers [34] developed a purification strategy for the isolation of the pectin lyase from Bacillus sp.DT7.The enzyme was precipitated with ammonium sulphate followed by DEAE-Sephacel and Sephadex G-150 column chromatographies.The pectin lyase produced by Aspergillus flavus was purified 58 fold with a recovery of 10.3 % of the initial activity in three steps: ammonium sulphate fraction, DEAE-Cellulose ion exchange and Sephadex G-100 gel filtration [77].Semenova and coworkers [30] isolated five pectinases produced by Aspergillus japonicus, PGI, PGII, PEI, PEII and PL, by hydrophobic and ion exchange column chromatographies.The polygalacturonase from Streptomyces lydicus was purified with 57.1-fold increase in the specific activity and a yield of 54.9 % after ultrafiltration followed by CM-Cellulose and Sephadex G-100 column chromatographies [78].
As shown, conventional chromatography techniques have been efficiently used to purify pectinolytic enzymes.

INDUSTRIAL APPLICATION
Pectinase production occupies about 10% of the overall manufacturing of enzyme preparations.These enzymes are widely used in the food industry in the production of juices, fruit drinks and wines [3].

Acid Pectinases
Acid pectinases, which are widely used in extraction, clarification, and removal of pectin in fruit juices, in maceration of vegetables to produce pastes and purées, and in winemaking, are often produced by fungi, especially Aspergillus niger .
The crushing of pectin-rich fruits results in high viscosity juice which stays linked to the fruit pulp in a gelatinous structure, hindering the juice extraction process by pressing.Pectinase addition in the extraction process improves the fruit juice yield through an easier process, decreases the juice vicosity and degrades the gel structure, thus improving the juice concentration capacity [5,9].In the case of fruit juice, extraction by enzymatic maceration can increase yields by more than 90% compared to conventional mechanical juicing, besides improving the organoleptic (color, flavor) and nutritional (vitamins) properties and technological efficiency (ease of filtering) [4].
In several processes, pectinolytic enzymes are applied associated with other cell wall degrading enzymes such as cellulases and hemicellulases [79].The mixture of pectinases and cellulases has been reported to improve more than 100 % juice extraction yields [5,9].Soares and coworkers [73] reported an improvement between three and four times in juice yields from papaya, banana and pear using enzymic extraction instead of the conventional pressing process.
The enzymic treatment can help decrease 62 % of the apple juice viscosity.When the depectinized apple juice is ultrafiltered, the permeate flux is much higher than when undepectinized juice is processed.The increase in the permeation rate is a result of both the reduction in apple juice viscosity and the reduction in total pectin content.Pectin is a fibershaped colloid that causes severe fouling of ultrafiltration membranes [80].
The commercially available pectinase preparations used in food processing are traditionally associations of polygalacturonases, pectin lyases and pectin methyl esterases.These preparations are usually derived from fungi, mainly the genera Aspergillus [3,10].

Alkaline Pectinases
Alkaline pectinases are generally produced by bacteria, particularly species of Bacillus, but are also made by some filamentous fungi and yeasts [34,45,81].They may be used in the pretreatment of waste water from vegetable foodprocessing that contains pectin residues; the processing of textile fibers such as flax, jute and hemp, coffee and tea fermentation, vegetable-oil extraction and the treatment of paper pulp [9,45,81,82].
Pectinolytic enzymes have been applied to the degumming of jute, sunn hemp, flax, ramie and coconut fibers for textile application [82][83][84].Degumming can be done by adding pectinolytic mixtures or by fiber fermentation (dewretting) using pectinase-producing microorganisms [85,86].In order to remove the non-cellulosic gummy material composed mainly of pectin and hemicellulose, Kapoor and coworkers [81] had run three treatments on ramie and sunn hemp bast fibers: enzymic, chemical and chemical associated with enzymic treatment.Of the three treatments, the third one was the most promising for degumming.The scanning electron microscopic studies revealed a complete removal of non-cellulosic gummy material from the surface of ramie and sunn hemp fibres.
Bioscouring is an alternative and more environmentally friendly method to remove non-cellulosic "impurities" from raw cotton by specific enzymes to make the surface more hydrophilic [87].Pectins are responsible for the hydrophobic properties of raw cotton and its degradation by pectinolytic enzymes was suggested to facilitate also removal of waxes and could thus lead to a considerable reduction rate of water and chemicals consumption and of effluent discharge.In contrast to drastic alkaline conditions conventionally used, this treatment with pectin degrading enzymes would not affect the cellulose backbone and thus avoid fiber damage [32,33,88].Klug-Santner and coworkers [33] reported up to 80 % of pectin removal from the outer layer of cotton by a purified endo-pectate lyase from Bacillus pumilus BK2.
During papermaking, alkaline peroxide bleaching of mechanical pulps solubilizes acidic polysaccharides which are troublesome interfering substances.Some of these acidic polysaccharides are pectins, or polygalacturonic acids.The ability of polygalacturonic acids to complex cationic polymers (cationic demand) depends strongly on their degree of polymerization, so monomers, dimers, and trimers of galacturonic acid did not cause measurable cationic demand, but hexamers and longer chains had high cationic demand.Pectinases can depolymerize polymers of galacturonic acid, and consequently lower the cationic demand of pectin solutions and the filtrates from peroxide bleaching [89,90].
Enzymes involved in the breakdown of plant cell wall polyssaccharides can be used to extract vegetal oils, coconut germ, palm, sunflower seed, rape seed olives and kernel oils which are traditionally produced by extraction with organic solvents, such as the potentially carcinogen hexane [9].By degrading cell wall components like pectin enzymes promote the oil liberation.
According to Angayarkanni and coworkers [91], adding pectinases in association with cellulases, hemicellulases and proteinases to the tea-leaf fermenting bath raises the tea quality index by 5 %.

CONCLUSION
Many studies have been conducted on the production, isolation and characterization of pectinases from various microorganisms, mainly enzymes specific to pectin smooth regions.But a few works have been published about hairy pectin region specific enzymes.The difficulties to obtain the appropriate substrate might be the biggest problem to develop studies with RG-degrading enzymes.
There are a lot of industrial processes to which pectinases can be applied to improve the quality and the yield of final products.In this way, it is important to investigate the production conditions and physico-chemical characteristics of new enzymes.Screening a large number of microorganisms for high active enzymes combined with protein engineering, direct evolution and metagenome approaches can lead to more efficient and stable enzymes.Enzymes having a set of biochemical and physical properties can be generated for each specific industrial process.
Pectinases structures have been solved by cristalography and nuclear magnetic resonance techniques allowing for the elucidation of the molecular basis behind substrate specificity.The future efforts into pectinase research should be concentrated on elucidation of the regulatory mechanism of enzymes secretion at the molecular level and the mechanism of action of different pectinolytic enzymes towards pectic substrates.These studies can provide valuable tools to manipulate microorganisms making them able to produce efficient enzymes in high amounts.

Fig. ( 1 ).
Fig. (1).The basic structure of pectin.Schematic representations of the conventional (A) and recently proposed alternative (B) structures of pectin.The polymers shown here are intended only to illustrate the some of the major domains found in most pectins rather than definitive structures.[7].

Table 1 . Properties of Some Purified Polygalacturonases
*Commercial names.