Applications of Pectinolytic Enzymes in Process Industries

Abstract

The manufacturing of industrial important products by using enzyme technology is sustainable method and much to offer as compared to using chemical catalyst. The enzyme can manage the industrial preparation under mild reaction conditions using specific substrate. Enzyme uses shorter time, produces limited or no wastes and eco-friendly in nature. Pectinase complex enzyme system catalyzes the breakdown of pectin polymers. Pectinase is a generic term used for a group of enzymes that catalyzes the degradation of pectin substances by hydrolysis, trans-elimination, as well as, de-esterification reactions. Pectinase is produced from various organisms including bacteria, fungi, yeast, insects, protozoa and nematodes. Microorganism is usually used for industrial production of pectinase because of its easily growth and cost-effective downstream process. Pectinase has various applications in different industrial process such as fruit juice extraction, treatment of wastewater, papermaking, degumming of plant-based fibers, coffee and tea fermentation. This chapter describes the importance of pectinase and its application in different industrial process. Furthermore, it gives detail review about the pectinase and opportunities for the future research.

1. Introduction

1.1 Pectin

The cell wall surrounds the plant cell and protects the cell and its distinctive components that are essential for plant survival from different environmental pressure. The plants cell wall supports the plant to survive under various climate conditions and the polysaccharides in most abundant material composition of cell as compared to protein, aromatic and aliphatic compounds. Pectin polymer plays significant role in the composition of cells and acts as cementing agent to structural organization and functionality of cell. Pectin is mostly present in the cell walls of higher plants. It is one of the major components of plants and responsible for the structure integrity and cohesion of plant tissues (Figure 1) [2, 3]. The term pectin was initially quoted in eighteenth century from tamarind fruit as a peculiar substance [4]. The basic characterization of pectin was done on nineteenth century and categorized it as active component of fruit and fruit-based products [5]. As an outcome, Nussinovitch suggested the word ‘pectin’ in Ref. to Greek work ‘pektikos’ which mean ‘congeal, solidify or curdle’ [6]. The actual chemistry of pectin began when Ehrlich [7] discovered that the D- galacturonic, an isomer of D-gluronic acid, is the central constituent of pectin, and the some of these D-galacturonic acid are partially esterified with methyl alcohol. Now, pectin is thought to be composed of at least seventeen kinds of different monosaccharides, in which D-galacturonic acid is usually the most abundant, followed by D-galactose or L-arbinose [8, 9].

Figure 1.

Cell wall structure of higher plants [1].

1.1.1 Structure of pectin

The pectin is a complex carbohydrate polymer of galacturonic acid residues having esterified carboxylic groups with methanol. The degree of esterification of carboxylic groups of the pectin has been changed from different sources. Pectin consisted of four polysaccharides including homogalacturonan, RGI (xylogalacturonan rhamnogalacturonan I) and RGII (rhamnogalacturonan II) (Figure 2) [11, 12]. The homogalacturonan is the main linear chain of galacturonic acids linked through α-1, 4 linked and makes the main chain of pectin polymer. RGI formed through the linkage of ragmnopyranose residues by α-1, 2 bond and makes the branches with the polymer. D-apiose, 2-O-methyl-D-xylose-and 2-O-methyl-L- fructose side chain generates the RGII region of pectin polymer [13]. The C-2 or C-3 position of galacturonic acid chain is acetylated in rhamnogalacturonan 1 region of pectin polymer, and most of the chains are consisted of D-galacturonic acids. In aqueous conditions the pectin polymer stand in pure form conformation with great flexibility and do not form straight conformation. This composition of pectin was varied among different sources [14]. The properties of pectin are strongly depending on methylation of galacturonic acids residues, which is usually 70%.

Figure 2.

Basic structure of pectin [10].

1.1.2 Classification of pectin

The pectin polymer is divided into protopectin, pectic acid, pectinic acid and pectin because of variation in backbone chain of galacturonic acid residues [15].

1.1.2.1 Protopectin

Protopectin term is used to illustrate the insoluble pectin. It is parent pectin and yield pectin or pectinic acid on restricted hydrolysis.

1.1.2.2 Pectic acid

Pectic acid contains insignificant amounts of methoxy groups. It is soluble form of pectin.

1.1.2.3 Pectinic acid

Pectinic acid is the polygalacturonan that contains various amounts of methoxy groups having the property to form gel with sugar and acid.

1.1.2.4 Pectin (Polymethylgalacturonate)

Pectin is the polygalacturonic acid in which the galacturonic acid residues are 75% methylated.

1.1.3 Distribution of pectin in nature

Pectin is widely disturbed in nature and mostly found in angiosperms and gymnosperms plants along with pteridophytes, bryophytes, lycophytes and carophytes [16]. The pectin represents 35% of total plant biomass and among the high percentage containing biomolecules in plants [17]. The concentration of pectin in plants varied according to the plant types, maturation time and its environmental conditions (Table 1). The 100 grams of citrus peels and apple pomaces contained 20 grams of pectin and have been used for commercial extraction of pectin [21].

Fruits	Botanical name	Pectin content (%)	References
Apple	Malus spp	0.5–1.6	[18]
Apple pomace		1.5–2.5	[19]
Banana	Musa acuminate L.	0.7–1.2	[18]
Beet pulp	Beta vulgaris	1.0	[19]
Carrot	Daucus carota	0.2–0.5	[19]
Giant granadilla	Passiflora quandrangularis L.	0.4	[20]
Carambola	Averrhoa carambola	0.66	[20]
Guava	Psidium guajava L.	0.77–0.99	[20]
Lemon pulp	Citrus limon	2.5–4.0	[19]
Lychee	Litchi chinesis S.	0.42	[18]
Mango	Mangifera indica L.	0.26–0.42	[20]
Orange peel	Citrus sinesis	3.5–5.5	[19]
Papaya	Caricia papaya	0.66–1.0	[20]
Passion fruit	Passiflora edulis L.		[20]
Passion fruit rind		2.1–3.0	[20]
Peaches	Prunus persica	0.1–0.9	[18]
Pineapple	Ananas comosus L.	0.04–0.13	[20]
Strawberries	Fragaria ananassa	0.6–0.7	[20]
Tamarind	Tamarindus indica L.	1.71	[20]
Thimbleberry	Rubus rosalfolius	0.72	[20]
Tomato fruit	Lycopersicon esculentum	0.2–0.6	[18]

Table 1.

Pectin content in various fruits.

1.1.4 Commercial utilization of pectin

Pectin has been used in various industrial processes and applications due to its biocompatibility, cost-effective, easily available and among the most abundant natural compounds on earth. The food additive, thickening agent, gelling agent, cosmetic texturizing agent, a good source of dietary fiber and also components of biodegradable films, adhesives, paper and antimicrobial food packaging are the major applications of pectin in industries [22]. The mechanism of enzymatic modification of pectin polymer needs to be understood to further increase the industrial scope of pectin polymer.

1.2 Pectinase

Pectinase is a complex group of enzyme that catalyzes the degradation of pectin polymer [23]. Pectinase has wide range of applications in fruit juices preparation, textile processing, papermaking, pectin containing wastewater treatment, degumming of plant’s bast fibers, wine clarification, oil processing and coffee and tea industry.

1.2.1 Classification of pectinase

On the basis of pectin degrading mechanism, the pectinase enzymes are classified into polygalacturonase, pectin lyase and pectin esterase (Figure 3).

Figure 3.

Mode of degradation of pectin by different pectinases. (A) Polygalacturonase (B) pectin esterase (C) pectin lyase [24].

1.2.1.1 Polygalacturonase

Polygalacturonase catalyzes the hydrolysis of pectin polymer into D- galacturonic acid monomer by addition of water molecules in α-1, 4 glycosidic linkages. Polygalacturonase is the most extensively studied enzyme among different pectinolytic enzymes and further classified into exo-polygalacturonase and endo-polygalacturonase by breaking of external and internal glycosidic bond of polymer chain, respectively.

1.2.1.2 Pectin lyase

Pectin lyase catalyzes the breaking of α- 1, 4 glycosidic bonds and generates galacturonide with unsaturated galacturonic acid. Pectin lyase is classified into exo-pectin lyase and endo-pectin lyase on the basis of catalyzing the breaking of α- 1, 4 glycosidic bonds sequentially and randomly, respectively.

1.2.1.3 Pectin esterase

Pectin esterase is also known as pectin methyl hydrolase that catalyzes the de-esterification of pectin molecule into pectic acid and methanol.

1.2.2 Structure of pectinase

The structure of pectinase (Figure 4) enables to understand the enzyme reaction mechanism on molecular basis and also provide information about the structural differences between enzymes that directly affect the catalytic properties of enzymes. Yoder et al. [26] characterized the three-dimensional structure of pectinase from E. chrysanthemis. The structure of pectinases composed of a domain of parallel β strands folded into a large right-handed cylinder. The inner cylinder contains seven to nine helical turns and due to distinctive arrangement of three β- strands in each turn of helix look like a prism shaped. The consecutive stands assemble to form three parallel β-sheets named PB1, PB2 and PB3. Even the mechanism of pectin degradation by three pectinases is different, the substrate binding figure out from structure, sequence similarity and site directed mutagenesis studies [27], are all observed in a similar location within cleft formed on the outer of the parallel β- helix among one side of PB1 and the overhanging loops [28].

Figure 4.

Three-dimensional structure of pectinase [25].

1.2.3 Sources of pectinase

Pectinase plays a very significant role in various biological processes across the entire field of living organism. The pectinase is widely distributed in nature and produced by different living organisms such as plants, microorganisms, insects, nematodes and protozoa. Microorganisms are considered as primary source for the production of industrial important enzymes.

1.2.3.1 Microbial pectinase

Pectinase is one of the important factors in the plant pathological process, plant-microbe symbiosis and in the decomposition of plant decay matters. The microbial pectinase is playing important role in nature by contributing natural recycling of carbon in the environment. Different microorganisms are known to produced pectinase with different molecular mass and catalytic properties. Aspergillus niger is mostly used for the industrial production of pectinase [29]. The pectinases from fungus sources are usually acidic in nature and only can work in acid conditions. Production of alkaline pectinase remains under developed, as only few reports are available on the production alkaline pectinase by bacterial strains [30, 31, 32]. Alkaline pectinase can be used for the treatment of pectineus substances containing wastewater from vegetable and food processing industries [33].

1.2.3.2 Bacillus

Bacillus is one of the large genera of bacterial strains. It is rod-shaped, endospore bearing bacteria and belongs to the family Firmicutes. The endospores of Bacillus are more resistant to heat, drying, disinfectants and other destructive agents. The genus Bacillus covered a great diversity of strains and some of them strictly aerobic, while the other are facultative anaerobic. The Bacillus especially Bacillus subtilis and Bacillus licheniformis are excellent candidates for large-scale production of commercially important enzymes.

1.2.3.3 B. licheniformis

B. licheniformis is a gram positive, rod-shaped and endospore forming bacterial strain, used for the production of different industrial important products. B. licheniformis is a saprophytic bacterium in nature and commonly found in soil and other natural environment. The B. licheniformis is capable of growing on a large diversity of nutrient sources because of synthesizing and secreting different hydrolytic enzymes, and this quality makes the B. licheniformis an industrial important microorganism.

Following are some of very important commercial applications of B. licheniformis.

1. B. licheniformis is widely used for production of commercially important thermostable enzymes like protease and α-amylase which are stable at 105–110°C for short period of times [34].

2. B. licheniformis is also used to produce commercially important antibiotics such as bacitracin and surfactin, as well as poly-gamma-glutamic acid in great numbers [34].

3. Thermostable α–amylase from B. licheniformis has been used for the liquefaction of wheat flour [35] and corn meal [36], and the hydrolysate was then saccharified to produce ethanol using Saccharomyces cerevisiae.

1.2.4 Production of pectinase

Fermentation technology has been effectively used in pectinase production by both fungus as well as bacterial strains. On the basis of production of pectinase, SSF fermentation provides higher productivity as compared to SmF fermentation [37]. Even with the advantage of high productivity, the industrial application of SSF is hard to visualize due to difficultly in product recovery. SmF is well developed fermentation technique used for large-scale production of metabolites and technically easier to perform as compared to SSF [38].

1.2.5 Biochemical properties of pectinase

Biochemical properties of pectinase are very important for their commercialization on industrial scale. The characteristics of pectinase produced from different microorganisms have been reported, and the biochemical properties of enzyme were varied from source to source [39, 40, 41]. The pectinase from various microorganism has different range of optimum temperature (30–60°C) and pH (3.0–9.0) for maximum enzymatic activities [40]. They also have different molecular weight, thermal stability and kinetic parameters [11]. Most of the pectinase have been reported to perform maximum activity in range of 40–50°C [42, 43, 44]. Mohamed et al. [45] reported that pectinase from Trichoderma harzianum showed 100% stability at 30°C after 60 minutes of incubation. The stability of enzyme provides valuable information about its structure and function. Bacillus sp. MG-cp-2 produced alkaline pectinase having stability at broad pH range and retained 80% of its initial activity at room temperature after 24 hours [46]. The activity of pectinase was increased in the presence of Fe²⁺, Mn²⁺, Ca²⁺ and Cu²⁺, but strongly inhibited by Mg²⁺ [41]. Hg²⁺, Zn²⁺ and Cu²⁺ were found to inhibit the pectinase activity, while Mn²⁺ and Co²⁺ enhanced the pectinase activity from S. sclerotinium [47].

1.2.6 Applications of pectinase

Pectinase is one of the very important enzymes in fruits and textiles industries as well as having different biotechnological application in various industries. Following are some important applications of pectinase in different aspects.

1.2.6.1 Fruit and vegetable juices industries

Pectinase has been widely used in fruit and vegetable juices industries. These industries commercially produced different juices including sparkling clear juices, cloudy juices and unicellular product. In sparkling clear juices, pectinase is added to enhance the production juices by hydrolyzing pectin particles [48].

1.2.6.2 Textile industry

Pectinase with the combination of other enzymes such as amylase, lipase, cellulase and hemicellulose has been used to degrade sizing agents from cotton in textile industry. Uses of enzymes ensured the low discharge of waste chemical in environment and improved both the environmental safety and the value of product. Traditionally the scouring of cotton was done by using 3–6% aqueous sodium hydroxide and high energy. Bioscouring is a novel process of using enzymes to specifically remove the non-cellulosic impurities such as pectin, protein and fats from the fiber. In addition, energy conservation and environmentally friendly bioscouring process also limit the fiber damage [49].

1.2.6.3 Degumming of plants bast fibers

Pectinase has been used in degumming process of plant fibers such as ramie, sunn hemp, jute, flax and hemp to remove the gum before subjecting them for textile making [31, 46, 50, 51]. The enzymatic degumming process using pectinase with combination of xylanase is environmentally friendly and economic and is excellent replacement of chemical degumming process, which is polluting, toxic and non-biodegradable [46].

1.2.6.4 Retting of plant fibers

In traditional retting process, mixed microbial cultures were used which produced pectinase that releases cellulosic fibers from fiber bundles. As compared to traditional retting, enzymatic retting is faster, controlled and produces fewer odors. Pectinase has been used to separate the fibers from jute and flax by eliminating pectin [49, 51].

1.2.6.5 Wastewater treatment

Typically multiples steps such as physical dewatering, spray irrigation, chemical coagulation and chemical hydrolysis are carried out for the treatment of wastewater from vegetable food industries that mostly contain pertinacious materials. Pectinases used in various industrial processes are a better choice as compared to traditional chemical methods because they are cost effective and ecofriendly. They can eliminate pectin containing substances and can make activated sludge treatment more feasible [33, 49, 52].

1.2.6.6 Coffee and tea fermentation

Pectinase has a significant role in tea and coffee fermentation. In addition to increased tea fermentation, pectinase also destroys the foaming character of instant tea powder by degradation of pectin [53].

1.2.6.7 Paper industry

The uses of different enzymes such as xylanase, ligninase, mannanase, pectinase and α-galactosidase are increasing for biobleaching and papermaking in the paper industry [54, 55]. Pectinase lowers the cationic requirement in papermaking processes [56, 57].

1.2.6.8 Animal feed

Pectinase has been used in feed preparation as multienzyme cocktail with glucanase, xylanase, protease and amylase that reduce the feed viscosity and increase the absorption nutrients, release nutrients either through hydrolysis of non-degradable fibers [49].

2. Conclusion

Pectinase is very important for sustainable industrial processes and can be applied for different industrial applications including food, textile, paper, wine production and research. Pectinase catalyzes the breakdown and modification of pectin-based substances through hydrolysis, trans-elimination and de-esterification reactions. Pectinase can be obtained from different organisms, and microorganisms are usually used for industrial pectinase productions. Microbial pectinases have tremendous applications in different industries and among the top most industrial enzymes that have significance role in the current biotechnological world.

Acknowledgments

The author acknowledged and is thankful to higher education commission Pakistan for financial support by providing local challenge fund to work on enzyme manufacturing.

Conflict of interest

The authors declare no conflict of interest.