APLIKASI BEBERAPA ENZIM HIDROLITIK

PROTEASE

Proteases, also known as proteinases or proteolytic enzymes, are a large group of enzymes that catalyze the hydrolysis of peptide bonds in proteins and polypeptides. They differ in properties such as substrate specificity, active site and catalytic mechanism, pH and temperature optima, and stability profile. There are several schemes for classifying proteases, which provide a wealth of relevant information about each protease. According to the Enzyme Commission (EC) classification, proteases belong to hydrolases (group 3), which hydrolyze peptide bonds (subgroup 4). Proteases can be classified into two major groups based on their ability to cleave N-or C-terminal peptide bonds (exopeptidases) or internal peptide bonds (endopeptidases). Although exopeptidases find commercial applications (such as leucine aminopeptidase in the debittering of protein hydrolysates), endopeptidases are industrially more important. By proteolytic mechanism, proteases are currently classified into six broad groups: serine proteases, threonine proteases, cysteine proteases, aspartic proteases, metalloproteases, and glutamic acid proteases. Alternatively, proteases may be classified into acidic, neutral, and alkaline (basic) proteases by the optimalpHin which they are active. The acid proteases have pHoptima in the range of 2.0–5.0 and aremainly fungal in origin. Proteases having pHoptima around 7.0 are called neutral proteases. They aremainly of plant origin and somebacteria and fungi also produce neutral proteases. Proteases with pHoptima in the range of 8.0–11.0 are grouped under the category of alkaline proteases. Some of the important alkaline proteases are those from Bacillus and Streptomyces species.

There are thousands of different protease molecules that have been isolated and characterized. Among them, several hundred proteases are commercially relevant, and have been used in laundry and dishwashing detergents, food processing, animal-feed additives, leather processing, waste treatment, pharmacology, and drug manufacture. The main industrial application of proteases is their use as detergent additives to remove protein deposits and stains, and the major player is subtilisins [7–9]. They are used in the detergents for dish washers and all types of powder and liquid laundry detergents as well as in laundry bleach additives. The subtilisin concentration in detergent and cleaning products is very low and depends on the type of product, typically ranging between 0.007% and 0.1%. Subtilisins (<10%) are also used in technical applications such as protein hydrolysate production, leather treatment, and in the textile and cosmetics industry.
According to the catalytic mechanism, subtilisins can be defined as serine proteases. The catalytic triad of subtilisins consists of aspartic acid, histidine, and serine, but their amino acid sequences and three-dimensional structures are apparently different from those of the other serine proteases, such as chymotrypsin and carboxypeptidase. Although the size of subtilisins varies from 18 to 90 kDa, all the subtilisins used in detergents are a globular protein with an average molecular weight of 27 kDa, consisting of 269–275 amino acids. The enzyme is active from pH 6 to 11, with an optimal activity in the pH range between 9 and 11.

The alkaline subtilisins from Bacillus species currently account for the major proteases used in the detergent industry [7, 10]. The following factors may explain this situation. First, the high stability and relatively low substrate specificity of these extracellular proteases make themselves excellent candidates for detergent additives; second, their production as extracellular enzymes greatly simplifies the separation of the enzyme from the biomass and facilitates other downstream processing steps. In addition, the ability of Bacillus strains to secrete enzymes over a very short period of time into the fermentation broth warrants a high production efficiency.
Over the past 20 years, enormous efforts to develop new and improved proteases for use in detergents – either through the protein engineering of traditional subtilisins or by searching the metagenome for new enzymes [11]. Hydrogen peroxide and peroxo acids are generated in the cleaning process of bleach-containing products. The oxidation of certain methionine residues to sulfoxides was known to be responsible for the inactivation by hydrogen peroxide. The first genetically engineered subtilisin was reported in 1985 to address the sensitivity of subtilisin to oxidation by peroxide [12]. Since then, most of the amino acid positions of subtilisin have been modified either by site-directed mutagenesis or by random mutagenesis. Several gene-shuffling approaches have been performed with subtilisins to improve their properties such as activity in organic solvents, temperature stability, and activity at high or low pH. In addition to classical microbiological screening methods, the exploitation of genomic data and metagenomic screening methods has been established and has enlarged the screening pool for search of new proteases. This search is not limited to subtilisins, but is also directed at finding completely new protease backbones, which has led to newer protease preparations with improved efficiency and better stability toward temperature, oxidizing agents, and changing wash conditions.
Proteases are a powerful tool for modifying the properties of food proteins, and they are widely used in the production of value-added food ingredients and food processing for improving the functional, nutritional, and flavor properties of proteins [13]. The function of proteases is to catalyze the hydrolysis of proteins, which has been exploited for the production of protein hydrolysates of high nutritional value from casein, whey, soy protein, and fish meat.
The most significant property of acidic proteases is the ability to coagulate proteins. Microbial acidic proteases have largely replaced the calf enzyme (rennet) in the dairy industry for their ability to coagulate milk protein (casein) to form curds from which cheese is prepared. A protease from Pseudomonas fluorescens R098 has found application as a debittering agent for its ability to hydrolyze the peptides responsible for the bitter taste in cheese [14].
Alkaline protease has also been exploited in the production of proteinaceous fodder from waste feathers or keratin-containing materials. For example, alkaline proteases from B. subtilis and B. licheniformis PWD-1 have shown keratinolytic activity and can be used in the hydrolysis of feather keratin to obtain a protein concentrate for fodder production [15, 16].
Alkaline proteases with elastolytic and keratinolytic activity can be used in leather-processing industries starting from soaking of hides to final products by replacing the hazardous chemicals involved in soaking, dehairing, and bating [17, 18]. The biotreatment of leather using proteases is preferable as it not only prevents pollution problems, but also is more effective in saving energy. For example, a keratinase from B. subtilis has shown the potential to replace sodium sulfide in the dehairing process [19]. As mentioned above, microbial proteases already play an important role in several industries. The pursuit of discovery strategies targeting new dimensions of molecular diversity and novel technologies to improve performance characteristics of existing proteases will certainly be the major field of development in coming years, and will lead to enzymes with much more efficient performance and novel applications [20, 21].

LIPASE

Lipases (triacylglycerol ester hydrolases EC 3.1.1.3) are a class of hydrolases that catalyze the hydrolysis of triglycerides to glycerol and free fatty acids [22]. Lipases are widely present in bacteria, fungi, plants, and animals. The active sites of lipases are generally characterized by the triad composed of serine, histidine, and aspartate. Acyl-enzyme complexes are the crucial intermediates in all lipase-catalyzed reactions. Lipases contain a helical oligopeptide unit that shields the active site of the enzyme. This, so-called lid, upon interaction with a hydrophobic interface such as a lipid droplet, undergoes movement in such a way that the active site is exposed to provide free access for the substrate. This phenomenon, called interfacial activation, of lipases occurs at the lipid–water interface. In fact, lipase reaction occurs at the interface between the aqueous and the oil phases, due to an opposite polarity between the enzyme (hydrophilic) and their substrates (hydrophobic). Recent studies have demonstrated that lipase activity as a function of interfacial composition is more attributed to substrate inaccessibility rather than to enzyme denaturation or inactivation [23]. In nonaqueous media, lipases can catalyze reversed ester-forming reactions, such as esterification, interesterification, and transesterification.
Therefore, lipases are the enzymes of choice for potential applications in numerous industrial processes including areas such as oils and fats, detergents, baking, cheese making, hard-surface cleaning, as well as leather and paper processing [24]. Furthermore, lipases are the most used enzymes in synthetic organic chemistry, catalyzing the chemo-, regio-, and/or stereoselective hydrolysis of carboxylic acid esters or the reverse reaction in organic solvents [25, 26].
In the fat and oil industries, several new enzyme-based processes have been introduced to replace conventional procedures. Cocoa butter fat is a high-value product in food, confection, and cosmetics industries, while palm oil is a low-value product. Conversion of palm oil into cocoa butter fat substitute has been achieved by lipase-catalyzed interesterification in organic solvent and is now a commercial process [24]. The process costs can be dramatically lowered by using immobilized lipases due to the high cost of the enzymes. Vegetable oils with nutritionally important structured triacylglycerols and tailored physicochemical properties have a big potential in the future market. Lipases are regiospecific and fatty acid specific, and could be exploited for upgrading of vegetable oils to low-caloric triacylglycerols and oleic acid-enriched oils. Another recently introduced process is the removal of phospholipids in vegetable oils (degumming), using a highly selective microbial phospholipase. Lipases have also been used to synthesize esters of short-chain fatty acids and alcohols, which are known as flavor and fragrance compounds and used as food additives to improve the flavor [27].
Nowadays, heavy-duty powder detergents and dishwasher detergents usually contain one or more enzymes including lipase, which can reduce the environmental impact of the detergent products. Lipases are used in detergent formulations for efficient removal of lipid stains, allowing better washing performances and energy savings. Laundering is generally carried out in alkaline media, and lipases active under such conditions are preferred. As early as 1988, the first lipase capable of dissolving fatty stains was produced by a selected strain of the fungus Humicola, but its production yield was too low for commercial application. In 1994, Lipolase, which originated from the fungus Thermomyces lanuginosus and was expressed in Aspergillus oryzae, was first commercialized by Novo Nordisk. One year later, two bacterial lipases, Lumafast from P. mendocina and Lipomax from P. alcaligenes, were introduced by Genencor International. Lipases used in detergents also include those from the species of the genera of Candida, Chromobacterium,and Acinetobacter.
Degreasing is an essential stage in the processing of fatty raw materials such as small animal skins and hides in leather industry. The conventional methods for degreasing are using organic solvents and surfactants, which resulted in serious environmental threats such as volatile organic compound emissions. Lipase can remove fats and grease from skins and hides by hydrolyzing triglyceride (the main form of fat stored in animal skins) to glycerol and free fatty acids. Both alkaline-stable and acid-active lipases can be used in skin and hide degreasing. Although alkaline-stable proteases are used to facilitate the degradation of fat cell membranes and sebaceous gland components, acid-active lipases can be used to treat skins in a pickled state. Lipases are also utilized in deliming and bating processing stages.
Pitch, composed of fatty acids, resin acids, sterols, glycerol esters of fatty acids, and other fats and waxes, causes major problems in pulp and paper industry. Different lipases have been used for removal of pitch. Lipases can control the accumulation of pitch during the production of paper from pulps with high resin content, such as sulfite and mechanical pulps from pine. This ecofriendly and nontoxic biotechnological method have been used in a large-scale papermaking process as a routine operation since the early 1990s. Lipases are also used to remove ink from recycled paper for higher brightness and lower residual ink than chemically deinked recycled pulps.
Biodiesel is an alternative diesel fuel consisting of short-chain alkyl (methyl or ethyl) esters, typically made by transesterification of vegetable oils or animal fats. The conventional method for producing biodiesel, involving acid or base catalysts to form fatty acid alkyl esters, results in high downstream-processing costs and serious environmental problems. Thus, lipase-catalyzed transesterification presents an excellent alternative for biodiesel production. As such, enzymatic processes using lipases have recently been developed, which not only produce high-purity product, but also enable the easy separation of the byproduct, glycerol [28, 29]. However, the cost of lipases remains a hurdle for their industrial implementation in the production of biodiesel. Therefore, considerable endeavors have been made to develop cost-effective enzyme systems. Protein engineering is being used to improve the catalytic efficiency of lipases for biodiesel production. Using the tools of recombinant DNA technology for lipase production warrants a sufficient supply of suitable lipases for biodiesel production. Immobilization of lipases on suitable support materials enables their reuse and further reduces their cost [30]. It can be expected that environmentally friendly, cost-effective processes for the industrial production of biodiesel are on the horizon.
Lipases catalyze the chemo-, regio-, and/or stereoselective hydrolysis of carboxylic acid esters or the reverse reactions in organic solvents. This makes lipases the most used enzymes in synthetic organic chemistry and can be used to resolve the racemic mixtures and to synthesize the chiral building blocks for pharmaceuticals, agrochemicals, and pesticides [26]. For example, the kinetic resolution of piperidine atropisomers can afford a chiral intermediate for the synthesis of the farnesyl protein transferase inhibitor SCH66336, an anticancer agent showing activity in the nanomolar range. The conventional kinetic resolution is limited by an inherent disadvantage that a maximum of 50% conversion cannot be exceeded. The combination of a lipase-catalyzed acylation and ruthenium-catalyzed racemization of the substrate allows for dynamic kinetic resolution of chiral alcohols, amines, and α-hydroxy esters in good yields and excellent enantiomeric excess (ee) values. Lipase-catalyzed chemo-and regioselective protective group incorporation and cleavage have found useful application in the chemical manipulation of multifunctional molecules.
The use of lipases in the preparation of optically pure alcohols, amines, and carboxylic acids constitutes a relatively mature and practical syntheticmethod in organic and pharmaceutical chemistry. In spite of this, novel developments in dynamic and parallel kinetic resolution, reactionmediumengineering, enzyme immobilization, protein engineering, and site-directed evolution of the enzymes still offer additional exciting opportunities that lead to further practical applications of lipases in synthetic organic chemistry [31].

AMILASE

Amylases are glycoside hydrolases that act on α-1,4-glycosidic bonds and break down starch into sugars [32]. Amylases of microbial origin are divided into exo-and endo-acting enzymes. Exo-amylases include glucoamylases and β-amylases. Glucoamylases (EC 3.2.1.3) catalyze the hydrolysis of α-1,4 and α-1,6 glucosidic linkages with lower rate for α-1,6 cleavage to release β-D-glucose from the nonreducing ends of starch and related poly-and oligosaccharides. β-Amylases (EC 3.2.1.2) cleave the α-1,4-glycosidic bonds in starch from the nonreducing ends to give maltose. Endo-amylases (α-amylases, EC 3.2.1.1) hydrolyze internal α-1,4 bonds and bypass α-1,6 linkages in amylopectin and glycogen at random in an endo-fashion producing malto-oligosaccharides of varying chain lengths [33].
α-Amylase is one of the most popular and important forms of industrial amylases, and has potential application in a wide number of industrial processes such as food, fermentation, textile, paper, detergent, and pharmaceutical industries [34]. α-Amylases also play a dominant role in carbohydrate metabolism and have been isolated from diversified sources including plants, animals, and microbes. The amylases of microorganisms have a broad spectrum of industrial applications as they are more stable than those of plant and animal origin. Although α-amylase has been derived from various species, enzymes from fungal and bacterial sources have dominated applications in industrial sectors [35]. Among bacteria, Bacillus sp. is widely used for the production of thermostable α-amylase to meet industrial needs. B. subtilis, B. stearothermophilus, B. licheniformis, and B. amyloliquefaciens are known to be good producers of thermostable α-amylases for various applications. The bacterial α-amylases have a broad profile of physicochemical properties. They display activity from pH 1.0 (Bacillus sp.) to approximately pH 11.5 (Bacillus No. A-40-2 α-amylase). The temperature-activity optima range from approximately 25 °C (Alteromonas haloplanctis α-amylase, AHA) to around 90 °C
(B. licheniformis α-amylase, BLA). The substrate specificity varies both in the preference for chain length and in the ability to cleave close to the α-1,6 branch points in amylopectin and other branched glucose polymers.
Starch consists of two glucose polymers: amylose and amylopectin. The former is exclusively α-1,4 linked, while the latter contains many α-1,6 branch points in addition to the α-1,4 linkages found in amylose. D-glucose (dextrose) used to be produced from starch by acid hydrolysis with a low yield of about 85% and concomitant formation of undesirable bitter sugar (gentiobiose) and coloring materials. The inevitable formation of large amount of salt from subsequent neutralization with alkali presented additional disadvantage. Enzymes have now largely replaced the use of strong acid and high-temperature processes. Two essential and distinct steps, liquefaction and saccharification, are usually involved in the enzymatic breakdown of starch to glucose [32, 33, 36]. During liquefaction, α-amylase hydrolyzes α-1,4 linkages of the gelatinized starch at random to a dextrose equivalent of 10–15. The optimal pH for the reaction is 6.0–6.5 and a structural factor Ca2+, which maintains the stability of the enzyme protein but does not participate in catalysis, is required. The Ca2+-binding site has been modified using protein engineering to improve its binding affinity and lower Ca2+ levels needed for the stabilization. Liquefied and partially hydrolyzed starches are known as maltodextrins and are widely used in the food industry as thickeners.
In the saccharification step, the reaction is usually carried out at 55–60 °C, pH 4.0–5.0. The amount of glucoamylase and reaction times (24–72 h) are dependent on the percent of glucose desired in the product as well as the dosage of the enzyme. Efficiency of saccharification with glucoamylase can be improved by adding pullulanase or isoamylase, and a glucose yield of 95–97.5% can be achieved. Isoamylases and pullulanases are debranching enzymes that hydrolyze only α-1,6 linkages. Addition of pullulanase or isoamylase can reduce the saccharification time from 72 to 48 h, allow for increased substrate concentrations (to 40%, dry solid (DS)), and lower the use of glucoamylase by up to 50%. The fermentable sugars obtained by these two enzymatic steps can be converted into ethanol using an ethanol-fermenting microorganism such as the yeast Saccharomyces cerevisiae.
As solubility of starch is highly dependent on temperature, the removal of stains that contain starch in low-and medium-temperature laundering becomes increasingly problematic. α-Amylases catalyze hydrolysis of starch into oligosaccharides that are soluble in water. Thus, the use of α-amylases in detergents formulations enhances their ability to remove tough starch stains [35, 36]. Detergents make rather severe demands on the enzymes added with respect to stability and activity, as they are often used under very alkalic and oxidizing conditions. Amylases maintain high activity at lower temperatures, alkaline pH, and oxidizing environment. These unique properties make amylases the second type of enzymes used in the formulation of enzymatic detergents for laundry and dishwashing to degrade the residues of starchy foods such as potatoes, gravies, custard, chocolate, dextrins, and other smaller oligosaccharides, and 90% of all liquid detergents contain these enzymes. α-Amylases used in the detergent industry are mainly derived from Bacillus or Aspergillus.
The viscosity of the natural starch gel is too high for paper sizing in the pulp and paper industry. α-Amylases have been used to partially degrade the polymer in a batch or continuous processes for the production of low-viscosity, high-molecular-weight starch gel, which is a good sizing agent for the finishing of paper, improving the quality, and erasability, besides being a good coating for the paper [35, 36]. The size enhances the stiffness and strength in paper and improves the writing quality of the paper. For example, some amylases obtained from microorganisms, Amizyme®, Termamyl®, Fungamyl, BAN®, and α-amylase G9995®, have been commercialized and used in paper industry.
α-Amylases are extensively employed in the bakery industry [37]. During baking, the α-amylase added in the dough of bread catalyzes the degradation of starch in the flour into smaller dextrins, which are subsequently fermented by the yeast to generate CO2. This process results in improvements in the volume and texture of the product. In addition, α-amylases in bread baking also generate additional sugar in the dough to improve the taste, crust color, and toasting qualities of the bread. Furthermore, α-amylases have an antistaling effect in bread baking and extend the softness retention time of baked goods, thus increasing the shelf life of these products. An example of commercial α-amylases currently used in the bakery industry is a thermostable maltogenic amylase of
B. stearothermophilus. α-Amylases are also used in other food-processing industries such as brewing, preparation of digestive aids, and production of fruit juices and starch syrups. Use of amylases in the pretreatment of animal feed improves the digestibility of fiber.
Amylases are used in textile industry for desizing process. Because starch is cheap, easily available in most regions of the world, it is widely employed to yarn before fabric production to prevent breaking of the warp thread during the weaving process. Starch is later removed from the woven fabric in a wet process, in which the addition of α-amylases selectively removes the size and does not attack the fibers. During the past decade or so, many well-proven strategies for stabilizing a protein have already been developed, and several stabilizing mutations of the α-amylases have been obtained. In particular, a number of mutations within domain B have improved the stability of B. licheniformis α-amylase to the extent that it can be used for almost any high-temperature application [36]. As the use of bacterial α-amylases has become more widespread in recent years, enzymes with specific pH-activity profiles and tailor-made substrate and product specificities will undoubtedly be welcome in many industrial applications. The initial studies have been made in both pH-activity profile engineering and substrate-specificity engineering to optimize α-amylases. It seems that theories and methods for addressing the engineering of both substrate specificities and pH-activity profiles are needed and such theories and methods will be rapidly developed, resulting in novel α-amylases with desired properties.

PEKTINASE

Pectinases or pectinolytic enzymes are a heterogeneous group of related enzymes that hydrolyze the pectic substances [40, 41]. Pectic substances are high-molecular-weight, negatively charged, acidic complex glycosidic macromolecules (polysaccharides) that are mostly present in the plant. The primary backbone of pectic substances consists of α-D-galacturonate units linked α-1,4, with 2–4% of L-rhamnose units linked β-1,2 and β-1,4 to the galacturonate units. The side chains of arabinan, galactan, arabinogalactan, xylose, or fucose are connected to the main chain through their C1 and C2 atoms. Pectic substances are classified into four main types: (1) protopectins, the water-insoluble pectic substances that are restrictedly hydrolyzed to yield pectins or pectic acids;
(2) pectic acids/pectates, the soluble polymers of galacturonans that contain negligible amount of methoxyl groups; (3) pectinic acids/pectinates, the polygalacturonans that contain less than 75% methylated galacturonate units; and (4) pectin (polymethyl galacturonate), the polymeric material in which at least 75% of the carboxyl groups of the galacturonate units are esterified with methanol.
Pectinases are divided into three broader groups as follows: (1) protopectinases that degrade the insoluble protopectin to give soluble pectin; (2) esterases that catalyze the de-esterifcation of pectin to remove methoxy esters; and (3) depolymerases that catalyze the cleavage of the α-1,4-glycosidic bonds in the D-galacturonic acid moieties of the pectic substances. Based on the substrate preference, the cleavage mechanism, and the splitting mode (random or endwise) of the glycosidic bonds, depolymerases can be subdivided into different categories: (1) polymethylgalacturonases (PMGs), which catalyze the hydrolytic cleavage of α-1,4-glycosidic bonds of pectin, including endo-PMG and exo-PMG; (2) polygalacturonases (PG), which catalyze the hydrolysis of α-1,4-glycosidic linkages in pectic acid including endo-PG and exo-PG; (3) polymethylegalacturonate lyases (PMGLs), which catalyze the breakdown of pectin by transeliminative cleavage, including endo-PMGL and exo-PMGL; (4) polygalacturonate lyases (PGLs), which catalyze the cleavage of α-1,4-glycosidic linkage in pectic acid by transelimination, including endo-PGL and exo-PGL [40].
The largest industrial application of pectinases is in fruit juice extraction and clarification [42]. The fruit juice extraction begins with washing, sorting, and crushing of the fruits in a mill. After the fruit pulp is stirred in a holding tank for 15–20 min, which removes the enzyme inhibitors (polyphenols) by oxidizing them with naturally occurring polyphenol oxidases present in the fruit or added oxidizing agent polyvinyl pyrolidone, it is treated with pectinase. During this incubation process, the pectinase degrades the soluble pectin in fruit pulp, which facilitates pressing, juice extraction, and the separation of a flocculent precipitate by sedimentation, filtration, or centrifugation. Otherwise, pectin makes juice extraction from the pulp difficult and blocks drainage channels in the pulp through which the juice must pass, thus hampering juice extraction. Treatment time with pectinase depends upon the nature and amount of the enzymes used, the reaction temperature, and the type of fruit. Pectinases in combination with other enzymes (e.g., cellulases, arabinases, and xylanases) have also been used in juice extraction to increase the pressing efficiency.
Pectins contribute to fruit juice viscosity and turbidity. A mixture of pectinases and amylases is used to clarify fruit juices, in which suspended matter is removed to give sparkling clear juices (free of haze). Pectinases also find some application in textile processing [42]. These enzymes have been used in conjunction with amylases, lipases, cellulases, and other hemicellulolytic enzymes to remove sizing agents before the fabric can be dyed. This enzymatic process has reduced the use of harsh chemicals in the textile industry, resulting in a lower discharge of waste chemicals to the environment, a safer working condition for the workers, and better fabric products for the customers. Pectin is like a powerful biological glue and binds the waxes and proteins together. These noncellulosic impurities together with ‘neps’ make the cotton basically undyeable. Scouring of cotton has traditionally been performed with caustic alkaline solution (3–6% aqueous sodium hydroxide) at high temperature to achieve uniform dyeing and finishing. The process is very water and energy consuming, and produces a large amount of waste. Bioscouring offers a cost-effective and ecofriendly strategy for cotton wet processing. This process is based on the idea of specifically targeting the noncellulosic impurities with specific enzymes, in which pectinases are used for the decomposition of pectinic substances, proteases for proteins, and lipases for fats. Pectinases promote efficient interruption of the matrix to achieve good water absorbance without the negative side effect of cellulose destruction, thus drastically limiting fiber damage. Selection of pectinases for bioscouring is based on their pH and temperature compatibility, process efficiency, and end-product quality.
Bast plant fibers, such as ramie, sunn hemp, jute, flax, and hemp, are excellent natural textile materials. They are formed in groups outside xylem in the cortex, phloem, or pericycle, and contain gum, which must be removed before their use for textile making. For example, decorticated ramie fibers contain 20–30% ramie gum, which consists mainly of pectic-and hemicelluloses. The traditional chemical degumming treatment is polluting, toxic, and nonbiodegradable. The enzymatic treatment of bast fibers using pectinases in combination with xylanases presents an ecofriendly and economic degumming process with no damage to the fibers. Vegetable food-processing industries release pectin-containing wastewaters as byproduct. The pretreatment of the wastewater with alkaline pectinase and alkalophilic pectinolytic microbes facilitates removal of pectinaceous material and renders it suitable for decomposition by activated sludge treatment [43]. For example, an extracellular endopectate lyase with optimal pH 10.0 from an alkalophilic soil isolate, Bacillus sp. GIR 621, was used effectively to remove pectic substances from industrial wastewater.
Pectinases are also used in the feed enzyme preparation containing glucanases, xylanases, proteinases, pectinases, and amylases. The enzymes facilitate the liberation of nutrients either by hydrolysis of nonbiodegradable fibers or by liberating nutrients blocked by these fibers [42]. This increases absorption of nutrients by animal and poultry, and reduces the amount of feces released by them, thus increasing animal weight gain with the same amount of barley, that is, higher feed conversion ratio.
The use of molecular biology techniques to study the biochemical, regulatory, and molecular aspects of pectinase systems used by microbes for metabolizing and for complete breakdown of pectin is the most important tools for elaborating the economical, ecofriendly, and green chemical technology for using pectin polysaccharide in nature. For pectinases to have a significant impact on industrial processes, much progress is required in understanding the basic mechanisms of pectinases and in engineering their properties. The future research will be directed toward the discovery or engineering of enzymes that are more robust with respect to pH and temperature tolerance, with emphasis on how they might point to new applications in ecofriendly processes.