Introduction

Pectinase enzymes today are an integral part of the food processing industry. These enzymes catalyze the breakdown of pectin, a key plant carbohydrate, and are used in diverse applications such as fruit juice processing, vinification, extraction of natural oils, waste water treatment, etc. [1, 2]. Pectinases are one of the highly sold enzymes and alone contribute to 40 % share of all the food enzymes [3, 4]. Today, most of the industrial enzymes including pectinase are either mesophilic or thermophilic in nature. The reason for developing meso or thermophilic enzymes was primarily enzyme tolerance or technology integration, because the processes in which these were supposed to be incorporated were necessarily conducted at elevated temperatures. Recently, there has been a growing trend, in the food industry and fruit processing segment, in particular, to replace high-temperature processes with low-temperature processes. The reason behind this change in trend is driven by certain economic and environmental advantages, such as, energy saving, retention of labile and volatile flavor compounds, prevention of contamination and elimination of any residual enzyme activity, which is inactivation of enzyme when temperature is raised [5, 6].

A huge interest has been seen in the area of cold active or psychrophilic enzymes. Enzymes which are able to function at or below 20 °C were termed as cold active or psychrophilic enzymes. This is a new emerging area where researchers have been exploring the psychrophilic microflora to find cold active replacement for the existing meso or thermophilic enzymes. There are reports of wide range of enzymes such as amylases, cellulases, pectinases, β-galactosidases, oxidases, proteases, lipases, etc., which are psychrophilic in nature [6, 10]. This article focuses on reviewing the cold active pectinases, its potential sources, and the advantage it can bring to the fruit processing industry.

Pectin

Pectin is the second most abundant carbohydrate in a plant tissue, apart from cellulose, and is responsible for conferring the rigidity to the fruits. It acts as a “mortar” or “cement” between the cellular “bricks” of cellulose (Fig. 1), which is broken down by naturally occurring pectinase, when the fruit ripes making it soft [7].

Fig. 1
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Arrangement of pectin fibres along with cellulose fibres in a typical plant cell wall

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Pectins are a complex high molecular weight heteropolysaccharides which itself, in pure form, has commercial value as a thickening agent and a food stabilizer in manufacturing of jams and marmalades. It is made up of four polysaccharide units, homogalacturonan, xylogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II, which are covalently linked with each other. The ratio of each unit varies with plant species, but typically homogalacturonan is the most abundant polysaccharide constituting about 65 % of the pectin, followed by rhamnogalacturonan I comprising 25–30 %, whereas the other two being minor components at less than 10 % of the total pectin [8]. Homogalacturonan is the main backbone and is made of galacturonic acid or methyl galacturonic acid residues linked with α 1–4 glycosidic linkage. These residues are substituted with xylose in xylogalacturonan unit and clusters of complex side chains of glycosyl residues, mainly rhamnose, galactose, and arabinose, in rhamnogalacturonan I and II unit, as a side chain. Surprisingly, rhamnogalacturonan II, though being a minor component, along with homogalacturonans is mainly responsible for the rigidity whereas rhamnogalacturonan I act as a plasticizer or a wall thickener (Fig. 2). A deep understanding of structural and functional aspects of pectin has been reported [8, 9].

Fig. 2
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Different sugar moieties in a pectin molecules and action of different subtypes of pectinase on it

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Mesophilic Pectinases

Today, mesophillic pectinase is one of the largely sold enzymes commercially. It comprises up to 7.5 % of the total industrial enzyme [10] or 40 % of the total food enzyme market [3, 4] and has been steadily growing at a rate of 10–15 % [11]. It has been used in fruit processing, since it started as a cottage industry and has become an integral part, as the fruit processing matured as a science. Juices are mostly derived from ripened fruits, which becomes soft due to the breakdown of pectins; it can be assumed that pressing the juice from ripe fruit would be easier than an unripe fruit. Surprisingly, it is not, because during the ripening, the partially broken-down pectins become soluble in water making the juice viscous. Such a juice is difficult to process and also the color and flavor compounds remain attached to the cell walls, leaving the juice of inferior quality [7]. Pectinases are especially used to overcome this problem of viscosity which is a bane in the various unit operations such as juice extraction, liquefaction, clarification, viscosity reduction, and filtration [12]. Apart from juice, it is used in retting and degumming of fibers, such as jute, flax, hemp, and coir, in wine making [13, 14], purification of natural oils [15], coffee and tea [14, 16], and paper making. It also finds application in treatment of effluent coming out of the fruit juice industry which inadvertently contains residual pectin and needs to be broken down [10]. In a way, it is used where ever pectin hinders with the normal processing and needs breakdown. Most of the commercially available pectinase today are known to function at the lowest 35 °C [17].

Pectinase is not a single enzyme but a heterogeneous group of enzymes, which collectively degrades pectin. It is generally classified on the basis of their action toward the galacturonan part of the pectin (Table 1) [10, 18].

Table 1 Classification of pectinases
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Additionally, pectinases are also classified as alkaline or acidic depending on the pH range in which pectinases function [10]. Very little has been known about rhamnogalacturonase and xylogalacturonase until now, except for Vlugt-Bergman et al. [19] and Zandleven et al. [20], because PGs, PLs, and PEs, which act on the smooth regions, also act on the hairy regions.

There are two basic mechanisms by which pectinase functions; fragmenting of the galacturonan backbone by (a) hydrolysis of β 1–4 glycosydic linkage, which is catalyzed by polygalacturonase (PG) and polymethylgalacturonase, and (b) transelimination which is catalyzed by pectin lyase and pentin methyl lyase. Debranching of the galacturonan side chains is catalyzed by esterases such as polymethyl esterases and polyacetyl esterases. A detailed mechanism of mode of action of pectinase has been reported [14].

Commercial pectinases are produced by both prokaryotes, which synthesize alkaline pectinases, and by eukaryotes which synthesize acidic pectinases [10, 12, 13, 15, 21, 22]. Many of these organisms are limited or specific in synthesizing one particular subtype of pectinase, while the other subtypes may be synthesized but in less quantity. So far, Aspergillus has been reported as the most well-established and extensively used microorganism for commercially producing pectinase [23], as they produce huge amounts of these enzymes and are considered to be generally recognized as safe (GRAS) microorganisms.

Structural Details of Pectinases

Research in the field of psychrophilic pectinases has not reached to the level of proteomics, hence most of the knowledge on the structure of pectinases has been derived from its mesophilic counterpart, mostly from the studies of the known pectinase producers such as Erwinia, Aspergillus niger and Bacillus subtilis. First pectinase to be crystallized was Lyase from Erwinia chrysanthemi and the gene responsible was pectate lyase C (PelC; Fig. 3) [24]. On further exploring, it was found that the same organism contained [25] PelA [26], PelE [27] and Pel9A [28] which were also responsible for expressing pectate lyase. Other pectinase producers, such as Aspergillus niger, contained PelA [29], B. subtilis contained BsPel [30], and alkaline pectatelyases [31] produced mostly by bacterial strains contained PelB [32]. Similar studies were conducted with polygalacturonse from Erwinia carotovora [33], endo-PG I and II from Aspergillus niger [34, 35] and pectin methylesterases from plants like Daucus carota [36] and Lycopersicon esculentum [37]. 3D structures of lyases, it molecular mechanism and the role of the active sites from various microorganisms has been widely studied and reported [25, 27, 29, 30, 32] (Figs. 3 and 4).

Fig. 3
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Structure of pectate lyase with trigalacturonic acid bound at the active site (mesh)

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Fig. 4
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Structure of pectate lyase showing β helix and loops psychrophilic pectinase

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Pectatelyase has a mass of ~30–40 kDa and has a parallel β-helix domain that is formed by parallel strands which fold into a large right-handed helix and a major loop region [38, 39]. After aligning a large number of sequences related to pectinases from plants, bacteria, and fungi, it was found that there are conserved motifs of pectin lyases positioned at 439–467, 715–816, and 829–910. A second type of pectin lyase expressed by PelB belonged to a multigene family of pectin lyases with a molecular mass of 37 kDa and consists of ~359 amino acids. When compared to other lyases, 46–65 % similarity was found [32]. Other pectin lyases, such as Bspel, PelC, and PelE, showed 30 % sequence identity. Even though these had lesser sequence similarity, the core structure of most proteins is similar [25].

Pectin esterase (PEs) has a molecular mass in between 30 and 50 kDa [40, 41]. These are made of parallel β-helix proteins with loops creating a substrate binding cleft that is located in a similar region as in PG. The core part of the enzyme is made of several conserved aromatic amino acids and the active site is located in the long shallow cleft having conserved aspartic acid residues Asp136 and Asp157 at the center. The mechanism involves two steps where the primary step is the attack of Asp157 nucleophile on the carboxymethyl carbonyl carbon, wherein Asp136 may act as an acid. Additionally, Asp136 acts as a base which extracts hydrogen from an incoming water molecule and results in cleaving the covalent bond between the substrate and Asp157.

PGs studied from E. carotovorum [42–44] has a molecular mass of 30–80 kDa. These have ten-turn parallel β-helix domains that have two loops formed as a cleft and have the active site placed in it. The cleft formed is more “tunnel-like” than in any other pectinases and is formed by aspartates and lysines that are conserved which create positive electrostatic potential. Four conserved regions, namely, Asn201-Thr202-Asp203, Gly222-Asp223-Asp224, Gly250-His251-Gly252, and Arg280-Ile281-Lys282, were reported by aligning 36 PG sequences. Endo-PGs are involved in inverting anomeric products during the processes where the mechanism starts with the hydrolysis followed by a donation of a proton to the glycosidic oxygen by an acid catalyst and nucleophilic attack of a water molecule onto anomeric carbon of galacturonate which is mediated by a catalytic base [33].

Generally, cold active or psychrophilic enzymes have high specific activity at low temperatures of 0–30 °C. There are two types of psychrophiles: strict psychrophiles which can function at temperatures less than 5 °C and facultative which function at temperatures between 5 and 30 °C. These are naturally found in organisms which have been colonizing the vast cold regions of the earth, especially oceans and snow-covered land masses. Recent research has revealed that such organisms have the ability to produce enzymes such as amylase, cellulase, galactosidase, oxidase, protease, lipase, and pectinase [6, 45]. So far, pectinase has been reported in organisms from deep oceans [5, 6, 45, 46], snow-filled land masses [47–49, 62], and special cold climactic zones [1, 50–52]. As these habitats are below 30 °C, it can be assumed that the pectinase produced from these should also be psychrophilic in nature.

Any cold active enzyme including pectinase needs to address two major aspects: stability and rate of reaction. At low temperature, the stability of a pectinase is lost as it gets deactivated due to protein unfolding, and all the structural factors responsible for enzyme stability, such as backbone strength, localized chain mobility, hydrophobicity, cofactor binding, and monomeric binding, are weakened [5]. Over the course of evolution, cold active microorganisms have under continuous structural and molecular adaptation to develop various cryo-defense strategies [6]. Some of these strategies are at cellular level, such as temperature-dependent membrane lipid composition changes [53], and others at the protein level such as cold shock domains (Csds) [54], cold acclimated proteins (Caps) [55] and antifreeze proteins [56]. The above factors contribute greatly to the stability of the psychrophilic enzyme in the nature and, if considered carefully, would give stability in man-made applications as well. However, it does not address the heat labile nature of the psychrophiles.

The rate of reaction of pectinase is significantly affected at low temperature, as it is a function of ΔG, and any decrease in temperature will exponentially decrease the activity [5]. Psychrophiles require lower ΔG which implies that not only these enzymes require less activation energy but also the rate of reaction would be slow. Gerday et al. reports a different view and observes that generally psychrophilic enyzmes may be thermolabile but have high specific activity, up to ten times more than their mesophilic counterparts at that temperature. This is because of the various evolutionary adaptations pshychrophiles have undergone over the course of evolution, and one of them being increased fluidity of the protein structure which allows the psychrophilic enzyme to be more flexible and requiring less activation energy [45]. This is exactly opposite of increased rigidity in mesophilic or thermophilic organisms, to withstand high temperatures [57–59]. Also because of fluidic active sites, the substrates are loosely bound to the enzyme, which leads to higher K m values and higher activity in the form of K cat. This has also been supported by X-ray crystallography studies on certain enzymes that the hydrophobic interactions which are typical of mesophilic enzymes are replaced by electrostatic interactions in psychrophiles. Electrostatic interactions are more stable at low temperature, resulting in an optimal K m values. One more adaptation, which supports lower activation energy, showed that the catalytic cavity seems to be larger and more accessible to substrates compared to mesophiles. This is inadvertently a result of deletion of stabilizing residues in the loop. Due to this, the substrate binding happens at very low energy cost [45]. All the above findings may not be directly related to pectinase but have been reported to be commonly observed in various cold active enzymes by various researchers. It can be assumed that the above-described factors will also contribute to stability and rate of reaction of psychrophilic pectinase. However, further detailed study needs to be done in regard to pectinase as most of the research is targeted mainly on identifying and isolating potential sources of cold active pectinases.

Current Research in Cold Active Pectinases

The most favored destination for scientists looking for psychrophilic enzymes has been the psychrophilic microflora which colonizes the cold regions of the earth. Of our planet, 75 % is cold and is exposed to temperatures less than 5 °C [16, 45]. In fact, NASA reports that the average temperature of the earth is 13–15 °C, which few decades earlier was further less. Oceans occupy most of the cold habitat (70 %), followed by snow-covered land masses (15 %), and the remaining are microclimatic cold zones and man-made cold areas [45]. Many cold active enzymes have been isolated from these habitats until now. But finding cold-adapted microorganism capable of producing pectinase in such a gigantic psychrosphere and enormous microbial population would be like finding a needle in a haystack. One has to apply unique philosophy to screen pectinase-producing organisms. The following section describes various reported psychrophilic pectinases isolated from different psychrophilic habitats.

Cabeza et al. [1] reports cold active pectinases from San Rafael, a Western Argentinean wine-making area which stands at the foothills of the Andes mountain range. It is not a permanently cold region, but presents a special cold microclimate that contributes to distinguished vineyards and wines. The grapes growing in this region may harbor a microflora capable of synthesizing pectinase. The group screened through various isolates from the microflora of grapes and shortlisted one bacterial strain which was identified as B. subtilis SC-H. This strain produced all three pectinases (PG, PE, and PL) and was active at 15 and 30 °C, which implies that this strain was not strictly psychrophilic in nature. The pectinase activity of the isolated strain was compared with some of the commercially available pectinase (mostly mesophilic) at 15 and 30 °C. It was observed that the isolated strain showed two to three times less activity compared to mesophilic pectinase from Aspergillus.. Exact details have not been reported, but such a pectinase, which has been termed as “psychrotolerant” mesophile, could be of greater significance to the local wine industry, as it is not very temperature-dependent and can easily be incorporated into the already established process.

Merin, Mendoza, Farías, and Morita de Ambrosini [51] screened for yeast strains capable for producing pectinase from the same region of San Rafael. The objective was to identify yeast strain which can produce pectinase at low temperature and which can used as an adjuvant in wine fermentation which is mostly conducted at low temperatures. This can be a big boon to the wine industry. Most of the wine fermentation is conducted at low temperature (15–20 °C) as it is responsible for flavor retention, but mesophilic pectinases, which is externally added to extract the colors from the grapes, are unable to function at their maximum. The author was successful in screening and isolating one yeast strain which was identified as Aureobasidium pullulans and which demonstrated maximum pectinase activity of 0.7–0.8 U/ml at 12 °C and 1.6–1.8 U/ml at 28 °C. This could be of significant interest to the local wine makers.

Margesin, Fauster, and Fonteyne [48] explored the snow-covered land masses such as the glaciers of the northern Siberia. The main objective was to screen for cold active pectinase which could be used in the treatment of effluent coming out of food processing units. This effluent contains residual pectin left after the processing which, unless degraded, leads to high chemical and biological oxygen demand and eventually leads to an environmental hazard. Pectin lyase, which is currently used in effluent treatments, becomes ineffective when such food processing units are located in cold regions. Significant amounts of energy are wasted to maintain optimum temperatures of sumps and lagoons so as to make these enzymes work. If there is a microorganism, preferably yeast because it can stand the adverse conditions of effluent, which can produce cold active pectin lyase, it would help overcome this issue and reduce energy consumption. The author was successful in identifying Mrakia frigida, which was the first reported yeast to produce cold active pectin lyase. This organism produced maximum activity at 15 °C, but was not strictly psychrophilic.

Birgisson, Delgado, Arroyo, Hatti-Kaul, and Mattiasson [47] isolated microorganisms from snow-covered soil, plants, leaves, and branches of Iceland. Yeast was selected due to the same reasons mentioned in previous section added with the fact that it could be declared as GRAS microorganism and can produce high levels of the enzymes. The strain isolated was Cryptococcus sp., which showed pectinolytic activity as high as 35–36 U/ml at 9 °C and which was capable of synthesizing pectinase on glucose as carbon substrate. Naga Padma, Anuradha, and Reddy [49] explored the Himalayan regions to look for a pectinase-producing yeast that adds another advantage of preferring yeast which is, faster fermentation and its ability to be immobilized. Cold soils from fruit yards and fruit dump sites of the Himalayan region were targeted as it could be a good source of pectinase-producing microorganism including yeast. The yeast strain identified here was a cold-adapted psychrotolerant Saccharomyces sp. and was able to produce 15–21 U/ml on different agricultural residues such as mango peel, orange peel, etc. This kind of strain which can grow on inexpensive pectin sources could be of great industrial significance.

Nakagawa, Nagaoka, Taniguchi, Miyaji, and Tomizuka [50] and Nakagawa, Yamada, Miyaji, and Tomizuka [52] isolated pectinolytic–psychrophillic yeast from the soil sources of Abashiri, Hokkaido (Japan). These strains were strictly psychrophilic as it demonstrated pectinolytic activity only at 5 °C and were unable to grow at mesophilic temperatures. Some of the predominant psychrophiles showing highest activity were identified to be Cryptococcus cylindricus, Cystofilobasidium capitatum, and Mrakia frigida along with species that have been already reported [47, 61].

Takasawa et al. [62] came across fungal pathogens which are responsible for spoilage of crops growing in cold regions. These pathogens must have pectin-producing capabilities as they survive on the plant material and would also be cold active as these crops are mostly snow-covered. While screening through wheat seedlings which had snow mold diseases, the group isolated one psychrophilic fungal strain which was identified as Sclerotinia borealis. Unlike the previous bacterial and yeast strains which were psychrotolerant, this strain was strictly psychrophillic as it showed maximum activity only at 5 °C. In another report Anuradha, Padma, Venkateshwar, and Reddy [63], while looking for fungal strains screened through, not pathogenic but fruit rots fungi, assumed that fungus rotting the fruits should also possess pectinase-producing ability. The chosen cold zone was Himalayan habitat and one fungal strain which showed maximum pectinase activity and was identified as Aspergillus awamori. This strain was capable of growing on inexpensive pectin sources, such as fruit peels, and not only produced cold active pectinases but also produced xylanases and celluloses. Its enzyme activity was similar to the commercially used Aspergillus used for manufacturing pectinase, which could be an encouraging fact to know that fungus could be more relevant in shifting from mesophilic to psychrophillic processing.

Of the earth's surface, 70 % is covered by oceans, which is at a constant temperature of 4–5 °C and harbors a diverse microflora. This has always attracted researchers for exploring microorganisms which can produce biochemical of potential interest. Unlike the terrestrial microflora, it would be very difficult to find pectinase producers in oceans, as it contains very little vegetation. Truong, Tuyen, Helmke, Binh, and Schweder [5] explained that though there is no vegetation in the ocean as it is on land, but there is abundant growth of phytoplankton at various depths of the ocean. This phytoplankton, similar to plant tissues, is composed of a variety of structural polymers such as cellulose, xylan and some amounts of pectin. Little is known about how it is hydrolyzed and what enzymes functions at such low temperatures, it is obvious that these polymers including pectin are being degraded by marine microorganisms [46]. This led to explore further and screen for pectin degraders in sea ice, sea water, and sediments from the Antarctic and the Arctic oceans. There was one isolate which showed that the highest activity in the preliminary screening was psychrotolerant in nature as it grew at 0–29 °C and was identified to be Pseudoalteromonas haloplanktis. They have also identified the gene responsible for pectinase activity which was found to be expressing pectin lyases. This is one step ahead as if one cannot optimize the growth of psychrophilic organisms, they can still express the gene in another host and produce cold active pectinases. Recently, cold active β- galactosidase enzyme production was found to be explored from Thalassospira frigidphilosprofundus, and its details were widely studied [65–67]. Apart from these findings, there has been very little citations on pectinase producers from marine environment mainly because it is inaccessible. Such a potential source from where many other cold active enzymes have been reported should be Explored further for pectinase producers.

Applications of Cold Active Pectinases: Refining Techniques in Food Processing

Any industry striving for excellence and sustainability has to continuously evolve and keep refining their techniques to sustain three important factors: cost, time, and quality. The food industry is not aloof to this and has been constantly evolving and the latest trend is to eliminate high-temperature treatment. This is still in its native hypothesis stage, but many advantages are being anticipated. One conspicuous advantage is that it would avoid spoilage, taste changes, and loss of nutritional value which are very common problems in the industry [50]. The existing commercial pectinases used to degrade pectin are all mesophilic in nature and require elevated temperatures of 40–60 °C.

Food industry which is the largest consumer of pectinase uses it to eliminate pectin in fruit juice processing. The raw juice obtained after tissue grinding and pressing, contains suspended pectin coming from the fruit. This result into increased viscosity, haziness, and inability to freely filter, hence leading to an overall drop in the efficiency. Pectinase is added to remove this suspended pectin and improve the process, but these being mesophilic in nature requires optimum temperatures which are normally ambient or higher. At this temperature, the juice becomes prone to contamination and loss of volatile aromatic compounds. If these are replaced by psychrophilic pectinases, the same steps can be conducted at lower temperature and one can control contamination [47, 50], retain the flavor of compounds [5], and increase the storage capacity [6]; as the processing is at low temperature, energy can be tremendously saved [47].

Another application of cold active pectinase is in winemaking. Winemaking does not require large quantities of pectinases, but offers a unique opportunity, where there is an absolute need of cold active pectinase [1]. Wine is produced by fermentation, but contrary to other fermentation processes, it is mostly conducted at low temperatures (10–15 °C). The unique flavor of wine is because of the low-temperature fermentation, which helps in retaining the original flavors of grapes. Pectinases are currently used for the following: (a) efficient juice extraction from the grapes, (b) settling the suspended particles in the fermentation mash [1], (c) releasing polymeric pigments responsible for the color [60], and terphenols for aroma [61]. The mesophilic pectinases currently used are unable to demonstrate maximum activity and the efficiency of the pectinases are severely affected below 10 °C [64], because of which one has to add more than required quantity. Psychrophilic pectinases could be an ideal alternative to this problem which can further improve production cost, apart from improving the organoleptic profile of wine [1, 51].

Wastewater treatment plants are associated with every industry, so is the case with food-processing industry. Effluents coming out of food processing units contain residual pectin carried over from the juices which require to be degraded by pectinases before subjecting to secondary and tertiary treatment. Even though this is an established practice, when it comes to plants located in cold regions, tremendous amounts of energy are consumed to maintain the temperatures of the larger sumps to make the mesophilic pectinase work. Cold active pectinases can bring dramatic changes in processing such effluents where energy inputs for maintaining temperature is not required, hence making it cost-effective and environment-friendly [48].

Until now, the above listed are the only few potential uses of cold active pectinases, but research is going on to find many more uses. Simultaneously, research is also being carried out to find a source of cold active pectinase from cold-adapted microorganism or the psychrophiles.

Conclusion

Cold active pectinase will have a definitive edge over the mesophilic pectinases currently used in the food-processing industry. It can add tremendous economic and environmental advantages to the industry. However, it is a long way to go before an ideal source of cold active pectinase is found and scaled up to an industrial process. The details of kinetics and enzyme stability also need to be evaluated before implementing low-temperature pectin degradation in the current food industry. However, the current state of the art suggests huge potential and the possibility of it being a reality.