Pullulanase (pullulan 6-glucanohydrolase, EC is an enzyme which is capable of hydrolyzing the a-l,6-glucosidic linkages of pullulan and produces maltotriose as the end product (Abdullah and French, 1970).

Pullulan is a polymer synthesized by the yeast-like fungus Aureobasidium pullulans. It is a linear a-D-glucan built of maltotriose subunits (Abdullah and French, 1966). Pullulanase, belongs to the a-amylase family, which is identified as glycoside hydrolase and break a-1,6 linkages in pullulan, starch, amylopectin, and related oligosaccharides (Duan and Wu, 2015). Pullulanasesare classified into five types based on the substrate specificity (Roy et al., 2003; Haki and Rakshit, 2003).

Types of Pullulanase (Based on substrate specificity)

  1. The glucoamylase type is an exo-acting carbohydrase (Reilly, 1979), which hydrolyzes pullulan from non-reducing ends to produce glucose.
  2. The pullulanase type I specifically hydrolyses a-1,6glycosidic linkages in pullulan or branched substrates such as amylopectin forming maltotriose.
  3. The isopullulanase type from Aspergillusniger, which hydrolyzes a-1,4 linkages of pullulan to produce isopanose.
  4. Pullulan hydrolase type I (neopullulanase) from Bacillus stearothermophilus (Ecel et al., 2015) which hydrolyzes a-1,4 linkages of pullulan to produce panose.
  5. Pullulanase type II (amylopullulanase) attacks a-1,6 linkages in pullulan and branched substrates in addition to a-1,4 links in polysaccharides other than pullulan (Bertoldo and Antranikian, 2002; Duffner et al., 2002).

Pullulanases belong to a group of glycosyl hydrolases that are widely distributed in nature and are produced by an extremely wide variety of species such as animals, plants, and microorganisms (Hyun and Zeikus, 1985; Zhang et al., 2013; Duan and Wu, 2015). Pullulanases have gained important in current saccharification processes as starchde branching enzymes. In the starch processing industry for the production of maltose syrups and high purity glucose and fructose (Jensen and Norman 1984). This occurs when pullulanase is used in combination with glucoamylase or a-amylase, respectively in the saccharification process.

Pullulanases that specifically attack the branching points of amylopectin are of special interest. The action of such enzymes would lead to the formation of linear oligosaccharides that can be attacked efficiently by other amylolytic enzymes, leading to high levels of glucose or maltose. Pullulanases are used in the detergent industry as effective additives in dishwashing and laundry detergents for the removal of starches under alkaline conditions (Van and Willem 1990) and in the manufacturing of low caloric beer (Olsen et al., 2000). It is also possible to use pullulanase as a dental plaque control agent (Marotta et al., 2002). Because of the various important uses of pullulanase, it has been the subject of various applications.

Pullulanase can be used with glucoamylase for the production of high glucose syrups or with beta-amylase for high maltose syrup production, where it functions to increase yield and decrease reaction times (Norman 1979; Saha and Zeikus 1987). Importance has been placed on the pullulanase property of environmental compatibility with glucoamylase or beta-amylase.

Many mesophiles can produce pullulanase (Fogarty 1983) but these pullulanases lack high thermostability. Recently, several thermostable pullulanases have been discovered from extreme thermophiles such as Clostridium thermohydrosulfuricum (Hyun and Zeikus 1985; Melasniemi 1987), Thermoanaerobiumbrockii (Coleman et al. 1987), Thermoanaerobium Tok6-B1 (Plant et al. 1987), a new Clostridium isolate (Antrani-kian et al. 1987), Thermusaquaticus YT-1 (Plant et al. 1986) and Thermus sp. (Nakamura et al. 1987).

Some novel pullulanase preparations display the unique property of not only degrading alpha-l,6-glucosidic linkages in pullulan but also degrading the alpha-l,4 linkages of soluble starch, amylose and amylopectin (Coleman et al. 1987; Plant et al. 1987; Saha and Zei- kus 1989). Although Suzuki and Chishiro (1983) and Imanaka and Kuriki (1989) reported that Bacillus stea-rothermophilus strains produce pullulan hydrolases, the enzymes were not able to cleave alpha-l,6-1inkages of pullulan and were not classified as true pullulanases.

We have isolated a pullulan-degrading thermophilic Bacillus species, strain 3183 (deposited with American Type Culture Collection, Rockville, Md, USA; ATCC 49341), which is able to grow at a pH lower than that of B. stearotherrnophilus and produces an extracellular thermostable pullulanase. In this paper, we report on the general physiological properties of this strain, and the general biochemical properties of its pullulanase activity.

Pullulanase proved to be a critical tool for the analysis of pullulan structure (Bender & Wallenfels, 1961). Pullulanase converted a yeast (A. pullulans) a-glucan containing a-(1 > 6) bonds into maltotriose, thereby describing pullulan as a polymer of (1 > 6) linked maltotriose subunit. Sometimes, partial acid hydrolysis of pullulan yields isomaltose, maltose, panose and isopanose (Bender et al., 1959, Bouveng et al., 1963 and Sowa et al., 1963).

Thus, pullulan is often viewed as a polymer of panose or isopanose. Panose [O-a-d-glucopyranosyl – (1 > 6)-O-a-d-glucopyranosyl – (1 > 4)-O-a-d-glucopyranose] and isopanose [O-a-d-glucopyranosyl-(1 > 4)-O-a-d-glucopyranosyl-(1 > 6)-O-a-d-glucopyranose] is the d-glucose containing trisaccharides having (1 > 4)- and (1 > 6)-a-d-glucosidic linkages.

According to Nakakuki et al.,(1982) production of pullulanase along with xo-maltohexahydrolase from UV irradiated culture of Aerobacteraerogenes. (Takasaki and Yagisawa (1985) produced heat resistant pullulanase by Klebsiella pneumonia which was cultured at 30 degree celcius

Sources of Pullulanase

In the last decade, a number of hyperthermophilic archaea, have been isolated which are able to grow around the high temperatures. Some organisms with the highest growth temperature are members of the genera Thermotoga maritima and Pyrococcusetc (Haki et al., 2003).

Pullulanases from Bacillus species:

Production of pullulanase and beta-amylase was observed by Bacillus cereus mycoides which was observed on alumina. The adsorption of pullulanase and beta-amylase was suppressed in presence of both sodium citrate and maltose (Yammanobe et al., (1979), liquefied corn, potato, sweet potato, topicoa, sago and wheat starchees by mixture of beta amylase of Bacillus subtilis and then sachharified by mixture of beta amylase and pullulanase by Bacillus cereus varmycoides, for the production of beta-amylase-pullulanase. Both enzyme were effective for the production of maltose from starch. Both the enzyme were purified and conditioned for sachharification of starch were optimized.

The enzyme combined with glucoamylase produced 97% sachharification of liquefied corn starch in 72 hours from Bacillus acidopullulyticus (Neilson et al., (1982). Bacillus circulans in a medium containing soyabean, polypeptone, dipottasium hydrogen phosphate, magnesium sulphate, soluble starch, ammonium sulphate and calcium chloride found to produce amylase along with the pullulanase.

Table 1. List of pullulan producing microorganisms

Table 1. List of pullulan producing microorganisms

Features and Structure of Pullulanase

Pullulanase (EC, pullulan 6-glucanohydrolase) is a debranching enzyme which hydrolyses the a-1,6- glucosidic linkages in pullulan and other amylaceous polysaccharides, and belong to a family of 13 glycosyl hydrolases, also called the a-amylase family (Janecek et al., 1997; Matzke et al., 2000; Singh et al., 2010). Pullulanases are widely distributed among animals, plants, fungi and bacteria. Detailed enzymatic mechanisms of substrate degradation and the resulting final products are different in each case (Domañ-Pytka and Bardowski, 2004; Singh et al., 2010).

The average molecular weight of pullulan is the range from 5.3×103 – 2×106 depending on the strain used and the culture medium (Ürküt, 2007). Pullulan produces a high viscosity solution at a relatively low concentration and can be used for oxygen impermeable films and fibers, thickening or extending agents, or adhesive or encapsulating agents (Ma et al., 2012; Mcneil and Kristiansen, 1990; Singh et al., 2008). Despite being a á-D-glucan, pullulan is resistant to á-D-amylolysis and may be used in lowcalorie food formulation. The chemical formula of pullulan is (C6H10O5).H2O (Gaur et al., 2010). Pullulan is produced by A. pullulans and some other microorganisms shown and summerised in Table 1.

Table-2: Quality parameters attributed to pullulan (Karim et al., 2009; Singh et al., 2008)

Table-2: Quality parameters attributed to pullulan (Karim et al., 2009; Singh et al., 2008)

Bauer (1938) made early observations on extracellular polymer formation by A. pullulans, and Bernier (1958) isolated and began to characterize the polysaccharide. Bender et al. (1959) studied the novel glucan and named it “pullulan.” During the 1960s, the basic structure of pullulan was resolved (Bouveng et al., 1962, 1963; Sowa et al., 1963; Ueda et al., 1963; Wallenfels et al., 1961, 1965). Bender and Wallenfels (1961) discovered the enzyme pullanase, which specifically hydrolyzes the a (1~6) linkages in pullulan and converts the polysaccharide almost quantitatively to maltotriose (Leathers, 2003). On this basis pullulan is commonly viewed as an a (1~6) linked polymer of maltotriose subunits (Figure 1). (Yatmaz and Turhan, 2012).

Pullulan undergo enzymatic hydrolysis by -D-pullulanases. The (1-6)-a-aboth (1-6)-a-D-and (1-4)- D-pullulanases cleave the (1-6)-a-D-glucopyranosidic linkages. Complete hydrolysis of pullulan using (1-6)-a-Dpullulanase yields maltotriose as a major product along with traces of maltotetraose. The (1-4)-a-D-pullulanases act on (1-4)-a-D-glucosidic linkages at their reducing ends adjacent to (1-6)-a-D linkages. Complete hydrolysis of pullulan with (1-4)-a-D-pullulanase gives isopanose as the main product. Products of enzymatic pullulandegrad-ation are used in food and pharmaceutical industry (Singh et al., 2010). Pullulan’s solubility can be controlled or provided with reactive groups by chemical derivatization.

Due to its excellent properties, pullulan is used as a low-calorie ingredient in foods, gelling agent, coating and packaging material for food and drugs, binder for fertilizers and as an oxidation-prevention agent for tablets. Other applications include contact lenses manufacturing, biodegradable foil, plywood, water solubility enhancer and for enhanced oil recovery (Israilides et al., 1998; Leathers, 2003; Schuster et al., 1993). It is water soluble, insoluble in organic solvents and non-hygroscopic in nature. Its aqueous solutions are stable and show a relatively low viscosity as compared to other polysaccharides. It decomposes at 250–280°C. It is moldable and spinnable, being a good adhesive and binder. It is also non-toxic, edible and biodegradable.

Utilization of Pullulanase in Food Industry

Pullulanase can be used effectively in edible films owing to both food ingredients and film-forming ability thanks to its features (Yatmaz and Turhan, 2012). Pullulan can be used as a coating of food and packaging material and starch replacer in low-calorie food formulations in food industry. Also can be used as spice and flavoring in seasoning agent for the microencapsulated (Israilides, 1994, 1998; Ürküt 2007). Pullulan is widely used in soups, sauces and beverages thanks to intensifier properties (Yatmaz and Turhan, 2012). It can also be used to stabilize the quality and texture of mayonnaise (Singh et al., 2008).

Pullulan can be used as a denture adhesive, a binder and stabilizer in food pastes, and to adhere nuts to cookies (Leathers, 2003). Pullulan can be employed as a binder for tobacco (Miyaka, 1979), seed coatings and plant fertilizers (Matsunaga et al., 1977, 1978; Singh et al., 2008). Pullulan, can be used appropriately in the production of low-calorie (diet) foods due to its take place in GRAS list and the slow digestion (Yatmaz and Turhan, 2012).

Underivatized films are readily dissolved in water, thus having property to melt in the mouth as edible food coatings (Conca and Yang, 1993; Singh et al., 2008). The oxygen resistance of pullulan films is suitable for the protection of readily oxidized fats and vitamins in food. Pullulan films can be employed as coating or packaging material of dried foods, including nuts, noodles, confectionaries, vegetables and meats (Krochta and De Mulder-Johnston, 1997; Singh et al., 2008).

Pullulan can be used directly to foods as a protective glaze. Pullulan substituted with cholesterol or fatty acids can be used to stabilize fatty emulsions (Leathers, 2003; Singh et al., 2008; Yamaguchi and Sunamoto, 1991). Singh et al. (2010), reported that maltotriose syrup can be produced by enzymatic hydrolysis of the polysaccharide ‘pullulan’ using the debranching enzyme, pullulanase.

This syrup possesses many excellent properties as low freezing point depression, mild sweetness, keeps in moisture, prevention of retrogradation of starch in foodstuffs, less color formation compared with maltose syrups, glucose syrups or sucrose, good heat stability, low solution pullulan obtained was used for preparing maltotriose syrup using pullulanase. These properties are useful in the food industry (Zoebelein and Böllert, 2001).

Industrial Applications of Pullulanase Enzyme

Starch Processing Industry

Pullulanases are used in sugar syrup industries to complete the hydrolysis of starch initiated by á-amylases. The combined application of pullulanase with other amylolytic enzymes increase the quality of sugar syrups. When starch is treated with amylase and pullulanase simultaneously this consequently increases the efficiency of a saccharification reaction. This method has the advantage of generating higher yields of the desired end product from starch.

Thermostable and acidophilus pullulanase from Bacillus stearothermophilus is useful here. (Nair et al., 2006), (Michaelis et al., 1985), (Saha et al., 1988).(Silva 2005). Pullulanase enzyme preparation intended for use as a processing aid in the food industry, in particular, pullulanase is useful in the debranching of corn starch in the production of certain corn sweeteners. (Modderman 1995).(Haki et al., 2003), (Bertoldo 2002).

Production of High-Maltose Corn Syrup

High-maltose syrups have mild sweetness, low viscosity in solution, low hygroscopicity, and good thermal stability. It is well suited for numerous applications in food processing such as in the manufacturing of high-quality candy and ice cream (J. F. Shaw and J. R. Sheu (1992). In recent years, there has been an increasing interest for pure maltose in the pharmaceutical industry.

Maltose may be used instead of D-glucose for intravenous feeding, where it can be administered at higher concentrations without elevating blood glucose levels (J. F. Shaw and J. R. Sheu (1992). Pure maltose may also be used as a starting material for the production of maltitol and crystalline maltitol (B. D. Jensen and B. E. Norman(1984).

Production of High-Fructose Corn Syrup

High-glucose syrup is used as a carbon source in fermentation and feed for making high-fructose syrups and crystalline glucose (H. S. Olsen, P. Goddard.,2000, P. Nigam and D. Singh., 1995]. High-fructose syrup is produced by the treatment of high-dextrose syrup, especially high-glucose syrup (DE 95–96) with immobilized glucose isomerase(M. J. E. C. Van Der Maarel, B. Van Der Veenet al., 2002). High-fructose corn syrup (HFCS) is a high-quality and clean-tasting caloric sweetener.

HFCS with very high DE value is required in the production of crystalline glucose. HFCS is also widely used in product formulation. It is cheaper and less caloric than sucrose but 1.2 to 1.8 times sweeter than sucrose on a dry weight basis (H. Guzman-Maldonado and O. Paredes-Lopez et al., 1995). Like high-maltose syrup, 90% HFCS is used in food for diabetics because it can be metabolized without insulin (J. D. Dziezak., 1989).

Saccharification of Starch

The primary application of pullulanase is in starch saccharification and the most important industrial application of pullulanase is in the production of high-glucose (30% to 50% glucose; 30% to 40% maltose) or high-maltose (30% to 50% maltose; 6% to 10% glucose) syrups (M. J. E. C. Van Der Maarel, 2002, I. Gomes, J. Gomes., 2003). In the saccharification process, pullulanase is normally used in combination with glucoamylase or a-amylase (Haki, G.D., Rakshit, S.K., (2003)., M. Chaplin., 2002 , I. Gomes, J. Gomes et al., 2003)

Use of Pullulanase Baking Industry

The baking industry is a large consumer of starch and starch-modifying enzymes. The staling effect is the major problem in the baking industry. Used in baking industry as the antistaling agent to improve texture, volume, and flavor of bakery products (M. J. E. C. Van DerMaarel, B. Van Der Veen., 2002). The staling effect includes an increase of crumb firmness, loss of crispness of the crust, decrease in the moisture content of the crumb and loss of bread flavor, leading to the deterioration of quality (Vander et al., 2002).

Although this problem can be overcome using chemical treatment, enzymatic treatment is more preferred because consumers nowadays demand for products without chemicals and higher acceptances by the consumers for enzymes, which are produced from natural ingredients, is seen. Pullulanase plays an important role in the enzymatic anti-staling treatment (Noorwez et al., 2006), (Bertoldo 2002).

Branched Cyclodextrin (CDs) Production

There is a very interesting and highly economical valued application of pullulanase in branched cyclodextrins (CDs) production. CDs and branched CDs, such as maltosyl-CDs and glucosylCDs are homogeneous cyclic.

Oligosaccharides, which are composed of only glucose units (Kitahata et al., 2000). Thus, CDs and branched CDs have been widely used for stabilizing labile materials, masking odors, and solubilizing insoluble or poorly soluble drugs (Bertoldo 2002), (Tanimoto et al., 2005), (Okada et al., 1988).

Glycogen and amylopectin-debranching enzyme such as pullulanase and isoamylase can be used as an effective additive in dish washing and laundry detergent as reported by for alkaline endoglucanase from some strain of Bacillus the properties of which also fulfill the essential requirements for enzyme to be used for such purposes (Ara, 1993).


Some alkaline pullulanases have been used as effective additives in dishwashing and laundry detergents for the removal of starches under alkaline conditions, (Y. Hatada, K. Saito (2001), J. F. Shaw and J. R. Sheu (1992) . The effectiveness of these alkaline debranching enzymes can be enhanced in washwater when the amylopullulanase is used in combination with alkaline á-amylase because one single amylopullulanase can catalyze both debranching (a-1,6 hydrolytic) and liquefying (a-1,4 hydrolytic) reactions.

Others Applications

Pullulanase also finds minor application in the manufacturing of low-calorie beer (H. S. Olsen, P. Goddard., 2000) It is also possible to use pullulanase as a dental plaque control agent (M. Marotta),( A. Martino et al., (2002).


It can be concluded from the present review that the pullulanase enzyme is a very potent enzyme for food processing applications and hence, there is a strong response for this enzyme everywhere in the world with its immense presence in the industries related to its usage. Pullulanase is inexpensive and easily available, hence it helps in the economical production of cyclodextrins, bioethanol and beer and other products related to different food commodities.

Resistant starch and substitute of natural gums are also produced by the action of pullulanase; these products have numerous pharmaceutical and neutraceutical applications. In the future, the application of pullulanase enzyme can be further enhanced by increasing its yield by optimizing the levels of different parameters.


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