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Pullulanase (pullulanase type I, pullulan 6-glucanohydrolase, EC 18.104.22.168), also known as limit dextrinase, is a debranching enzyme that specifically breaks up Î±-1,6-glycosidic linkages of pullulan, starch and amylopectin. Depending on their inability or ability to hydrolyze Î±-1,4 glucosidic linkages in other polysaccharides, pullulanases are divided into two categories based on substrate specificity: (i) pullulanase type I, which specifically cleaves the Î±-1,6-glycosidic linkages in pullulan and branched oligosaccharides, forming maltotriose and linear oligosacharides, respectively, and (ii) pullulanase type II, or amylopullulanase, which hydrolyzes both Î±-1,6-glycosidic linkages and Î±-1,4-glycosidic linkages in branched and linear oligosaccharides (Bertoldo & Antranikian, 2002). In addition, based on amino-acid sequence similarities these pullulanases are classified into glycoside hydrolase (GH) family 13 together with Î±-amylases and isoamylases (Henrissat, 1991; http://afmb.cnrs-mrs.fr/CAZY/GH_13.html).
History of the Enzyme
Pullulanase type I (referred to here simply as pullulanase) was first isolated from a culture of
Aerobacter aerogenes (Klebsiella pneumoniae) by Wallenfels et al. in 1966. The enzyme was then isolated from several microorganisms, including Streptococcus mitis,9 Bacillus no. 202-1,10 and Klebsiella aerogenes W70, and characterized.11
Structure of the enzyme
Pullulanase has a crystal structure. The structure of pullulanase comprises five domains: N1 from residues 39-172, N2 from 32-38 and 173-287, N3 from 288-395, A from 396-966, and C from 967-1083. The N1 domain, having the highest average B-factor, could be refined only in the complexes with G2, G3, and G4. The average deviation of the Ca position in the N1 domain between the G4 and G3 complexes reached 4.1A ° after the whole Ca was superimposed (r.m.s. Z 0.33 A ° for 946 Ca atoms within 2A ° ), indicating the mobility of the N1 domain. The average B-factors of the five domains were: N1 54.0 A ° 2, N2 36.7 A ° 2, N3 21.4 A ° 2, A 18.5 A ° 2 and C 26.1 A ° 2 in the structure of the G4 complex. Figure 1 shows the whole structure of pullulanase complexed with G4. The enzyme has dimensions of 102 A° Ã-65A° Ã-71A°.
Fig 1 Overall Structure of Pullulanase
Pullulanases can be found in higher plants (Nakamura et al., 1996; Renz et al., 1998; Kristensen et al., 1999; Beatty et al., 1999), as well as in bacteria, particularly the genera Bacillus (Jensen
& Norman, 1984; Kuriki et al., 1990; Suzuki et al., 1991; Ara et al., 1992; Kim et al., 1993), Fervidobacterium (Bertoldo et al., 1999), Klebsiella (Michaelis et al., 1985; Eisele et al., 1972; Dupuy et al., 1992), Thermotoga (Kriegshauser & Liebl, 2000) and Thermus (Tomiyasu et al., 2001). The presense of pullulanase has been reported from mesophiles, e.g., Bacillus sp. strain KSM-1378 (Ara et al., 1995), Bacillus sp. strain S-1 (Lee et al., 1997), Bacillus sp. strain KSM-
1876 (Hatada et al., 2001), etc. Besides, pullulanase also has been found from thermophiles, e.g. Anaerobranca gottschalkii (Bertoldo et al., 2004), Bacillus thermoleovorans US105 (Messaoud et al., 2002), and also from hyperthermophiles Fervidobacterium pennavorans Ven5 (Bertoldo et al., 1999), Rhodothermus marinus (Gomes et al., 2003), etc.
Pullulanase is also a starch debranching enzyme, which can cleave the Î±-1,6 glycosidic linkage. Moreover, pullulanase type II are also able to hydrolyze Î±-1,4 glycosidic linkages despite of Î±-1,6 glycosodic linkages. The molecular weight of pullulanase varies widely from different sources. Generally, the molecular weight of pullulanase type I is 70-80 kDa, and 100-210 kDa for pullulanase type II (Kim et al., 2000). Few studies have found that Ca2+ ion have no effect on the stabilization and activity of pullulanase enzyme (Stefanova et al., 1999; Lee et al., 1997, Ara et al., 1995). However, it was found that in the presence of increasing amounts of Ca2+ ions, similar significant increases are observed in the enzyme thermoactivity and thermostability ( Erra-Pujada et al., 2001; Gantelet & Duchiron, 1998; Antranikian et al., 1987). According to Saha et al. (1988), pullulanase enzyme may not require Ca2+ ions for its activity, but Ca2+ ions may play an important role in thermal stability and may maintain the conformation of the enzyme. According to Lévêque et al. (2000), there was no conclusion that can be drawn out about the mechanism by which Ca2+ ions stabilize and activate the pullulanase enzyme.
The optimum pH of most pullulanase occurs between pH 5.0 and 7.0, and optimum temperature between 45 to 60 °C (Saha et al., 1988; Ara et al., 1995; Kelly et al., 1983). But the optimum pH for alkaliphilic enzyme is between pH 8.0 to 10.0 (Bertoldo et al., 1999).The optimum temperature of archaeal pullulanases are typically between 80 to 100 °C (Lévêque et al., 2000), and some thermophiles and hyperthermophiles pullulanase are generally above 70 °C.
The divalent ions or some reagents may affect the activity of pullulanase. Different sources of pullulanase may have different degrees of divalent ions effect. Clostridium thermohydrosulfuricum pullulanase is inhibited by cyclodextrins, EDTA and N-bromosuccinimide, but not by acarbose and p-chloromercuribenzoate (Saha et al., 1988). Kim et al (2000) showed that the activity of pullulanase from Thermus IM6501 was strongly inhibited by Mn2+, Ni2+, Cu2+, Zn2+, Fe2+ and Ag2+, while enhanced slightly by Ca2+, Ba2+, Li2+ and Mg2+. EDTA also inhibited the pullulanase activity by 70%. On the other hand, Zn2+, Cu2+, Fe2+, Nbromosuccinimide and EDTA were able to inhibit the pullulanase from Desulfurococcus mucosus, while Ca2+, Mn2+ and cyclodextrins had no inhibition effect on the enzyme activity (Duffner et al., 2000). According to Ara et al. (1995), the pullulanase from Bacillus sp. KSM-1378 was inhibited by diethyl pyrocarbonate, phenylmethanesulphonyl fluoride, Nbromosuccinimide, Î±-CD and Î²-CD, meanwhile N-ethylmaleimide, 4- chloromercuribenzoate and monoiodoacetate had no effect on the enzyme activity. For the effect of divalent ions, Bacillus sp. KSM-1378 pullulanase was strongly inhibited by Hg2+, Cd2+, Pb2+ and Mn2+ ions, and Co2+ ions slightly stimulated the pullulanase activity.
Pullulanase production from wild microorganism usually faces many difficulties, such as low yields of enzymes, low enzyme activity and tedious downstream purification procedure especially for the intracellular enzymes. Genetic engineering techniques can overcome all the difficulties above. Firstly, large quantities of specific gene can be isolated in pure form by molecular cloning and the target DNA or enzyme can be produced in large amounts under the control of the expression vector (Madigan et al., 2000). Besides, the overexpression following the cloning step can significantly increase the enzyme yield by subcloning the target gene into a suitable expression vector. Through all the techniques above, the production cost for pullulanase enzyme can be significantly reduced and the improved enzyme properties may also meet the requirements for industrial use.
Starch Processing Industry
Some pullulanases are used in industries to complete the hydrolysis of starch initiated by Î±-amylases. Amylases hydrolyze Î±-1,4 glycosidic linkages in starch to produce a mixture of glucose, maltooligosaccharides and Î±-limit dextrins. All the remaining Î±-1,6 glycosidic branches in the products are hydrolyzed by pullulanase. Therefore, dextrin does not remain in the hydrolysate when starch is treated with amylase and pullulanase simultaneously, and consequently increase the efficiency of a saccharification reaction. This method has an advantage of generating higher yields of a desired end product from starch (Kim et al., 2000). Beside, the combined application of pullulanase with other amylolytic enzymes may increase the quality of sugar syrups.
The baking industry is a large consumer of starch and starch-modifying enzymes. Staling effect is the major problem in the baking industry. The staling effect includes increase of crumb firmness, loss of crispness of the crust, decrease in moisture content of the crumb and loss of bread flavor, leads to the deterioration of quality (van der Maarel et al., 2002). Although this problem can be overcome using chemical treatment, enzymatic treatment is more preferred due to the consumers nowadays demand for products without chemicals and higher acceptance by the
consumers for enzymes, which are produced from natural ingredients, are found. Some amylolytic enzymes act as anti-staling agent to solve the staling problem. Pullulanase can specifically remove the compound responsible for the gumminess associated with Î±-amylase treated bakery products. Pullulanase is able to rapidly hydrolyze the branched maltodextrins of DP20-100 produced by the Î±-amylase (van der Maarel et al., 2002). Pullulanase play an important role in the enzymatic antistaling treatment.\
Branched Cyclodextrins (CDs) Production
There is a very interesting and high economical valued application of pullulanase enzyme in branched cyclodextrins (CDs) production. CDs and branched CDs, such as maltosyl-CDs and glucosyl-CDs are homogeneous cyclic oligosaccharides, which are composed of only glucose units (Kitahata et al., 2000). These saccharides have a hydrophobic region and a hydrophilic region in their structures, and have the ability to form inclusion complexes with various kinds of
compounds (Hamayasu et al., 1999). Thus, CDs and branched CDs have been widely used or stabilizing labile materials, masking odours, and solubilizing insoluble or poorly soluble drugs (Tanimoto et al., 2005; Okada et al., 1988).
Due to the specific debranching ability, the use of pullulanase in starchprocessing industry is mostly promoted. Pullulanase is employed in the saccharification process to enhance the efficiency of the process. Beside, the combined application of pullulanase with other amylolytic enzymes may increase the quality of sugar syrups. The products from the starch-processing have wide application in various industries, such as beverages, confectionary, canning, icecream etc. Pullulanase can also be applied in baking industry and detergent industry. Recently, there are some reports on the application of pullulanase in the synthesis of branched-CD. Those reports also stated that the branched-CDs, especially heterobranched-CDs, can contribute to the pharmaceutical field as drug-carrier due to its higher aqueous solubility and cell-targeting ability.
In the food industry, pullulanases are used in the brewing process and starch hydrolysis together with Î²-amylases in order to produce a starch syrup that is high in maltose content (Belitz & Grosch, 1999). Pullanase is used as a detergent in biotechnology.
The wide application has encouraged studies on pullulanase from various microorganisms isolated from different location. Medium development is one of the important aspects to enhance pullulanase production. The culture condition and medium composition will greatly influence the pullulanase production and also the production cost. For the industrial purpose, high pullulanase production but involving lower cost is an important aspect to be considered in production studies.
Besides, the studies using molecular biology techniques may also improve the pullulanase enzyme and the production. The molecular studies also provided better hunderstanding on the reaction mechanism.