Pullulanase ( pullulanase type I, pullulan 6-glucanohydrolase, EC 18.104.22.168 ) , besides known as bound dextrinase, is a debranching enzyme that specifically breaks up I±-1,6-glycosidic linkages of pullulan, amylum and amylopectin. Depending on their inability or ability to hydrolyse I±-1,4 glucosidic linkages in other polyoses, pullulanases are divided into two classs based on substrate specificity: ( I ) pullulanase type I, which specifically cleaves the I±-1,6-glycosidic linkages in pullulan and branched oligosaccharides, organizing maltotriose and additive oligosacharides, severally, and ( two ) pullulanase type II, or amylopullulanase, which hydrolyzes both I±-1,6-glycosidic linkages and I±-1,4-glycosidic linkages in bifurcate and additive oligosaccharides ( Bertoldo & A ; Antranikian, 2002 ) . In add-on, based on amino-acid sequence similarities these pullulanases are classified into glycoside hydrolase ( GH ) household 13 together with I±-amylases and isoamylases ( Henrissat, 1991 ; hypertext transfer protocol: //afmb.cnrs-mrs.fr/CAZY/GH_13.html ) .
History of the Enzyme
Pullulanase type I ( referred to here merely as pullulanase ) was foremost isolated from a civilization of
Aerobacter aerogenes ( Klebsiella pneumoniae ) by Wallenfels et Al. in 1966. The enzyme was so isolated from several micro-organisms, including Streptococcus mitis,9 Bacillus no. 202-1,10 and Klebsiella aerogenes W70, and characterized.11
Structure of the enzyme
Pullulanase has a crystal construction. The construction of pullulanase comprises five spheres: 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 sphere, holding the highest mean B-factor, could be refined merely in the composites with G2, G3, and G4. The mean divergence of the Ca place in the N1 sphere between the G4 and G3 composites reached 4.1A A° after the whole Ca was superimposed ( r.m.s. Z 0.33 A A° for 946 Ca atoms within 2A A° ) , bespeaking the mobility of the N1 sphere. The mean B-factors of the five spheres were: N1 54.0 A A° 2, N2 36.7 A A° 2, N3 21.4 A A° 2, A 18.5 A A° 2 and C 26.1 A A° 2 in the construction of the G4 composite. Figure 1 shows the whole construction of pullulanase complexed with G4. The enzyme has dimensions of 102 AA° A-65AA° A-71A° .
Fig 1 Overall Structure of Pullulanase
Pullulanases can be found in higher workss ( Nakamura et al. , 1996 ; Renz et al. , 1998 ; Kristensen et al. , 1999 ; Beatty et al. , 1999 ) , every bit good as in bacteriums, peculiarly the genera Bacillus ( Jensen
& A ; 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 & A ; Liebl, 2000 ) and Thermus ( Tomiyasu et al. , 2001 ) . The presense of pullulanase has been reported from mesophiles, e.g. , Bacillus sp. strive KSM-1378 ( Ara et al. , 1995 ) , Bacillus sp. strive S-1 ( Lee et al. , 1997 ) , Bacillus sp. strive KSM-
1876 ( Hatada et al. , 2001 ) , etc. Besides, pullulanase besides has been found from thermophiles, e.g. Anaerobranca gottschalkii ( Bertoldo et al. , 2004 ) , Bacillus thermoleovorans US105 ( Messaoud et al. , 2002 ) , and besides from hyperthermophiles Fervidobacterium pennavorans Ven5 ( Bertoldo et al. , 1999 ) , Rhodothermus marinus ( Gomes et al. , 2003 ) , etc.
Pullulanase is besides a amylum debranching enzyme, which can split the I±-1,6 glycosidic linkage. Furthermore, pullulanase type II are besides able to hydrolyse I±-1,4 glycosidic linkages despite of I±-1,6 glycosodic linkages. The molecular weight of pullulanase varies widely from different beginnings. By and large, the molecular weight of pullulanase type I is 70-80 kDa, and 100-210 kDa for pullulanase type II ( Kim et al. , 2000 ) . Few surveies have found that Ca2+ ion have no consequence on the stabilisation 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 sums of Ca2+ ions, similar important additions are observed in the enzyme thermoactivity and thermostability ( Erra-Pujada et al. , 2001 ; Gantelet & A ; Duchiron, 1998 ; Antranikian et al. , 1987 ) . Harmonizing to Saha et Al. ( 1988 ) , pullulanase enzyme may non necessitate Ca2+ ions for its activity, but Ca2+ ions may play an of import function in thermic stableness and may keep the conformation of the enzyme. Harmonizing to Leveque et Al. ( 2000 ) , there was no decision that can be drawn out about the mechanism by which Ca2+ ions stabilise and trip the pullulanase enzyme.
The optimal pH of most pullulanase occurs between pH 5.0 and 7.0, and optimal temperature between 45 to 60 °C ( Saha et al. , 1988 ; Ara et al. , 1995 ; Kelly et al. , 1983 ) . But the optimal pH for alkaliphilic enzyme is between pH 8.0 to 10.0 ( Bertoldo et al. , 1999 ) .The optimal temperature of archaeal pullulanases are typically between 80 to 100 °C ( Leveque et al. , 2000 ) , and some thermophiles and hyperthermophiles pullulanase are by and large above 70 °C.
The bivalent ions or some reagents may impact the activity of pullulanase. Different beginnings of pullulanase may hold different grades of bivalent ions consequence. Clostridium thermohydrosulfuricum pullulanase is inhibited by cyclodextrins, EDTA and N-bromosuccinimide, but non 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 somewhat by Ca2+ , Ba2+ , Li2+ and Mg2+ . EDTA besides inhibited the pullulanase activity by 70 % . On the other manus, Zn2+ , Cu2+ , Fe2+ , Nbromosuccinimide and EDTA were able to suppress the pullulanase from Desulfurococcus mucosus, while Ca2+ , Mn2+ and cyclodextrins had no suppression consequence on the enzyme activity ( Duffner et al. , 2000 ) . Harmonizing to Ara et Al. ( 1995 ) , the pullulanase from Bacillus sp. KSM-1378 was inhibited by diethyl pyrocarbonate, phenylmethanesulphonyl fluoride, Nbromosuccinimide, I±-CD and I?-CD, meanwhile N-ethylmaleimide, 4- chloromercuribenzoate and monoiodoacetate had no consequence on the enzyme activity. For the consequence of divalent ions, Bacillus sp. KSM-1378 pullulanase was strongly inhibited by Hg2+ , Cd2+ , Pb2+ and Mn2+ ions, and Co2+ ions somewhat stimulated the pullulanase activity.
Pullulanase production from wild micro-organism normally faces many troubles, such as low outputs of enzymes, low enzyme activity and boring downstream purification process particularly for the intracellular enzymes. Familial technology techniques can get the better of all the troubles above. First, big measures of specific cistron can be isolated in pure signifier by molecular cloning and the mark DNA or enzyme can be produced in big sums under the control of the look vector ( Madigan et al. , 2000 ) . Besides, the overexpression following the cloning measure can significantly increase the enzyme output by subcloning the mark cistron into a suited look vector. Through all the techniques above, the production cost for pullulanase enzyme can be significantly reduced and the improved enzyme belongingss may besides run into the demands for industrial usage.
Starch Processing Industry
Some pullulanases are used in industries to finish the hydrolysis of amylum initiated by I±-amylases. Amylases hydrolyze I±-1,4 glycosidic linkages in amylum to bring forth a mixture of glucose, maltooligosaccharides and I±-limit dextrins. All the staying I±-1,6 glycosidic subdivisions in the merchandises are hydrolyzed by pullulanase. Therefore, dextrin does non stay in the hydrolysate when amylum is treated with amylase and pullulanase at the same time, and accordingly increase the efficiency of a saccharification reaction. This method has an advantage of bring forthing higher outputs of a coveted terminal merchandise from amylum ( Kim et al. , 2000 ) . Beside, the combined application of pullulanase with other amylolytic enzymes may increase the quality of sugar sirups.
The baking industry is a big consumer of amylum and starch-modifying enzymes. Staling consequence is the major job in the baking industry. The staling consequence includes addition of crumb soundness, loss of crispness of the crust, lessening in wet content of the crumb and loss of bread spirit, leads to the impairment of quality ( van der Maarel et al. , 2002 ) . Although this job can be overcome utilizing chemical intervention, enzymatic intervention is more preferable due to the consumers presents demand for merchandises without chemicals and higher credence by the
consumers for enzymes, which are produced from natural ingredients, are found. Some amylolytic enzymes act as anti-staling agent to work out the staling job. Pullulanase can specifically take the compound responsible for the cohesiveness associated with I±-amylase treated bakeshop merchandises. Pullulanase is able to quickly hydrolyse the bifurcate maltodextrins of DP20-100 produced by the I±-amylase ( van der Maarel et al. , 2002 ) . Pullulanase play an of import function in the enzymatic antistaling treatment.
Branched Cyclodextrins ( Cadmiums ) Production
There is a really interesting and high economical valued application of pullulanase enzyme in bifurcate cyclodextrins ( Cadmiums ) production. Cadmiums and branched Cadmiums, such as maltosyl-CDs and glucosyl-CDs are homogenous cyclic oligosaccharides, which are composed of merely glucose units ( Kitahata et al. , 2000 ) . These carbohydrates have a hydrophobic part and a hydrophilic part in their constructions, and have the ability to organize inclusion composites with assorted sorts of
compounds ( Hamayasu et al. , 1999 ) . Therefore, Cadmiums and branched Cadmiums have been widely used or stabilising labile stuffs, dissembling smells, and solubilizing indissoluble or ailing soluble drugs ( Tanimoto et al. , 2005 ; Okada et al. , 1988 ) .
Due to the specific debranching ability, the usage of pullulanase in starchprocessing industry is largely promoted. Pullulanase is employed in the saccharification procedure to heighten the efficiency of the procedure. Beside, the combined application of pullulanase with other amylolytic enzymes may increase the quality of sugar sirups. The merchandises from the starch-processing have broad application in assorted industries, such as drinks, confectionary, canning, ice cream etc. Pullulanase can besides be applied in baking industry and detergent industry. Recently, there are some studies on the application of pullulanase in the synthesis of branched-CD. Those studies besides stated that the branched-CDs, particularly heterobranched-CDs, can lend to the pharmaceutical field as drug-carrier due to its higher aqueous solubility and cell-targeting ability.
In the nutrient industry, pullulanases are used in the brewing procedure and amylum hydrolysis together with I?-amylases in order to bring forth a amylum sirup that is high in maltose content ( Belitz & A ; Grosch, 1999 ) . Pullanase is used as a detergent in biotechnology.
The broad application has encouraged surveies on pullulanase from assorted micro-organisms isolated from different location. Medium development is one of the of import facets to heighten pullulanase production. The civilization status and medium composing will greatly act upon the pullulanase production and besides the production cost. For the industrial intent, high pullulanase production but affecting lower cost is an of import facet to be considered in production surveies.
Besides, the surveies utilizing molecular biological science techniques may besides better the pullulanase enzyme and the production. The molecular surveies besides provided better hunderstanding on the reaction mechanism.