Bonds In Pullulan Research Articles

Pullulanase: unleashing the power of enzyme with a promising future in the food industry.

Pullulanases are the most important industrial group of enzymes in family 13 glycosyl hydrolases. They hydrolyze either α-1,6 and α-1,4 or both glycosidic bonds in pullulan as well as other carbohydrates to produce glucose, maltose, and maltotriose syrups, which have important uses in food and other related sectors. However, very less reports are available on pullulanase production from native strains because of low yield issues. In line with the increasing demands for pullulanase, it has become important to search for novel pullulanase-producing microorganisms with high yields. Moreover, high production costs and low yield are major limitations in the industrial production of pullulanase enzymes. The production cost of pullulanase by using the solid-state fermentation (SSF) process can be minimized by selecting agro-industrial waste. This review summarizes the types, sources, production strategies, and potential applications of pullulanase in different food and other related industries. Researchers should focus on fungal strains producing pullulanase for better yield and low production costs by using agro-waste. It will prove a better enzyme in different food processing industries and will surely reduce the cost of products.

Structure and function of the type III pullulan hydrolase from Thermococcus kodakarensis.

Pullulan-hydrolysing enzymes, more commonly known as debranching enzymes for starch and other polysaccharides, are of great interest and have been widely used in the starch-saccharification industry. Type III pullulan hydrolase from Thermococcus kodakarensis (TK-PUL) possesses both pullulanase and α-amylase activities. Until now, only two enzymes in this class, which are capable of hydrolysing both α-1,4- and α-1,6-glycosidic bonds in pullulan to produce a mixture of maltose, panose and maltotriose, have been described. TK-PUL shows highest activity in the temperature range 95-100°C and has a pH optimum in the range 3.5-4.2. Its unique ability to hydrolyse maltotriose into maltose and glucose has not been reported for other homologous enzymes. The crystal structure of TK-PUL has been determined at a resolution of 2.8 Å and represents the first analysis of a type III pullulan hydrolyse. The structure reveals that the last part of the N-terminal domain and the C-terminal domain are significantly different from homologous structures. In addition, the loop regions at the active-site end of the central catalytic domain are quite different. The enzyme has a well defined calcium-binding site and possesses a rare vicinal disulfide bridge. The thermostability of TK-PUL and its homologues may be attributable to several factors, including the increased content of salt bridges, helical segments, Pro, Arg and Tyr residues and the decreased content of serine.

Cloning and Characterization of a Novel Thermophilic Amylopullulanase with a Type I Pullulanase Structure From Anoxybacillus sp. WB42

Pullulanase is an important starch‐debranching enzyme that is used in the food, chemical, and pharmaceutical industries. In this study, pulWB42 is cloned from a novel pullulan‐degrading thermophile (Anoxybacillus sp. WB42) by homology cloning and genome walking. PulWB42 has the signature sequences of type I pullulanases, which cleave only α‐1,6‐glycosidic bonds in pullulan, and shows 74% identity with a type I pullulanase from A. tepidamans. Homology modeling suggests that PulWB42 has a typical type I pullulanase structure, which is distinct from that of known amylopullulanases. The recombinant PulWB42 is expressed in Escherichia coli and identified as an amylopullulanase by thin layer chromatography. rPulWB42 exhibits optimal activity toward pullulan at pH 5.8 and toward amylose at pH 5.5 over a temperature range of 55–65 °C, with a Km value of 5.3 ± 1.0 mg ml−1 for pullulan (0.09‐fold lower than that for amylose) and kcat value of 152.6 ± 13.2 s−1 for both pullulan and amylose. Moreover, the initial activity is completely retained after a 24‐h incubation at 60 °C and pH 5.5. Calcium ions activate the α‐amylase activity of rPulWB42, while weakly impacting the pullulanase activity. These catalytic properties make PulWB42 an important candidate for use in starch bioprocessing.

Cloning, expression and characterization of the recombinant cold-active type-I pullulanase from Shewanella arctica

An activity-based screening approach led to the identification of a novel glycoside hydrolase family-13 pullulanase gene (pul13A) from a psychrophilic bacterium. The recombinant enzyme exhibited a deduced peptide sequence of 1440 amino acid residues and was produced in a heterologous host in Escherichia coli. Purification from inclusion bodies was achieved by a six-step dialysis protocol enabling mild refolding of the urea-denaturated protein followed by affinity- and size-exclusion chromatography under native conditions. Pul13A is a type-I pullulanase, which is capable of hydrolysing α-1,6-glycosidic bonds in pullulan to produce maltotriose, while maltose and intermediate oligosaccharides are produced from soluble starch and amylopectin. The recombinant enzyme exhibited typical properties of cold-adapted proteins including low thermostability at elevated temperatures. It showed a temperature optimum at 35°C, while at 10°C residual activity (25%) remained. The optimal pH was in the range of 6.0–7.0, with Pul13A being stable at neutral and basic pH, but not in the acidic range. Catalytic activity was increased in the presence of divalent cations calcium and cobalt and both metal ions were also able to restore catalytic activity of EDTA-chelated enzyme samples. Pul13A represents the first type-I pullulanase from a psychrophile that has been produced in recombinant form. Moreover, its favourable enzymatic properties make this enzyme a potential candidate for industrial applications such as starch degradation for ethanol based biofuel production.

Substrate Specificity and Detailed Characterization of a Bifunctional Amylase-Pullulanase Enzyme from Bacillus circulans F-2 Having Two Different Active Sites on One Polypeptide

Bacillus circulans F-2 amylase-pullulanase enzyme (APE) displayed dual activity with respect to glycosidic bond cleavage. The enzyme was active on alpha-1,6 bonds in pullulan, amylopectin, and glycogen, while it showed alpha-1,4 activity against malto-oligosaccharides, amylose, amylopectin, and soluble starch, but not pullulan. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as two competing substrates was used to distinguish the dual specificity of the enzyme from the single-substrate specificity known for pullulanases and alpha-amylases. Enzyme activities were inhibited by some metal ions, and by metal-chelating agents with a different mode. The enzyme-inhibitory results of amylase and pullulanase with Hg2+ and Co2+ ions were different, indicating that the activation mechanisms of both enzyme activities are different. Cyclomaltoheptaose inhibited both alpha-amylase and pullulanase activities with inhibition constants (Ki) of 0.029 and 0.06 mg/ml, respectively. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide confirmed a carboxy group at the active sites of both enzymes. The N-terminal sequence of the enzyme was: Ala-Asp-Ala-Lys-Lys-Thr-Pro- Gln-Gln-Gln-Phe- Asp-Ala-Leu-Trp-Ala-Ala-Gly-Ile-Val-Thr-Gly-Thr-Pro-Asp-Gly-Phe. The purified enzyme displayed Michaelis constant (Km) values of 0.55 mg/ml for amylose, and 0.71 mg/ml for pullulan. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which the two substrates are hydrolyzed at different active sites. These results suggest that amylopullulanases, which possess both alpha-1,6 and alpha-1,4 cleavage activities at the same active site, should be distinguished from APEs, which contain both activities at different active sites on the same polypeptide. Also, it is proposed that the Enzyme Commission use the term 'amylase-pullulanase enzyme' to refer to enzymes which act on starch and cleave both alpha-1,6-bonds in pullulan and alpha-1,4 bonds in amylose at different active sites.

Chemiluminescence related to degradation of thermally oxidized pullulans. Comparison with cellulose and dextran

The degradation of pullulan, cellulose and dextran in the presence of oxygen was examined by chemiluminescence and by size-exclusion chromatography (SEC) methods. While 1,4-α glycosidic bonds in pullulan are split thermally only with a slight effect of oxygen, thermolysis of 1,4-β bonds in cellulose and 1,6-α bonds in dextran is preceded by free radical oxidation of carbon 6 of glucopyranose unit with formation of carboxyl groups and water contributing to the subsequent induced degradation of polysaccharide. MgO which remains in polysaccharide after its impregnation by methoxy magnesium methyl carbonate (MMMC) promotes the reaction of terminal semiacetal groups and/or hydroperoxides which has considerably higher quantum yield of light emission.

Purification and properties of the catalytic domain of the thermostable pullulanase type II from Thermococcus hydrothermalis

A pullulanase type II was produced in Escherichia coli using the relevant gene from Thermococcus hydrothermalis. This protein was purified and its pullulanolytic and amylolytic activities were characterised. The optimum temperature and Ca2+ concentration for each activity were identical (105 °C and 0.09 mM), whereas the optimum pH (pHpullulan 5.75, pHamylose 5) and the influence of Ca2+ ions on the kinetic parameters were different. Further analyses revealed that this enzyme exhibits an endo-processive-like action and specifically cleaves α-1,6 bonds in pullulan.

The amylopullulanase of Bacillus sp. DSM 405.

The amylopullulanse produced by Bacillus sp. DSM 405 was purified to homogeneity. It exhibited dual activity, cleaving the alpha 1-4 bonds in starch, releasing a range of malto-oligosaccharides, and also cleaving the alpha 1-6 bonds in pullulan, releasing maltotriose as the sole end-product. The enzyme was a glycoprotein and had a relative molecular mass of 126,000 and an isoelectric point of 4.3. While the enzyme was optimally active on starch at pH 6.5 and at pH 6.0 on pullulan, activity on both substrates was maximal at 70 degrees C. Kinetic analyses of the enzyme in a system that contained both starch and pullulan as two competing substrates demonstrated the dual specificity of the enzyme. Chemical modification of the carboxyl groups within the active centre of the protein showed that one active site was responsible for hydrolysis of the alpha 1-4 and alpha 1-6 bonds in starch and pullulan respectively. This is the first comprehensive investigation of an amylopullulanse produced by an aerobic bacterium, showing a single active site responsible for both activities.

Substrate specificity and detailed characterization of a bifunctional amylase-pullulanase enzyme from Bacillus circulans F-2 having two different active sites on one polypeptide.

Bacillus circulans F-2 amylase-pullulanase enzyme (APE) displayed dual activity with respect to glycosidic bond cleavage. The enzyme was active on alpha-1,6 bonds in pullulan, amylopectin, and glycogen, while it showed alpha-1,4 activity against malto-oligosaccharides, amylose, amylopectin, and soluble starch, but not pullulan. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as two competing substrates was used to distinguish the dual specificity of the enzyme from the single-substrate specificity known for pullulanases and alpha-amylases. Enzyme activities were inhibited by some metal ions, and by metal-chelating agents with a different mode. The enzyme-inhibitory results of amylase and pullulanase with Hg2+ and Co2+ ions were different, indicating that the activation mechanisms of both enzyme activities are different. Cyclomaltoheptaose inhibited both alpha-amylase and pullulanase activities with inhibition constants (Ki) of 0.029 and 0.06 mg/ml, respectively. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide confirmed a carboxy group at the active sites of both enzymes. The N-terminal sequence of the enzyme was: Ala-Asp-Ala-Lys-Lys-Thr-Pro- Gln-Gln-Gln-Phe- Asp-Ala-Leu-Trp-Ala-Ala-Gly-Ile-Val-Thr-Gly-Thr-Pro-Asp-Gly-Phe. The purified enzyme displayed Michaelis constant (Km) values of 0.55 mg/ml for amylose, and 0.71 mg/ml for pullulan. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which the two substrates are hydrolyzed at different active sites. These results suggest that amylopullulanases, which possess both alpha-1,6 and alpha-1,4 cleavage activities at the same active site, should be distinguished from APEs, which contain both activities at different active sites on the same polypeptide. Also, it is proposed that the Enzyme Commission use the term 'amylase-pullulanase enzyme' to refer to enzymes which act on starch and cleave both alpha-1,6-bonds in pullulan and alpha-1,4 bonds in amylose at different active sites.

Purification and general biochemical properties of thermostable pullulanase from Bacillus stearothermophilus G-82.

Thermostable extracellular pullulanase, produced by Bacillus stearothermophilus G-82 was purified to homogeneity from supernatants of continuous culture by ultrafiltration, ammonium sulphate precipitation, chromatography on Sephadex G-100, and DEAE cellulose. A mol wt of 53,000 was determined by gel filtration and 56,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point (pI) was 4.2. The pullulanase contained predominantly acidic amino acids. The enzyme was optimally active at a temperature of 60 degrees C and pH 7.0. It preserved 100% of its activity after 10 min treatment at 60 degrees C. The thermostability was considerably increased in the presence of pullulan. Ca2+ did not increase activity or thermostability. Enzyme activity was fully inhibited by N-bromosuccinimide and partially by phenylmethylsulfonyl fluoride. Bacillus stearothermophilus G-82 pullulanase was able to hydrolyze alpha 1-6 as well as alpha 1-4 glucosidic bonds in pullulan, amylopectin, amylose, glycogen, and dextrin. The enzyme showed highest affinity to pullulan (Km = 0.14).

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1

Previously, we constructed a gene disruption in the pullulanase I gene of Bacteroides thetaiotaomicron 5482A. This mutant, designated B. thetaiotaomicron 95-1, had a lower level of pullulanase specific activity than did wild-type B. thetaiotaomicron but still exhibited a substantial amount of pullulanase activity. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an alpha(1----4)-D-glucosidic bond cleaving pullulanase which has been tentatively designated a neopullulanase. The neopullulanase (pullulanase II) is a 70-kDa soluble protein which cleaves alpha(1----4)-D-glucosidic bonds in pullulan to produce panose. The neopullulanase also cleaved alpha(1----4) bonds in amylose and in oligosaccharides of maltotriose through maltoheptaose in chain length. An alpha-glucosidase from B. thetaiotaomicron 95-1 was characterized. The alpha-glucosidase was partially purified to a preparation containing three proteins of 80, 57, and 50 kDa. Pullulan and amylose were not hydrolyzed by the alpha-glucosidase. alpha(1----4)-D-Glucosidic oligosaccharides from maltose to maltoheptaose were hydrolyzed to glucose by the alpha-glucosidase. The alpha-glucosidase also hydrolyzed alpha(1----6)-linked oligosaccharides such as panose (the product of the pullulanase II action on pullulan) and isomaltotriose.

Substrate competition and specificity at the active site of amylopullulanase from Clostridium thermohydrosulfuricum

A highly thermostable pullulanase purified from Clostridium thermohydrosulfuricum strain 39E displayed dual activity with respect to glycosidic bond cleavage. The enzyme cleaved α-1,6 bonds in pullulan, while it showed α-1,4 activity against malto-oligosaccharides. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as the two competing substrates were used to distinguish the dual specificity of the enzyme from the single substrate specificity known for pullulanases and α-amylases.