Hydrolysis Of Pullulan Research Articles

Purification and biochemical properties of an alkaline pullulanase from alkalophilic Bacillus sp. S-1.

A novel extracellular pullulanase (PUL-E, pullulan 6-glucanohydrolase, EC 3.2.1.41) has been purified from the alkalophilic Bacillus sp. S-1. The purified enzyme had a molecular mass of about 140 kDa on denaturated and natural conditions. The pI was 5.5. The pullulanase, when resolved by SDS-PAGE, was negative for Schiff staining, suggesting that the enzyme is not a glycoprotein. The N-terminal amino acid sequence of the enzyme was Phe-Leu-Asn-Met-Ser-(Trp-Phe). The enzyme displayed a temperature optimum of around 60 degrees C and a pH optimum of around pH 9.0. The enzyme was stable to incubation from pH 4.0 to pH 11.0 at 4 degrees C for 24 h. The presence of pullulan protected the enzyme from heat inactivation, the extent depending upon the substrate concentration. The activity of the enzyme was stimulated by Mn2+ ions. Ca2+ ions and EDTA did not inhibit the enzyme activity. The enzyme hydrolyzed the alpha-1,6-linkages of amylopectin, glycogens, alpha,beta-limited dextrin, and pullulan. The enzyme had an apparent Km of 7.92 mg/ml for pullulan, a Km of 1.63 mg/ml for amylopectin, and a Km of 3.1 mg/ml for alpha,beta-limited dextrin, when measured at pH 9.0 and 50 degrees C. The enzyme caused the complete hydrolysis of pullulan to maltotriose. The activity was not inhibited by alpha, beta, or gamma-cyclodextrins. The western blotting analysis with mouse anti-serum against PUL-E showed that PUL-E is produced as a single enzyme form during bacterial cultivation.

Dilute solution properties of pullulan by dynamic light scattering

Pullulan, produced by the fungus Aureobasidium, has been studied in dilute solution by viscometry, static low angle light scattering and dynamic light scattering at temperatures of 25, 40, 60 and 80 °C. The samples used were fractions obtained by partial hydrolysis of native pullulan and successive fractionation with ethanol. The mass-average molar mass, MW, of the studied pullulans were in the range of 1.67*105 to 1.0*106 g/mol. The translational diffusion coefficient at infinite dilution, Do, was obtained from the reduced first cumulant of the intensity autocorrelation function of the light scattered by extrapolation to zero scattering angle and zero polymer concentration. The activation energy of diffusion, ED, of pullulan macromolecules was calculated from Do=Doexp(-ED/RT). It was found that in the given temperature rangeED = 16.5 ± 0.5 kJmol−1 for all samples.

Purification and characterization of a thermostable α-glucosidase from aBacillus subtilis high-temperature growth transformant

Ap-nitrophenyl-α-d-maltoside-hydrolyzing α-glucosidase was purified and characterized from aBacillus subtilis high-temperature growth transformant (H-17), previously generated by transformation ofBacillus subtilis 25S withBacillus caldolyticus C2 DNA. The enzyme showed endo-oligo-1,4-glucosidase activity owing to its hydrolysis of linear malto-oligosaccharides to maltose and glucose, and pullulan hydrolase activity owing to its hydrolysis of pullulan to glucose, maltose, and (iso)panose. The enzyme was inactive againstp-nitrophenyl-α-d-glucopyranoside, maltose, isomaltose, isomaltotriose, and panose, but slightly hydrolyzed starch. The native structure of the enzyme is a dimer composed of two identical subunits of Mr 55,000. The enzyme had a pI of 4.8, pH optimum of 7.5, was 80% inhibited by 5 mM Tris-HCl, and had a Km value of 1.46 mM for the chromogenic substratep-nitrophenyl-α-d-maltoside. The enzyme showed optimal activity between 65° and 68°C, and retained 100% of initial activity after incubation at 65°C for 1 h. A minimum concentration of 0.02% 2-mercaptoethanol or 0.005 mM EDTA was required for thermostability. These physiochemical characteristics are similar to those for the previously described corresponding enzyme fromB. subtilis 25S, except that the same enzyme from the transformed strain was thermolabile. Amino acid analysis showed higher levels of alanine, glycine, and proline residues in the H-17 enzyme, compared with 25S. This may account for the enhanced thermostability, owing to increased internal hydrophobicity.

Purification, characterization, and comparison of anα-glucosidase (endo-oligo-1, 4-glucosidase) from the mesophileBacillus subtilis and the obligate thermophileBacillus caldolyticus

Thep-nitrophenyl-α-d-maltoside hydrolyzingα-glucosidase from the mesophileBacillus subtilis 25S and the obligate thermophileBacillus caldolyticus C2 was purified, characterized, and compared in order to determine the molecular mechanisms that may confer thermostability of starch-degrading enzymes. Both enzymes showed endo-oligo-1,4-glucosidase activity owing to their identical hydrolysis of linear malto-oligosaccharides to maltose and glucose as determined by thin-layer chromatography. Neither enzyme showed activity againstp-nitrophenyl-α-d-glucopyranoside, maltose, isomaltose, isomaltotriose, or panose. The enzymes may tentatively be classified as a panose-producing pullulanase owing to their hydrolysis of pullulan. The 25S and C2 enzymes were composed of two identical subunits of Mr 55,000 and 60,000 respectively. Both the 25S and C2 enzymes have a pI of 4.85, pH optimum of 7.5 and 7.0, and Km values for the chromogenic substratep-nitrophenyl-α-d-maltoside of 2.96 mM and 1.31 mM respectively. The 25S enzyme exhibited optimal activity between 35 and 37°C, and complete inactivation after 10 min at 45°C, while the C2 enzyme showed optimal activity at 60°C and retained 100% of initial activity at 60°C for 2 h. The C2 enzyme required a minimum of 0.02% 2-mercaptoethanol or 0.01 mM EDTA for thermostability. A comparison of the amino acid compositions showed an increase in the number of proline, alanine, and leucine residues for the thermostable C2 enzyme. These alterations in hydrophobicity may influence enzyme thermostability; this may be a factor in the design of engineered proteins for industrial use.

Extracellular constitutive production of pullulan-hydrolysing activity by Sclerotium rolfsii

Extracellular constitutive pullulan-hydrolysing activity was detected in Sclerotium rolfsii (1.05 μmol glucose equivalents liberated∙mL−1∙min−1) when cultivated in a synthetic medium containing starch as the carbon source. The influence of various inorganic nitrogen and carbon sources, surfactants, and organic supplements on the production of pullulan-hydrolysing activity was studied. In an optimized medium supplemented with 1% corn steep liquor, increased yields of enzyme activity (1.8 μmol∙mL−1∙min−1) were obtained. The enzyme was most active at pH 4.2 and was stable in the pH range of 3.5–5.5. The optimum temperature for pullulan hydrolysis was 50 °C and the activity was stable in the temperature range of 25–60 °C at pH 4.2 for 30 min. The Ea for the native enzyme from the Arrhenius plot with pullulan as a substrate was 22.6 kJ∙mol−1. The Km value for the native enzyme with pullulan as a substrate was 8.33 mg∙mL−1. The enzyme did not produce glucose or maltose from pullulan. Inability of the enzyme to rapidly decrease the specific viscosity (ηsp) of a 1% pullulan solution indicated susceptibility of α-(1 → 6) and not α-(1 → 4) linkages.

Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis.

The gene for an enzyme with single or dual specificity on complex carbohydrates has been transferred from its native host (Thermoanaerobium brockii), a thermophilic anaerobe, into Escherichia coli and Bacillus subtilis. Most of the gene coding region is in a 2.2-kilobase PstI fragment that is common to the E. coli and B. subtilis chimeric vectors pCPC902 and pCPC903, respectively. Although the T. brockii debranching enzyme secreted from B. subtilis was unglycosylated and had less thermostability, more enzyme was secreted from B. subtilis (0.80 to 1.0 U/ml) than from T. brockii (0.23 U/ml). E. coli did not export any measurable enzyme. From the fermentation broth of B. subtilis containing pCPC903, three active species of the debranching enzyme were separated; two species are possibly protease digestion products of the larger protein (105,000 molecular weight). Whereas the enzyme can cleave all of the alpha-1----6 glucosidic linkages (and none of the alpha-1----4 bonds) in pullulan, it hydrolyzed mostly alpha-1----4 and very few of the alpha-1----6 linkages in starch. Upon hydrolysis of pullulan by the enzyme, only maltotriose was produced, while starch was digested to various-sized oligomers.

Active-site- and substrate-specificity of Thermoanaerobium Tok6-B1 pullulanase.

Thermoanaerobium Tok6-B1 pullulanase (EC 3.2.1.41) was active on alpha 1-6-glucosidic linkages of pullulan, amylopectin and glycogen and the alpha 1-4 linkages of amylose, amylopectin and glycogen but not of pullulan. Hydrolysis of short-chain-length malto-oligosaccharides (seven or fewer glucose residues) yielded maltose as product. Pullulan hydrolysis was pH-dependent and a plot of log(V/Km) versus pH implied a carboxy group with pKa 4.3 at the active site. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide (EDAC) confirmed this view, and analysis of the order of reaction and inactivation kinetics suggested the presence of a single carboxy group at a catalytic centre of the active site. EDAC-mediated inhibition of pullulan alpha 1-6-bond hydrolysis was relieved by amylose or pullulan. Similarly both pullulan and amylose protected the activity directed at alpha 1-4 bonds of amylose from EDAC inhibition. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which both substrates were hydrolysed at the same active site.

A highly stable pullulanase from Thermus aquaticus YT-1

A cell-associated pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) from Thermus aquaticus YT-1 has been detergent solubilized and purified by a method involving dispersion into ethanediol followed by gel filtration and ion-exchange chromatography. Molecular weight was determined as 83 000 by SDS-PAGE and 80 000 by steric exclusion chromatography. The enzyme had an optimum pH of 6.4 and exhibited debranching activity towards starch ( K m = 3.4 × 10 −3%) and amylopectin. ( K m = 2.5 × 10 −3%). Hydrolysis of pullulan was specific to α(1→6) glycosyl linkages ( K m = 6.3 × 10 −3%). Activity was highly thermostable with over 80% activity remaining after 10 h at 85°C and t 1 2 = 4.5 h at 95°C. Thermostability was Ca 2+ dependent and influenced by pH ( t 1 2 at 100°C = 13.5 min at pH = 7.0, 6.5 min at pH 5.9 and 1.5 min at pH 4.7 in the presence of Ca 2+).

Observations on the structure of the fungal extracellular polysaccharide, pullulan

Hydrolysis of pullulan, derived from strain P50 of Aureobasidium pullulans, by amyloglucosidase leaves a major portion of the glucan untouched, suggesting that the polysaccharide does not contain the simple linear sequence of α-(1→6)-linked maltotriosyl residues hitherto ascribed.

Synthesis from pullulan of spacer-arm, lipid, and ethyl glycosides of a tetrasaccharide [α- d-Glc-(1→6)-α- d-Glc-(1→4)-α- d-Glc-(1→4)- d-Glc] found in human urine; preparation of neoglycoproteins

Enzymic hydrolysis of pullulan, followed by acetylation and chromatography, gave acetylated α- d-Glc p-(1→6)-α- d-Glc p-(1→4)-α- d-Glc p-(1→4)- d-Glc p which, with 2-bromoethanol and boron trifluoride etherate in dichloromethane, gave the 2-bromoethyl glycoside. The reactions of the glycoside with methyl 3-mercaptopropionate, methyl 11-mercaptoundecanoate, and octadecanethiol are described, and also its hydrogenolysis to give an ethyl glycoside. The mercaptopropionate-derived, spacer-arm glycoside has been coupled to bovine serum albumin and keyhole limpet haemocyanin.

A cultural method for the detection of pullulan--degrading enzymes in bacteria and its application to the genus Bacillus.

Pullulan in a solid growth medium can be precipitated selectively with ethanol. Clear zones due to pullulan hydrolysis surround microbial colonies which produce the appropriate enzyme. When this procedure was applied to 297 strains representing 26 species of the genus Bacillus, 138 (46%) of the organisms were shown to hydrolyze pullulan.

Mode of action of a rice debranching enzyme on pullulan and its related oligosaccharides.

In the hydrolysis of pullulan by a rice debranching enzyme, oligosaccharides intermediately produced were more rapidly degraded than expected from the random cleavage (relative maximum velocities: hexaose 3.0, nonaose 1.7, oligosaccharides of higher polymerizations 1.2, pullulan 1.0), and the final product maltotriose accumulated extraordinarily even at early stages of the hydrolysis. Therefore, the hydrolytic action on pullulan was confirmed to proceed in an endo-fashion with the incomplete random cleavage.

Hydrolysis of Pullulan by a Novel Enzyme fromAspergillus niger

Journal Article Hydrolysis of Pullulan by a Novel Enzyme from Aspergillus niger Get access Yoshiyuki Sakano, Yoshiyuki Sakano Department of Agricultural Chemistry, Tokyo Noko University, Fuchu, Tokyo Search for other works by this author on: Oxford Academic Google Scholar Noriko Masuda, Noriko Masuda Department of Agricultural Chemistry, Tokyo Noko University, Fuchu, Tokyo Search for other works by this author on: Oxford Academic Google Scholar Tsuneo Kobayashi Tsuneo Kobayashi Department of Agricultural Chemistry, Tokyo Noko University, Fuchu, Tokyo Search for other works by this author on: Oxford Academic Google Scholar Agricultural and Biological Chemistry, Volume 35, Issue 6, 1 June 1971, Pages 971–973, https://doi.org/10.1080/00021369.1971.10860023 Published: 01 June 1971 Article history Received: 23 March 1971 Published: 01 June 1971

Hydrolysis of Pullulan by a Novel Enzyme from Aspergillus niger

Hydrolysis of Pullulan by a Novel Enzyme from Aspergillus niger