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No pullulanase activity could be detected in maltose-grown cultures of 11 different strains of Escherichia coli. Neither these strains, nor 40 other strains of the same species would grow on pullulan, while all of them grew on maltose and some were shown to grow on maltotriose the exclusive product of pullulanase action on pullulan. These results are in contradiction with those reported last year in this journal by Palmer, Wober and Whelan. On the other hand pullulanase activity could be detected, as reported by Bender and Wallenfels in 1966, in two strains of Aerobacter aerogenes.

An extracellular α-glucan, pullulan, elaborated by a strain of Pullularia pullulans , contains 0.6% of a maltotetraose subunit, as well as the already known major component, maltotriose. The majority, at least, of the maltotetraose is contained within the polymeric chains and is linked, through its terminal glucose units, by α-1→6— bonds.

Numerous polysaccharides of diverse monomeric constituents and modes of linkage (such as galactan, isolichenan, laminaran, levans, lichenan, pullulan, and yeast mannan) combine with gelatin to form macromolecular complexes soluble in 0.83% trichloroacetic acid −83% ethanol. In contrast, inulin, nigeran, and yeast d -glucan do not complex with gelatin. Dextrans having various contents of α- d -(1→6), α- d -(1→4), and α- d -(1→3) linkages appear to bind gelatin more effectively than either glycogen or soluble starch. Decreased stoichiometric binding has been correlated with increased branching of the dextran polymers; conversely, the branched-chain levans bind gelatin, whereas inulin, a linear polymer, does not. With the exception of starch acetate, esterification of neutral glycans with formic, acetic, or phosphoric acid either enhances, or has no effect on, formation of complex.

Measurements of optical rotatory dispersion (ORD) and circular dichroism (CD) were made in the range of 400–205 nm for polysaccharide tribenzoates such as 2,3,6-tri-O-benzoyl amylose (I), 2,3,4-tri-O-benzoyl dextran (II), tri-O-benzoyl pullulan (III), 2,3,6-tri-O-benzoyl cellulose (IV), 2,3,6-tri-O-benzoyl mannan (V), and polyglycan dibenzoates such as 2,3,-di-O-benzoyl amylose (VI), cellulose (VII), and mannan (VIII). All compounds exhibit Cotton effects in the region of their UV absorption bands (206–285 nm). Comparison of the corresponding di- and tribenzoyl polysaccharides shows a qualitative agreement in number, position and sign of the CD bands but differences in ellipticity magnitude. The disubstituted derivatives exhibit smaller amplitudes than the trisubstituted ones. The contribution of the C(6) chromophore (linked by a CH2-group to the asymmetric C(5) atom) was determined to be of the same sign as the combined contribution of the C(2) and C(3) substituents. The CD bonds of the individual polysaccharide derivatives, which differ in number, sign, and position, were discussed in terms of the steric position of the single chromophores and the steric arrangement and interaction caused by the configuration of the polysaccharides. The optical behavior of these polysaccharide derivatives was found to be not strongly influenced by a definite chain conformation in solution.

R-Enzyme and limit dextrinase of malted barley have been separated by continuous electrophoresis. The preparation of R-enzyme, which was free of α-amylase, hydrolysed the outermost inter-chain linkages of amylopectin and glycogen which had interior chain lengths exceeding five glucose residues. It had no action on glycogen with shorter interior chains, on pullulan, or on oligosaccharides containing α-1, 6-glucosidic linkages.

Using pullulan as the substrate, the limit dextrinase activity of protein extracts from higher plants and from commercial malts has been measured. Extracts of ungerminated barley, rye, maize and wheat show low levels of activity which increase markedly on germination. By contrast, extracts of ungerminated oats have a high level of activity which does not increase during germination. Gibberellic acid has a significant effect on the rate of development of limit dextrinase activity in barley. Commercial samples of malt show varying amounts of limit dextrinase activity which does not correlate with the diastatic power. For comparative purposes, the α-amylase and maltotriase activities of all the protein extracts have also been measured.

Pullulanase (EC 3.2.1.9) prepared from a culture of Acrobacter aerogeneshas been covalently bound to an inert crosslinked copolymer of aerylamide-acrylic acid by using a water-soluble carbodi-imide. The binding yield based on the amount of added pullulanase was 34%. The residual enzymic activity was 43%, of that of free enzyme. Coupling in the presence of the substrate pullulan gave a 5-fold increase in activity over that obtained when substrate was lacking. The effect of different carbodi-imide concentrations on the coupling has been investigated. The isoelectric point of the pullulanase preparation (3.5–4.0) was determined using isoelectric, focusing, in order to find optimal pH conditions for the coupling procedure. The immobilized pullulanase in a packed bed column was used to debranch amylopeetin to low molecular weight amylose.

Die Permethylierung von Amylose, Dextran, Pullulan und Mannan gelingt in Gegenwart von Methylsulfinylmethylcarbanion (I) mit Methyljodid in Dimethylsulfoxid (DMSO) unter N2 und Feuchtigkeitsausschlus in einem Schritt und die von Cellulose nach einer Anmethylierung mit Dimethylsulfat in Natronlauge, also in zwei Schritten; bei dieser Reaktion betragt der Abbau 10−20%. Die Trimethylather der genannten Polysaccharide konnen in guten Ausbeuten (70−90%) in einem weiten Molekulargewichtsbereich hergestellt werden. The permethylation of polysaccharides such as amylose, dextran, pullulan, and mannan can be achieved with methyljodide in dimethylsulfoxide (DMSO) unter N2 in the presence of methylsulfinylmethylcarbanion (I) in one step and that of cellulose after a preliminary methylation, with dimethylsulphate in NaOH, in two steps. The trimethylpolyglycans of various degree of polymerisation can be prepared in this way in good yield (70−90%) with only 10−20% degradation.

Extracts of sweet corn contain a mixture of debranching enzymes which have been separated to give a typical R-enzyme (which acts only on amylopectin) and an isoamylase (which acts on both glycogen and amylopectin). The action of isoamylase on phytoglycogen gives a mixture of maltosaccharides and a residual polysaccharide which had a glycogen rather than an amylopectin-type structure. This suggests that, in vivo , amylopectin is not formed by the enzymic debranching of a glycogen precursor. Sweet-corn extracts also contain a third α-(1→6)-glucosidase which hydrolyses pullulan.

Pullulanase from Aerobacter aerogenes is a glucosidase specifically splitting α-1,6-glycosidic bonds. A polymeric substrate for the pullulanase is the linear α-glucan pullulan from Pullularia pullulans. This α-glucan consists of maltotriose units linked together by α-1,6-glycosidic bonds. This means that the polysaccharide is degraded by pullulanase to yield exclusively maltotriose. There are two possibilities of enzyme action: a) either the hydrolysis starts from one end of the linear chain directly setting free maltotriose; b) or random splitting occurs to give larger units of varying molecular weight which will finally be degraded to maltotriose. The decision between these two alternatives could be made by gas liquid chromatography. The quantity of liberated maltotriose rises linearly with the reaction time, which proves that hydrolysis occurs from the end of the substrate chain. This method using gas liquid chromatography as an analytical tool may also be applied to monitor the action of other hydrolytic enzymes, as it could be shown for α- and β-amylase.

Publisher Summary This chapter discusses the preparation of pullulanase from Aerobacter aerogenes. Pullulan is a linear α-glucan produced by the yeastlike fungus Pullularia pullulans, in which about, 480 maltotriose units are linked by 1→ 6 glycosidic bonds. By pullulanase it is exclusively degraded to maltotriose units. The assay method for pullulanase is based on the determination of maltotriose split off from pullulan by the enzyme. The increase in reducing power is measured with Nelson reagent. The pullulanase unit is defined as that amount of enzyme, which in 1 minute at 30 ° and a pH of 5.0 liberates 1 micromole of maltotriose. The cultivation of Aerobacter and preparation of the pullulanase is also described in the chapter. Batchwise-cultivated Aerobacter excretes the pullulanase into the culture filtrate while continuously cultivated cells contain the enzyme cell-bound. Pullulanase produced under the conditions of continuous cultivation is tightly bound. Even prolonged shaking of the resuspended cells in water, different buffers, or hypertonic solutions removed only one-third of the cell-bound activity.

Summary: During the growth of the yeast-like fungus Pullularia pullulans, the pattern of glucose utilization by the cell varied from a complete inability to elaborate extracellular polysaccharide to the diversion of almost two-thirds of metabolized glucose to pullulan elaboration. The effects of carbon and nitrogen levels on this changing pattern during growth were examined. These investigations indicated that the elaboration of pullulan, and the onset of a morphological change of Pullularia from a filamentous to a yeast-like form, were related to the availability of nitrogen, and not carbon, in the growth medium.

We have isolated a Pseudomonas sp. (strain SB15) which produces an isoamylase (EC 3.2.1.9). Highest yields of this enzyme were obtained when the bacterium was grown in shaken culture in a medium containing maltose, dextrin, starch, or isomaltose. Specific carbon and nitrogen sources were required for growth. The most satisfactory medium consisted of 2% maltose, 0.4% sodium glutamate, 0.3% diammonium hydrogen phosphate, and other inorganic salts. The optimal p H for enzyme production was 5 to 6. The enzyme is stable between p H 3 and 6 but is extremely labile above p H 7. It splits amylopectin completely by combined action with β-amylase but does not attack pullulan.

Pullulan 4-glucanohydrolase, a novel pullulan-hydrolyzing enzyme from Aspergillus niger, was highly purified by means of acetone precipitation, chromatography on P-cellulose and DEAE-cellulose, and gel filtration on Sephadex G-150. More than 430-fold purification was achieved through these procedures from crude extract of wheat bran culture. The enzyme can liberate a large amount of isopanose and a small amount of tetrasaccharide from pullulan. The optimum pH of the enzyme action on pullulan was 3.0–3.5 and the optimum temperature was 40 °C at pH 3.5. The enzyme activity remained intact after heating at 50 °C for 30 min at pH 3.7–4.5. The enzyme was stable at pH 2.0–8.0 on storage at 5 °C for 24 hr. The purified enzyme attacked reducing end α-1,4-glucosidic linkages adjacent to α-1,6-glucosidic linkages in pullulan, 63-α-glucosylmaltotriose, 62-α-maltosylmaltose and panose, to liberate isopanose, isomaltose and maltose, isopanose and glucose, and isomaltose and glucose, respectively. The molecular weight of the enzyme determined by gel filtration on Bio-Gel P-150 was about 74,000.

During the growth of the yeastlike fungus Pullularia pullulans on glucose as sole carbon source, the cell-mass does not increase concomitantly with the elaboration of the extracellular polysaccharide pullulan, but precedes it. The conditions generated in the culture medium which activate the secretion of polysaccharide have been sought for, and, in particular, the roles of extracellular p H and nitrogen limitatation are examined.

THE enzyme pullulanase1 is a bacterial R-enzyme in that it is capable of cleaving certain α 1→6 glucosidic bonds where these occur together with α 1→4 glucosidic bonds as in pullulan, starch and dextrins. The substrate specificity of this enzyme has recently been reported2 to embrace oligosaccharides and dextrins containing as few as two 1→4 linked α-D-glucopyranoside units in the ‘A’ chain and two such units in the ‘B’ chain. Thus, a tetra-saccharide linked 2 on 2 is the smallest known substrate for the hydrolytic action of this enzyme2.

The specific activities of Pseudomonas isoamylase (amylopectin 6-glucano-hydrolase, EC 3.2.1.9) and Aerobacter pullulanase were measured as cleavage of glycosidic bonds per min per mg of protein at substrate concentrations of 2%. Those of Pseudomonas isoamylase were 110–280 μmoles for amylopectins and glycogens and 1.1 μmoles for pullulan. Those of Aerobacter pullulanase were 3–5 μmoles for amylopectins 0.5–1.2 μmoles for glycogens and 53 μmoles for pullulan. The Km values (g/ml) of the isoamylase were about 1·10−4–2·10−4 for amylopectins and glycogens and 2·10−3 for pullulan while those of the pullulanase were about 8·10−3-1·10−2 for amylopectins, about 2·10−2–5·10−2 for glycogens and 1.7·10−5 for pullulan. The molecular activities of the isoamylase were about 9500 to 26 700 for amylopectins and glycogens but only about 110 for pullulan.