Maltotriose from pullulan

A strain of Bacillus cereus synthesizes a pullulanase and a -amylase, which together convert starch into maltose in high yield. The homogeneous pullulanase was obtained by fractional precipitation, adsorption onto starch and Celite, and gel filtration. The purified enzyme (pH optimum 6.0—6.5, mol. wt. 1.10 0.20 X 10 ) released maltose, maltotriose, and maltotetraose from -limit dextrin and maltotriose from pullulan, but it did not release amylose-like substances from amylopectin. The enzymic activity was inhibited by mercuric chloride and 4-chloromercuribenzoate, although the activity in the latter instance could be restored by cysteine. 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

N. Sakairi, M. Hayashida. and H. Knzuhara, 1,6-Anhydro-p-maltotriose Preparation from pullulan, and regioselective partial protection reactions, Carbokydr. Res. 765 91 (1989). 

Pullulanase is an extracellular enzyme of Aerobacter aerogenes that causes essentially quantitative hydrolysis of pullulan to maltotriose. The enzyme is readily prepared in a crude form that is free from other carbohydrases, and is important in structural studies because it debranches amylopectin and glycogen. When the A. aerogenes is grown in continuous culture, the enzyme is bound to the cells, but it can be released by detergents, and purified by ion-exchange chromatography. This purified enzyme has been crystallized. ... 

Data on pullulan gathered by Wallenfels, Lindberg, and Adams and their coworkers are consistent with a linear polymer containing mainly maltotriose residues connected by a-D- l- 6)-linkages (1). Pullulan prepared directly from solution or through... 

Pullulan is a glucan of (1 6)-a-D-linked maltotriose residues produced by Aureobasidium pullulans from starch or sucrose feedstockJ It is well soluble in aqueous medium and forms films that possess thermal stability and are antistatic and elastic. Pullulan has adhesive properties and is directly compressible under heat with moisture. The polysaccharide is produced on an industrial scale and is used for multiple applications, including food, pharmaceuticals, and cosmetics. It has recently been classified as GRAS by the U.S. FDA. 

The discovery, shortly thereafter, of the enzyme pullulanase from the bacterium Aerobacter aerogenes helped considerably in further structural analysis of the polysaccharide. Hydrolysis of pullulan by diis enzyme showed that essentially no products containing (l->6)-ff-i>-giucosidic linkages were formed, the preponderant product being maltotriose. It was thus clear that the enzyme hydrolyzes (l->6)-a-D-glucosidic link-... 

Pullulanase (EC 3.2.1.41) is the enzyme which hydrolyses pullulan, a polymer of 1,6-linked maltotriose units, to maltotriose. This distinguishes it from other enzymes that can act on pullulan to give isopanose, panose or glucose, i.e. isopullulanase (EC 3.2.1.57), a-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) respectively. Plant et al. [231,232] studied the pullulanase from Thermoanaerobium strain Tok 6-Bl and found it to hydrolyse not only the al,6 bonds in pullulan... 

A pullulanase in E. coli appears to be bound to the cell wall its properties are very similar to those of a pullulanase from Klebsiella (Aerobacter) aerogenes. However, no pullulanase activity was detected in cultures of E. coli grown on maltose. Neither this nor forty other strains of E. coli grew on pullulan, whereas all the strains grew on maltose, and some on maltotriose. The synthesis of pullulanase by various strains of Klebsiella (Aerobacter) aerogenes using maltose both as an inducer and as a source of carbon has been studied." Conditions leading to the synthesis of pullulanase were reported. 

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