Activity on pullulan

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. 

The concurrent action of sucrose a-D-glucohydrolase and oligo-l,6-D-gluco-sidase active sites of the hybrid oligo-l,6-D-glucosidase-sucrose a-D-glucohydrolase ( sucrose-isomaltase ) from rat intestine on an a-limit dextrin was studied by use of 6 -maltotriosylmaltotriose isolated from the products of controlled action of pullulanase on pullulan (see p. 522). ... 

At the request of the Codex Committee on Food Additives and Contaminants at its thirty-eighth session (Codex Alimentarius Commission, 2006), the Committee evaluated the enzyme isoamylase (glycogen a-1,6-glucanohydrolase EC 3.2.1.68). Isoamylase catalyses the hydrolysis of 1,6-a-D-glucosidic branch linkages in glycogen, amylopectin and their beta-limit dextrine. It has no or only limited activity on linear polysaccharides linked by a-1,6-glycosidic bonds (e.g. pullulan) and on alpha-limit dextrine. 

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. 

Duan X, Chi Z, Wang L, Wang X (2008) Influence of different sugars on pullulan production and activities of a -phosphoglucose mutase, UDPG-pyiophosphorylase and glucosyltransferase involved in pullulan synthesis in Aureobasidium pullulans Y68. Carbohydr Polym 73 587—593 Duboc P, Mollet B (2001) Applications of exopolysaccharides in the dairy industry. Int Dairy J 11 759—768... 

Information on the hydrolytic activity in marine sediments has been obtained from the use of model substrates labeled with fluorescent dyes such as methylumbelliferone (MUF) or fluorescein. These substrates may be small dimeric molecules, the hydrolytic cleavage of which releases the fluorescence signal, which is then indicative of the activity of specific enzymes such as glucosidase, chitobiase, lipase, ami-nopeptidase or esterase (Chrost 1991). Also large fluorescently labeled polymers such as the polysaccharides laminarin or pullulan have been used in experiments to demonstrate the mechanism and kinetics of bacterial degradation (Amosti 1996). 

K.I. Shingel, Current knowledge on biosynthesis, biological activity, and chemical modification of the exopolysaccharide, pullulan. Carbohydr. Res. 339(3), 447-460 (2004). 

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