Maltotriose action

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 data in Table I show that the enzyme acts with highest velocity on maltotetraose and oligosaccharides of higher molecular weight. The rate of action on maltotriose is one-half of the rate for the tetrasaccharide, and on maltose the rate is only one-tenth of the rate for the tetrasaccharide. Oligosaccharides in which the glucose units are joined by linkages other than a-1,4 are hydrolyzed at much slower rates than the 1,4... 

Figure 9.72 Chromatograms of the action patterns of maltoheptaose after the indicated periods of incubation with a-amylase and a-glucosidase. Peaks 1, glucose 2, maltose 3, maltotriose 4, maltotetraose 5, maltopentaose (x) compound A 6, maltohexaose 7, maltoheptaose. (A) Pure maltoheptaose used for the assay. (B) Blank sample before the addition of substrate. (C-H) Chromatograms after 1, 5, 10,15, 20, and 30 minutes, respectively, of incubation. Chromatographic conditions column, 10 jum Nucleosil SA (250 mm X 4 mm) solvent, acetonitrile-water (72.527.5) flow rate, 0.7 mL/min temperature, 27°C detection, differential refractometer, full scale = 2 X 10-6 refractive index units. (From Haegel et aL, 1981.)...

The /8-amylase starts from the non-reducing end group and attacks chains with maltose linkages. Only maltose is liberated. Anomalies in the constitution of the chain molecule, such as isomaltose linkages, stop the enzyme action. The length of the chain plays no significant role. Maltotriose and maltose are not attacked. The enzyme is an exo amylase. ... 

Fig. 9.—Radioautograph of paper chromatogram showing radioactive Schardinger dextrins produced from radioactive glycogen by B. macerans amylase. Small amounts of linear oligosaccharides together with unreacted residues from the glycogen were removed by beta amylase action and converted to maltose and maltotriose.

With this method, for each bond hydrolyzed by AMY, two molecules of NADH are produced. This holds true only if neither glucose nor maltotriose is produced by the action of AMY. Later work has shown that these products are present in the reaction mixture in significant quantities. Thus the assay underestimates AMY activity. 

After the action of salivary and pancreatic a-amylases on dietary starch and glycogen, the carbohydrate content of the small intestine consists of newly formed maltose ingested monosaccharides dietary disaccharides, such as lactose, sucrose, maltose, and trehalose oligosaccharides, such as dextrins and maltotriose and indigestible oligosaccharides and polysaccharides, such as cellulose, agar, and other oligosaccharide dietary fibers. 

In addition to their actions on disaccharides, the brush border enzymes further hydrolyze the products of amylase action, including maltose, maltotriose, and a-limit dextrins. The brush border enzymes appear to act in an integrated manner in that there is a flow of substrate from glucoamy-lase and isomaltase to sucrase with the production of the monosaccharides glucose, galactose, and fructose. These monosaccharides are transported into the ehterocyte by... 

Enzyme action on maltosaccharides is not entirely random, and the above specificity requirements may be altered. For example, salivary a-amylase yields small quantities of D-glucose and maltotriose from malto-tetraose, although maltose is the major product and, with maltopentaose, the linkage penultimate to the reducing end is hydrolyzed faster than that penultimate to the nonreducing end. ... 

In addition to its in vivo importance, R-enzyme is extremely useful for structural analysis. For example, the release of maltose and maltotriose from amylopectin jS-dextrin by R-enzyme provides good evidence of multiple branching, since the yield (12.8%) is near to that calculated (10.4%) for a tree-type structure having equal numbers of A- and B-chains. In addition, the simultaneous action of R-enzyme and 8-amylase can be used for the determination of CL (for details, see Ref. 144). 

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