Pullulanase

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.  

The enzyme has not yet been examined in detail. The crude enzyme often contains traces of alpha-amylase. The presence of contaminating alpha-amylase can only be accurately tested for by its effect on the viscosity of linear amylose, and application of this test is essential before the enzyme is used for structural determinations. The crude pullulanase appears to be particularly heat-stable, and, on gel chromatography, two molecular forms, of estimated size 150,000 and 50,000, were obtained. The significance of the two forms is not yet known. The crystallized enzyme has been reported to have a molecular weight of 145,000. 

Pullulanase has great potential in structural investigations. In the case of amylose and its jS-limit dextrin, treatment with pullulanase has shown unambiguously (for the first time) that a-D-(l- 6) branch-points form the natural barrier to heta-amylase action, that is, some amylose fractions have limited branching.  

Pullulanase has also been used for investigating the structure of the phosphorylase limit-dextrin of glycogen. -  

No kinetic or mechanistic studies of pullulanase action have yet been reported. 

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This paper will review our studies on 1) B-amylase of C. thermosulfUrogenes 2) a-glucosidase of C. thermohydrosulfuricum 3) glucose isomerase of C. thermosulfurogenes and Thermoanaerohacter strain B6A and, 4) endoxylanase of Thermoanaerohacter strain B6A. A separate paper by Saha et al., in this symposium, will report on the properties of the novel pullulanase present in these thermoanaerobes. 

The B-amylase has been used to produce extremely high maltose syrups for confectionery industries and high conversiem syrups used in brewing (77-79). Figure 2 shows the effect of B-amylase and pullulanase on the time course of maltose... 

In comparison with a pullulanase, the purified amylopullulanase displayed 3-fold higher specificity towards soluble starch and amylopectin and 7-fold higher specificity towards glycogen (12). The amylopullulanase hydrolyzed various high molecular weight starch substrates (Table II). Even the mammalian glycogen which... 

SAHA ET AL. Amylopullulanase, a Amylase, and Pullulanase Literature cited... 

Saccharification. Saccharifications were performed with amyloglucosidase (AMG) or amyloglucosidase/pullulanase (Dextrozyme) at a dose of 0.18 AG/units/gram DS for 48 hours at 60 C, pH 4.3-4.5. One AG unit is the amount of enzyme which hydrolyzes 1.0 micromole of maltose per minute at 25 C, pH 4.3. 

Roy, I. and Gupta, M.N., Hydrolysis of starch by a mixture of glucoamylase and pullulanase entrapped individually in calcium alginate beads, Enzyme Microbial Tech., 34 (2004) 26-32. 

Mixed enzyme solution - either make up a mixed enzyme solution containing 5000 units of a-amylase and 5 units of pullalanase per millilitre acetate buffer (original method), or, as is usual, separately add 0.5 ml a-amylase reagent and 0.1 ml pullulanase reagent per sample. 

Thermostable pullulanase Clostridium thermosuifurogenes SV2 (Rama Mohan Reddy et al., 1999)... 

Rama Mohan Reddy, P, Reddy, G., Seenayya, G. (1999). Produetion of thermostable pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation optimization of nutrients levels using response surfaee methodology. Biopro. Engg., 21,497-503. 

Dextrose yield, however, can be increased by conducting saccharification at a lower solids level where the reverse reaction is minimized. For instance, dextrose yields of 98.8, 98.2, 97.5, and 96.9% dry basis can be achieved at solids levels of 10, 15, 20, and 25%, respectively (10). Low solids operation, however, is not used commercially owing to problems associated with microbial contamination and cost of water removal. Dextrose level can be increased by 0.5—1.5% at normal reaction solids by using an enzyme such as pullulanase (11) or a B. megaterium amylase (12) in conjunction with... 

There have been several examinations of the structure of Nageli dextrin,405407 which is prepared by the prolonged action of acid on granular starch. In one study,405 there was separated from waxy maize a branched fraction that was resistant to pullulanase action. As this fraction contained some molecules having two branch points that were in close proximity, it was considered that this may have hindered hydrolysis, and that it could be of relevance to studies on the structure of the original amylopectin. 

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