Pullulanases

Intracellular and extracellular pullulanases accelerated the hydrolysis of waxy corn starch by the glucoamylase of a Rhizopus species, but they are less active towards non-waxy corn starch.  

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. 

A study of the metabolism of exogenous and endogenous carbohydrates by E. coli showed that pullulanase and other enzymes are induced by maltodextrins. The pullulanase, which appears to be bound to the cell wall, has properties similar to those of A. aerogenes pullulanase. It is suggested that E. coli uses pullulanase for debranching extracellular polysaccharides. 

Pullulanase was converted into an active immobilized form when it reacted with a copolymer of acrylic acid and acrylamide.  

An extracellular form and a cell-bound form of pullulanase (mol. wts. 1.26 x 10 and 1.41 x 10 , respectively) from Klebsiella aerogenes have been purified by ion-exchange chromatography, gel filtration, and affinity chromatography. The cell-bound form was also released by proteolysis. Comparisons of their physicochemical properties and amino-acid compositions suggested that the enzymes are derived from the same gene product, which can be cleaved selectively by endogenous or added proteases. 

An assay reported for pullulanase levels in the presence of other carbohydrases is dependent upon the three-fold increase in release of reducing groups by the enzyme acting upon pullulan when glucoamylase is present in excess.  

The purification of pullulanase from Aerobacter aerogenes via affinity chromatography on an agarose-immobilized cyanogen bromide-activated cyclohexa-amylose has been reported. Elution from the affinity column was achieved using cyclohepta-amylose. Crossed immunoelectrophoresis was used to follow the various steps of the purification. 

Some properties of crystalline extracellular pullulanase from Aerobacter aerogenes were compared with those of crystalline intracellular pullulanase. The amino-acid compositions of these enzymes were found to be alike on the 

Rexova-Benkova and M. MraCkovd, Biochim. Biophys. Acta, 1978, 523, 162. 

Pullulanase activity in a Klebsiella species, and which is of course an enzyme of starch catabolism, has been shown to be associated with the outer membrane of the cells.  

A viscosimetric assay of debranching enzymes of the pullulanase type has been reported interference by a-D-glucosidase is avoided.  

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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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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