Pullulanase: role in starch hydrolysis and potential industrial applications

Abstract

The use of pullulanase (EC 3.2.1.41) has recently been the subject of increased applications in starch-based industries especially those aimed for glucose production. Pullulanase, an important debranching enzyme, has been widely utilised to hydrolyse the α-1,6 glucosidic linkages in starch, amylopectin, pullulan, and related oligosaccharides, which enables a complete and efficient conversion of the branched polysaccharides into small fermentable sugars during saccharification process. The industrial manufacturing of glucose involves two successive enzymatic steps: liquefaction, carried out after gelatinisation by the action of α-amylase; saccharification, which results in further transformation of maltodextrins into glucose. During saccharification process, pullulanase has been used to increase the final glucose concentration with reduced amount of glucoamylase. Therefore, the reversion reaction that involves resynthesis of saccharides from glucose molecules is prevented. To date, five groups of pullulanase enzymes have been reported, that is, (i) pullulanase type I, (ii) amylopullulanase, (iii) neopullulanase, (iv) isopullulanase, and (v) pullulan hydrolase type III. The current paper extensively reviews each category of pullulanase, properties of pullulanase, merits of applying pullulanase during starch bioprocessing, current genetic engineering works related to pullulanase genes, and possible industrial applications of pullulanase.

Figure 1

Section of the amylose molecule showing the repeating anhydroglucose unit (modified from [8]).

Figure 2

Section of the amylopectin molecule showing the α-1, 4 and α-1,6 chain linkages in starch (modified from [8]).

Figure 3

A diagram showing how the side-branching chains are clustered together within the amylopectin molecule (modified from [7]).

Figure 4

Schematic presentation of the action of amylases. Black circles indicate reducing sugars (modified from [17]).

Figure 5

Action of amylo-1,6-glucosidase (modified from [18]).

Figure 6

Generalized structure of pullulan from Aureobasidium pullulans strain CH-1 (modified from [2]).

Figure 7

Partial hydrolysis of starch by α-amylase (modified from [11]).

Figure 8

Action of glucoamylase on liquefied starch (modified from [11]).

Figure 9

The percentage (%) of glucose formed from maltodextrin using various enzyme solutions. Symbols: A, 200 U/kg Aspergillus niger glucoamylase; B, 400 U/kg Aspergillus niger glucoamylase; C, 200 U/kg Aspergillus niger glucoamylase plus 200 U/kg Bacillus acidopullulyticus pullulanase (modified from [20]).

Figure 10

Effect of pullulanase during saccharification (modified from [11]).

Figure 11

The effect of pullulanase activity on maximum D-glucose concentration (modified from [11]).

Figure 12

The effect of substrate concentration on maximum D-glucose concentration. Symbols: a, without pullulanase; b, with pullulanase (modified from [11, 18]).

Figure 13

The effect of pullulanase dosage on reaction time. Symbols: a, without pullulanase addition; b, 0.04 unit of pullulanase/g dry solid; c, 0.08 unit of pullulanase/g dry solid; d, 0.16 unit of pullulanase/g dry solid (modified from [11]).