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. 1. Introduction Starch is a major industrial raw material and is chemically and/or enzymatically processed into variety of products for subsequent use in various industries, ranging from food (especially high-fructose and glucose syrups) to washing detergent industries [1–3]. Starch is, after cellulose, one of the most abundant heterogeneous polysaccharide produced by plants in the form of water insoluble granules. It is a polymeric carbohydrate, composed of C, H, and O atoms in the ratio of 6？:？10？:？5, (C6H10O5)？？. Molecules of starch are made of hundreds or thousands of glucose, corresponding to values of ？？ that range from 50 to several thousands. Glucose units are linked to one another through C1 oxygen as glucosidic bond. Glucosidic bonds are stable under alkaline conditions while treatment of starch with acids or certain enzymes breaks the polymer into its constituent glucose molecules. The end unit of the polymeric chain has a latent aldehyde group and is known as the reducing end group. Most starches are mixture of two polymers with high molecular weight: (i) a linear chain molecule—amylose, and (ii) a branch
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