Starch is an important food ingredient and a substrate for the production of many industrial products. Biological and industrial processes involve hydrolysis of raw starch, such as digestion by humans and animals, starch metabolism in plants, and industrial starch conversion for obtaining glucose, fructose and maltose syrup or bioethanol. Raw starch degrading α-amylases (RSDA) can directly degrade raw starch below the gelatinization temperature of starch. Knowledge of the structures and properties of starch and RSDA has increased significantly in recent years. Understanding the relationships between structural peculiarities and properties of RSDA is a prerequisite for efficient application in different aspects of human benefit from health to the industry. This review summarizes recent advances on RSDA research with emphasizes on representatives of glycoside hydrolase family GH13. Definite understanding of raw starch digesting ability is yet to come with accumulating structural and functional studies of RSDA.

Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.

α-Amylase from Bacillus licheniformis ATCC 9945a (BliAmy) was proven to be very efficient in hydrolysis of granular starch below the temperature of gelatinization. By applying two-stage feeding strategy to achieve high-cell-density cultivation of Escherichia coli and extracellular production of BliAmy, total of 250.5 U/mL (i.e. 0.7 g/L) of enzyme was obtained. Thermostability of amylase was exploited to simplify purification. The hydrolysis of concentrated raw starch was optimized using response surface methodology. Regardless of raw starch concentration tested (20, 25, 30 %), BliAmy was very effective, achieving the final hydrolysis degree of 91 % for the hydrolysis of 30 % starch suspension after 24 h. The major A-type crystalline structure and amorphous domains of the starch granule were degraded at the same rates, while amylose-lipid complexes were not degraded. BliAmy presents interesting performances on highly concentrated solid starch and could be of value for starch-consuming industries while response surface methodology (RSM) could be efficiently applied for the optimization of the hydrolysis.

Bacillus licheniformis 9945a α-amylase is known as a potent enzyme for raw starch hydrolysis. In this paper, a mixed mode Nuvia cPrime™ resin is examined with the aim to improve the downstream processing of raw starch digesting amylases and exploit the hydrophobic patches on their surface. This resin combines hydrophobic interactions with cation exchange groups and as such the presence of salt facilitates hydrophobic interactions while the ion-exchange groups enable proper selectivity. α-Amylase was produced using an optimized fed-batch approach in a defined media and significant overexpression of 1.2 g L−1 was achieved. This single step procedure enables simultaneous concentration, pigment removal as well as purification of amylase with yields of 96% directly from the fermentation broth.

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