Application of microbial α-amylase in industry – A review

Abstract

Amylases are essential enzymes broadly utilized in various industries. These enzymes catalyze the hydrolysis of starch molecules, breaking them down into polymers of glucose units. As noted in several industry reviews, microbial α-amylases have emerged as critical components in food, fermentation, and pharmaceutical applications. Deriving primarily from fungi and bacteria, these enzymes facilitate the conversion of starches into oligosaccharides, which are vital in numerous industrial processes. Starch plays a pivotal role in human nutrition and is the predominant storage form in several economically important crops, including wheat, rice, and corn. Microbial α-amylases find applications in producing maltodextrin, modified starches, glucose, and fructose syrups, being integral to multiple sectors such as food, textiles, paper production, and detergents. The predominant production methods for α-amylases are submerged fermentation, while solid-state fermentation is gaining recognition as a viable alternative. The varying properties of each α-amylase—thermostability, pH profile, stability, and calcium independence—are crucial for optimizing fermentation processes. This review provides a comprehensive analysis of both bacterial and fungal α-amylases, covering their distribution, structure-function attributes, physical and chemical parameters, and industrial applications.

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Keywords: α-Amylases, enzyme production, bacterial and fungal amylases, starch

INTRODUCTION

α-Amylases (E.C.3.2.1.1) are crucial enzymes that facilitate the hydrolysis of internal α-1,4-glycosidic linkages in starch, resulting in low molecular weight products like glucose, maltose, and maltotriose (29, 42, 66). As one of the most significant enzymes within the biotechnology sector, they represent approximately 25% of the global enzyme market (66, 68). These enzymes can be sourced from various origins, including plants, animals, and microorganisms. However, microbial amylases have largely supplanted chemical hydrolysis in starch processing due to their wider availability and greater stability compared to plant and animal enzymes (81). Among microorganisms, α-amylases are predominantly derived from fungi and bacteria, offering a broad array of industrial applications (29).

The versatility of α-amylases allows for their application across numerous industrial processes, encompassing the food, fermentation, textile, paper, detergent, and pharmaceutical industries. Their potential extends into clinical, medicinal, and analytical chemistry due to advancements in biotechnology, alongside their widespread use in starch saccharification within food, brewing, and distillation industries (29, 42, 61).

This review delves into the microbial sources of α-amylases, highlighting their structural and functional characteristics.

STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF α-AMYLASE

α-Amylase (α-1,4-glucan-4-glucanohydrolase) is visible in numerous organisms, including microorganisms, plants, and higher animals (42). This enzyme belongs to the endo-amylase family, initiating starch hydrolysis into shorter oligosaccharides by cleaving α-D-(1,4) glycosidic bonds (9, 36, 42, 80). Notably, α-amylase does not cleave terminal glucose residues or α-1,6 linkages (88). The action of α-amylase produces oligosaccharides of varying lengths, comprising an α-configuration and α-limit dextrins (86) constituted by maltose, maltotriose, and branched oligosaccharides containing both α-1,4 and α-1,6 linkages (88). Although other amylolytic enzymes contribute to starch breakdown, α-amylase plays an essential role in initiating this process (80).

The enzyme exhibits a three-dimensional structure, enabling substrate binding and facilitating the cleavage of glycosidic links via catalytically specific groups (36). Human α-amylase is a calcium-dependent enzyme consisting of a single oligosaccharide chain of 512 amino acids, with a molecular weight of 57.6 kDa (88). The enzyme comprises three domains: A, B, and C (Figure 1). Domain A comprises a barrel-shaped (β/α)8 superstructure, while domain B, positioned between A and C, is attached to domain A via disulfide bonds. Domain C possesses a β-sheet structure and is linked to domain A by a polypeptide chain of unknown function. The active site, responsible for substrate binding, is found in a cleft formed by the carboxyl end of the A and B domains. Calcium ions play a stabilizing role in the enzyme’s three-dimensional structure and may also act as an allosteric activator. Substrate analogs reveal that Asp206, Glu230, and Asp297 are involved in catalysis (56). The substrate-binding site consists of five subsites, with the catalytic site located at subsite three, allowing substrates to bind at various glucose residues for cleavage (88).

Figure 1.

α-Amylase Structure. Domain A is shown in red, domain B in yellow, and domain C in purple. The catalytic center's calcium ion is depicted as a blue sphere, with the chloride ion represented as a yellow sphere, and the green structures are bound to active and surface-binding sites (62).

STARCH

Starch serves as a critical component of the human diet and undergoes chemical and enzymatic processing to yield numerous products like starch hydrolysates, glucose syrups, fructose, maltodextrin derivatives, or cyclodextrins, especially in the food industry. The resulting sugars may also be fermented to produce ethanol. While many plants produce starch, only a select few are essential for industrial starch processing, primarily sourced from maize, tapioca, potatoes, and wheat. However, limitations related to shear resistance, thermal resistance, thermal decomposition, and retrogradation may restrict starch's industrial food applications (1, 28, 86). Among carbohydrate polymers, starch is gaining increasing attention due to its diverse applications across food products. It contributes significantly to the textural properties in many foods and is widely employed in both food and industrial applications as a thickener, colloidal stabilizer, gelling agent, bulking agent, and water retention agent (37).

Starch consists of glucose polymers bonded via glycosidic bonds, encompassing two types: amylose and amylopectin (Figure 2a and 2b). Amylose is a linear polymer of glucose units linked by α-1,4 glycosidic bonds, while amylopectin features branched chains with both α-1,4 and α-1,6 linkages. Granule-bound starch synthase and soluble starch synthase play pivotal roles in synthesizing these polymers, respectively. α-Amylase's ability to cleave the α-1,4 glycosidic bonds within amylose or amylopectin chains emphasizes its significance in various industrial processes (56, 78, 84, 86).

Figure 2.

Two types of glucose polymers exist in starch: amylose (A) is a linear polymer of α-1,4 glycosidic bonds, while amylopectin (B) features short α-1,4 linked to linear chains and side chains of glucose units (56).

Through hydrolysis, starch is converted into smaller oligosaccharides by α-amylase, marking one of the most critical commercial enzymatic processes. Applications of amylases span across food, detergent, textile, and paper industries for starch hydrolysis (29, 47, 81). The saccharide composition obtained from starch depends on temperature conditions and enzyme origin. For industrial use, enzyme specificity, thermostability, and pH response are vital properties (42).

α-AMYLASE PRODUCTION

The production of α-amylase through submerged fermentation (SmF) and solid-state fermentation (SSF) has been extensively studied, relying on various physicochemical factors. Traditionally, SmF has been favored for producing industrially significant enzymes due to its controllable parameters like pH, temperature, aeration, and oxygen transfer (16, 22).

SSF systems are becoming increasingly favored due to their natural advantages. They mimic the microorganisms' natural habitat, promoting high productivity, simpler techniques, and more cost-effective production compared to SmF. Historically, fungi and yeast have been deemed suitable for SSF based on water activity theory, whereas bacterial contributions received limited attention. Nonetheless, bacterial cultures can also be effectively managed for SSF processes (60). The merits of SSF include superior yields, straightforward procedures, reduced capital investment, lowered energy requirements, better product recovery, and minimal foam buildup, positioning this method as ideal, especially for developing regions. Recent studies confirmed that SSF yields higher amounts of enzymes compared to SmF (16, 82).

Optimizing fermentation conditions, especially physical and chemical parameters, is crucial for developing effective processes, affecting both economic viability and practicality (21). Various factors—including pH, temperature, metal ions, carbon and nitrogen sources, surface-active agents, phosphate, and agitation—have been explored to enhance α-amylase production. Attributes such as thermostability, pH profile, stability, and calcium independence are essential for matching applications; for instance, α-amylases for the starch industry must function efficiently at lower pH levels, while those for detergent applications require higher pH stability. The composition of growth media, medium pH, phosphate concentration, inoculum age, temperature, aeration, carbon source, and nitrogen source are significant variables (16, 69). Renowned studies have profiled the physical and chemical characteristics of α-amylases from bacterial and fungal sources (29). Table 1 summarizes properties of specific amylases sourced from microorganisms.

Table 1.

Properties of bacterial and fungal α-amylases

  Microorganism                         Fermentation     pH optimal/stability   Temperature optimal/stability  Molecular weight (kDa)  Inhibitors
Bacteria
Bacillus amyloliquefaciens              SmF               7.0                  33 °C                        -                            -
...

BACTERIAL AMYLASES

α-Amylase can be sourced from diverse microorganism species; however, for commercial endeavors, it predominantly originates from the genus Bacillus. Specific species, like Bacillus licheniformis, Bacillus stearothermophilus, and Bacillus amyloliquefaciens, have effective applications across multiple industries, including food, fermentation, textiles, and paper (46, 61).

Thermostability is a desired trait for industrial enzymes. Thermostable enzymes obtained from thermophilic organisms are beneficial for various commercial applications, particularly in the enzymatic liquefaction and saccharification of starch performed at elevated temperatures (100-110 °C). Studies on thermostable amylolytic enzymes reveal their potential to enhance industrial starch degradation processes, producing valuable products like glucose, dextrose, maltose, and maltodextrins (6, 26, 79). Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis, and Bacillus amyloliquefaciens are recognized for their prolific thermostable α-amylase production, extensively utilized in commercial enzyme production (64). Thermostable α-amylases from various bacterial strains can be produced through both SmF and SSF, though SSF is more cost-effective (83). Production of α-amylase via SSF remains confined to Bacillus species. B. subtilis, B. polymyxia, B. mesentericus, B. vulgarus, B. megaterium, and B. licheniformis represent common strains for SSF-based α-amylase production (8). Industries utilize thermostable amylases from Bacillus stearothermophilus or Bacillus licheniformis in starch processing (26).

Halophilic microorganisms produce enzymes active at high salinities, making them suitable for numerous industrial processes involving concentrated salt solutions that may otherwise inhibit enzymatic conversions (4, 63). Moreover, many halobacterial enzymes demonstrate significant thermotolerance and maintain stability at room temperatures over extended periods (51). Halophilic amylases have been largely characterized from halophilic bacteria such as Chromohalobacter sp. (63), Halobacillus sp. (4), Haloarcula hispanica (34), Halomonas meridiana (15), and Bacillus dipsosauri (18).

FUNGAL AMYLASES

Reports on fungi producing α-amylase primarily focus on a few species of mesophilic fungi, emphasizing the need for optimizing culture conditions and selecting superior fungal strains for commercial production (29). Most fungal sources are terrestrial isolates, predominantly within Aspergillus and Penicillium species (43).

Aspergillus species generate a broad range of extracellular enzymes, among which amylases hold significant industrial importance (32). Filamentous fungi, namely Aspergillus oryzae and Aspergillus niger, produce substantial enzyme quantities extensively utilized within industry. Notably, A. oryzae has garnered attention as a favorable host for heterologous protein production due to its ability to secrete abundant high-value proteins and industrial enzymes, including α-amylase (39). This species finds applications in food production (e.g. soy sauce), organic acids synthesis (e.g. citric and acetic acids), and as a source of commercial enzymes (41). Aspergillus niger is known for its hydrolytic capacities in α-amylase production and has the advantage of tolerating acidic environments (pH < 3), facilitating reduced bacterial contamination risk (19).

Filamentous fungi are particularly suitable for solid-state fermentation (SSF), thanks to their morphology which enables effective colonization and penetration of solid substrates (65). Fungal α-amylases are often preferred over others due to their Generally Recognized As Safe (GRAS) status (29).

The thermophilic fungus Thermomyces lanuginosus stands out as an exceptional α-amylase producer, with studies by Jensen (38) and Kunamneni (48) confirming its thermostability.

PURIFICATION OF α-AMYLASE

Although industrially produced enzymes typically require minimal downstream processing, thus remaining relatively crude preparations, α-amylases intended for pharmaceutical and clinical applications necessitate high purification levels. The purified form is also essential for investigating structure-function relationships and biochemical characteristics (29). Various purification strategies exploit specific biomolecule attributes, with laboratory-scale purification methods incorporating approaches like ion exchange, gel filtration, hydrophobicity interactions, and reverse-phase chromatography. α-Amylase extraction protocols employing organic solvents (e.g. ethanol, acetone) and ammonium sulfate precipitation (25, 31, 44) have also been proposed, along with ultrafiltration (53). However, conventional multi-step methodologies can be labor-intensive, time-intensive, and may lead to decreased yields (5). Liquid-liquid extraction methods present a promising alternative, allowing the consolidation of early processing steps into one operation, thereby enhancing efficiency, reducing costs, and facilitating scale-up. This method has garnered attention in the chemical industry due to its simplicity and reduced chemical costs. The dynamic behavior of these systems warrants investigation to bolster continuous liquid-liquid extraction's efficiency and assess associated safety and environmental risks at the design stage (49).

INDUSTRIAL APPLICATION OF α-AMYLASE

Starch conversion

One of the most prevalent applications of α-amylases is within the starch industry, specifically for starch hydrolysis in the liquefaction process that transforms starch into fructose and glucose syrups (57). The enzymatic conversion of starch comprises gelatinization—where starch granules dissolve, forming a viscous suspension—followed by liquefaction, which partially hydrolyzes starch to reduce viscosity and finally saccharification, producing glucose and maltose through further hydrolysis (29, 64). Initially, Bacillus amyloliquefaciens served as the primary α-amylase source, succeeded by Bacillus stearothermophilus or Bacillus licheniformis with advantages pertaining to thermostability and effective biotechnological applications (86).

Detergent industry

The detergent manufacturing sector stands as the leading consumer of enzymes, both in terms of quantity and economic value. Enzymatic formulations within detergents enhance their capability in removing persistent stains, contributing to environmental safety. Amylases rank as the second most utilized enzymes in liquid detergent formulations, comprising 90% of these products (29, 33, 50). They function in laundering and automatic dishwashing scenarios by degrading residues from starchy foods (e.g., potatoes, gravies, chocolate) into dextrins and smaller oligosaccharides (54, 58). These enzymes exhibit activity at reduced temperatures and alkaline pH levels while maintaining stability within harsh detergent environments. The oxidative stability of amylases stands as a principal criterion in detergent applications, as oxidative conditions are often prevalent during washing (13, 45). Additionally, starch removal from surfaces plays a critical role in enhancing whiteness, owing to starch's tendency to attract particulate soils. Notably, amylases for the detergent industry derive from Bacillus or Aspergillus sources (50).

Fuel alcohol production

Ethanol constitutes the most widely used liquid biofuel, with starch serving as the principal substrate due to its abundance and economic viability across diverse regions (14). In the ethanol production process, starch undergoes solubilization before two enzymatic steps convert it into fermentable sugars. The bioconversion of starch into ethanol entails liquefaction and saccharification, where α-amylase or amylolytic microorganisms convert starch into sugar, followed by fermentation—converting sugars into ethanol via microbes like Saccharomyces cerevisiae (53, 59). The economic climate of Brazil relies heavily on ethanol production from yeast fermentation (17). Research encouraging new yeast strains capable of directly producing ethanol from starch—eliminating the saccharification stage—was performed through protoplast fusion within amylolytic yeast and S. cerevisiae (14). Among bacterial sources, α-amylase derived from thermoresistant species (e.g., Bacillus licheniformis and engineered strains of Escherichia coli or Bacillus subtilis) is actively employed during starch hydrolysis (70).

Food industry

Amylases are instrumental in various segments of the processed food industry, notably baking, brewing, digestive aids, cake production, fruit juice clarification, and starch syrup formulation (16). Within the baking domain, α-amylases enhance dough quality by hydrolyzing starch into smaller dextrins, which yeast subsequently ferments. This enzymatic addition accelerates fermentation rates and reduces dough viscosity, thereby improving final bread structure and texture. Additionally, α-amylase contributes extra fermentable sugars, enhancing flavor, crust color, and toasting qualities. Notably, these enzymes exhibit anti-staling properties in bread, promoting texture retention and prolonging shelf life (29, 86). Currently, a thermostable maltogenic α-amylase sourced from Bacillus stearothermophilus is commercially utilized in the bakery sector (86). Amylases also play a role in clarifying beer and fruit juices, and animal feed pretreatment to improve fiber digestibility (23, 24, 86).

Textile industry

Within the textile sector, amylases facilitate the desizing process, involving starch application as a sizing agent on yarn before fabric manufacturing, ensuring efficient weaving. Starch serves as a cost-effective sizing agent due to its availability and ease of removal post-weaving. The ensuing desizing process necessitates selective amylase application to remove starch without damaging the fibers (3, 20, 29). Bacillus-derived amylases have been traditionally employed in textile industries.

Paper industry

In the pulp and paper industry, α-amylases modify starch for producing low-viscosity, high molecular weight starch used in coated paper manufacturing (29, 86). Coating treatments improve paper surface quality and strength, enhancing writing performance. Given the high viscosity of natural starch, α-amylases partially degrade the polymer in batch or continuous processes for effective sizing. Starch remains an efficient sizing agent, elevating paper stiffness and strength (10, 29). Examples of microbial-derived amylases in the paper industry include Amizyme® (PMP Fermentation Products, Peoria, USA), Termamyl®, Fungamyl, BAN® (Novozymes, Denmark), and α-amylase G® (Enzyme Biosystems, USA) (72).

CONCLUSION

Over decades, α-amylases have become ubiquitous in starch-based industries, with numerous microbial sources available for efficient enzyme production. However, only select strains of bacteria and fungi align with commercial production standards. The continuous search for new microorganisms demonstrating promising amylase production is ongoing. Recent findings have shown substantial advancements in purification techniques that allow for higher purity levels, particularly suited for pharmaceutical and clinical applications.

ACKNOWLEDGEMENTS

This research was supported by grants from the Coordination for Higher Level Graduate Improvements (Capes — Brazil), National Council for Scientific and Technological Development (CNPq — Brazil), and the State of Distrito Federal Research Support Foundation (FAPDF — Brazil).