β-Galactosidase: Catalytic Mechanism and Conventional Applications

General Description

β-Galactosidase, a versatile enzyme also known as lactase, plays a crucial role in catalyzing the hydrolysis of β-galactosides. Its discovery dates back to the late 19th century and has since found widespread applications in various industries. Apart from lactose hydrolysis in dairy products, β-Galactosidase is used in diverse applications such as the production of galactosyllactose to enhance nutritional value in food products and convert lactose-rich waste into valuable by-products. Its catalytic mechanism, structural basis, and industrial significance highlight its importance in biotechnology and food technology, shaping fields from microbiology to enzyme engineering.

Figure 1. β-Galactosidase

Overview

Definition and Catalytic Activity

β-Galactosidase, formally known as β-D-galactoside galactohydrolase (E.C. 3.2.1.23), is an enzyme that catalyzes the hydrolysis of the glycosidic bond linking a terminal β-D-galactoside unit to an aglycone moiety. While commonly referred to as lactase due to its role in lactose hydrolysis, not all β-galactosidases can perform this function. Therefore, the term "lactase" should be used judiciously to avoid confusion. β-Galactosidase is ubiquitous across microorganisms, plants, insects, and animal cells, with microbial variants being particularly significant due to their economical production capabilities. β-Galactosidase plays crucial roles beyond lactose hydrolysis, including in cellular metabolism and as a tool in biotechnology. Its ability to break down β-galactosides makes it valuable in various industrial applications, such as food processing and the pharmaceutical industry. Microbial β-Galactosidase variants, produced through cost-effective fermentation processes, are pivotal in these applications due to their high yield and productivity.

Historical Development and Discovery

The discovery of β-galactosidase traces back to Martinus W. Beijerinck's work in 1889, initially termed as lactase. Beijerinck's experiments with Photobacterium phosphoreum and Kluyveromyces marxianus highlighted the enzyme's role in lactose fermentation, later confirmed by Emyl Fischer in 1894 through chemical analyses. Fischer's findings solidified the understanding of β-Galactosidase as an intracellular enzyme produced by microorganisms like K. marxianus, establishing him as a pivotal figure in its discovery. This enzyme's journey from early controversies to its modern applications underscores its significance in both scientific research and industrial processes, shaping fields from microbiology to biotechnology. ¹

Catalytic Mechanism

β-Galactosidases exhibit a catalytic mechanism crucial for their function in glycoside hydrolysis and transgalactosylation reactions. These enzymes belong to various glycoside hydrolase families, including GH1, GH2, GH35, GH42, GH59, and GH147, based on structural homology rather than traditional Enzyme Commission classifications. A defining feature of β-Galactosidases is their ability to retain the β-anomeric configuration of substrates during catalysis, which is particularly advantageous in the synthesis of glycosides resistant to hydrolysis by digestive enzymes in the upper gastrointestinal tract. This property allows β-Galactosidases to facilitate the production of prebiotic compounds like GOS, fGOS, and lactulose, which promote beneficial microbial fermentation in the large intestine, thereby contributing to gastrointestinal health.

Mechanistic Insight

The catalytic mechanism of β-galactosidases follows a double-displacement scheme, facilitated by two glutamic acid residues strategically positioned approximately 5.5 Å apart within the enzyme's (β/α)8 barrel structure. Initially, one glutamic acid residue acts as a nucleophile, attacking the anomeric center of the β-galactoside substrate, forming a galactosyl-enzyme intermediate. Concurrently, the second glutamic acid residue serves as an acid, protonating the glycosidic oxygen of the aglycone to facilitate its departure. In the subsequent step, a hydroxyl-containing nucleophile accepts the galactose moiety, leading to product liberation and enzyme regeneration. Notably, the retention of the β-anomeric configuration throughout this process is crucial for the enzymatic activity and specificity of β-Galactosidases.

Structural Basis

The (β/α)8 barrel fold, characteristic of clan GH-A enzymes to which most β-Galactosidases belong, provides a supportive structural framework for these catalytic activities. This structural motif consists of eight alternating β-strands and α-helices, with the catalytic residues typically located in β-strands 4 and 7, where the glutamic acid residues act as both acid/base catalysts and nucleophiles. Understanding this mechanism not only elucidates the biochemical basis of β-galactosidase function but also underscores its industrial and biological applications in glycoside synthesis and digestive health modulation. ²

Conventional Applications

Lactose Hydrolysis

β-Galactosidases are enzymes known for their ability to catalyze both transgalactosylation and hydrolysis reactions, with the predominance of each reaction influenced significantly by lactose concentration in the reaction medium. At lower lactose concentrations (below 300 mM), βGs predominantly catalyze hydrolysis due to the high water activity (aw) conditions favoring this reaction. This characteristic makes β-Galactosidases particularly effective in hydrolyzing lactose present in milk and cheese whey, typically containing around 130 mM lactose.

Lactose hydrolysis using β-Galactosidases represents a conventional and commercially established application of these enzymes. Compared to acid catalysis, enzymatic hydrolysis by β-Galactosidases offers several advantages, including the ability to operate under milder conditions, compatibility with protein-containing substrates like milk, and the production of high-quality, low-lactose or lactose-free dairy products. This method eliminates the need for extensive purification steps required in acid hydrolysis to remove undesirable by-products and ensures the preservation of sensory and nutritional qualities in dairy products.

Additional Applications

Beyond lactose hydrolysis, β-galactosidases find diverse applications in the food industry. They are crucial in the production of galactosyllactose through transgalactosylation reactions, particularly favored at higher lactose concentrations (980 mM). This process enhances the nutritional value and functionality of dairy products, improving their sensory attributes and bioavailability. Moreover, β-Galactosidases play a pivotal role in dairy waste management by converting lactose-rich waste streams into valuable products such as whey syrups, thus reducing environmental impact and enhancing economic efficiency in dairy processing. These applications underscore the versatility and importance of β-galactosidases in modern food technology, contributing to both product innovation and sustainability in the dairy sector. ³

Reference

1. Rouwenhorst RJ, Pronk JT, Dijken JP. The discovery of β-galactosidase. Trends Biochem. Sci. 1989; 14: 416–418.

2. Kötzler MP, Withers SG, Glycosidases: functions, families and folds. ELS, John Wiley & Sons, Ltd, Chichester. UK, 2014: 1-14.

3. Vera C, Guerrero C, Aburto C, Cordova A, Illanes A. Conventional and non-conventional applications of β-galactosidases. Biochim Biophys Acta Proteins Proteom. 2020; 1868(1): 140271.