The enzyme, named BWbg1, belongs to a class of proteins known as beta-galactosidases, which catalyze the cleavage of lactose, the disaccharide sugar that makes up a large fraction of the solids in milk, into its two component monosaccharides, glucose and galactose. Beta-galactosidases have long occupied a central place in dairy biotechnology because they support two distinct and commercially significant applications. The first is the production of low-lactose and lactose-free milk and dairy goods for the large population of people who cannot properly digest lactose. The second is the synthesis of galacto-oligosaccharides, or GOS, through transgalactosylation reactions in which the enzyme, instead of simply splitting lactose, stitches galactose units together into short chains that humans cannot digest but that beneficial gut bacteria eagerly consume.

What makes BWbg1 stand out in a crowded field of known beta-galactosidases is its thermal profile. The purified enzyme exhibits maximum activity at 80 degrees Celsius and at a neutral pH of 7, a combination that immediately distinguishes it from the mesophilic enzymes traditionally used in dairy processing. It also demonstrates impressive resilience, retaining more than 72 percent of its activity after six hours of incubation at 55 degrees Celsius. For industrial operators, these are not merely laboratory curiosities. Elevated operating temperatures increase the initial productivity of enzymatic reactions, improve the solubility of substrates such as concentrated lactose solutions, and dramatically reduce the risk of contamination by spoilage and pathogenic microorganisms, which struggle to proliferate at temperatures that the enzyme tolerates with ease.

The path to BWbg1 illustrates the power of functional metagenomic screening, a technique that has become one of the most productive strategies in modern bioprospecting. Rather than trying to culture the microorganisms from the As Burgas hot spring, an approach that would capture only a small fraction of the community because most environmental microbes resist laboratory cultivation, the researchers extracted the collective DNA of the entire microbial ecosystem and cloned it into a metagenomic library. They then screened that library for clones exhibiting beta-galactosidase activity, allowing the function of the gene itself, rather than any prior knowledge of the organism that carried it, to guide the discovery. Sequence analysis of the protein encoded by the recovered gene revealed that BWbg1 belongs to glycoside hydrolase family 35, a well-characterized group of enzymes, yet its properties proved distinctive enough to warrant the label of novelty.

The enzyme’s performance in GOS synthesis is arguably its most commercially compelling feature. Working with a concentrated lactose solution of 40 percent weight per volume at 70 degrees Celsius, BWbg1 converted the substrate into a product mixture containing up to 48 percent galacto-oligosaccharides by weight over a four-hour reaction. That yield, achieved at a temperature at which most conventional enzymes would rapidly denature, positions BWbg1 as a serious candidate for industrial prebiotic production. GOS are among the most widely added prebiotics in infant formula and functional foods, valued for their ability to selectively stimulate the growth of beneficial bacteria such as Bifidobacteria in the gut. A thermostable enzyme that can sustain high-yield synthesis at 70 degrees Celsius offers manufacturers both faster throughput and a substantially lower microbial contamination risk during extended production runs.

Perhaps the most surprising finding, however, concerns what happens when the enzyme is exposed to heat before it is asked to work. The researchers observed a remarkable heat activation effect: after two hours of incubation at 65 degrees Celsius, BWbg1’s ability both to hydrolyze lactose and to produce GOS was enhanced rather than diminished. This behavior has direct implications for how the enzyme could be deployed in real dairy plants. Milk destined for the market is routinely pasteurized, and the two dominant methods are HTST, high-temperature short-time pasteurization, and VAT, the older vat pasteurization approach that uses lower temperatures over longer holding periods. The study demonstrated that BWbg1 retains its activity after both HTST and VAT pasteurization of commercial milk, and that the heat activation it experiences during these treatments actually improves its subsequent performance on the lactose in the milk.

This compatibility with pasteurization regimes is a genuinely practical advantage. In current industrial practice, lactose hydrolysis is typically performed as a separate step after pasteurization, requiring additional equipment, time, and careful hygiene controls. An enzyme that can be added to milk, survive the pasteurization step itself, and then go on to work more effectively afterward could allow manufacturers to merge two processing stages into one streamlined operation. The enzyme’s tolerance of the exact thermal conditions used to make milk safe for consumers means that the same heat treatment that protects public health simultaneously primes the biocatalyst for action, a coincidence of process requirements that enzyme engineers usually struggle to achieve by design.

Equally noteworthy is the enzyme’s behavior at the opposite end of the temperature scale. The study reports that BWbg1 remains capable of hydrolyzing lactose from commercial milk and producing GOS even at 8 degrees Celsius, a temperature within the range of standard refrigerated storage. This cold activity opens the door to applications in which milk is treated during chilled logistics or storage, extending the enzyme’s usefulness well beyond the hot processing floor. A single biocatalyst that functions productively from refrigeration temperatures up to 80 degrees Celsius spans a thermal range that few, if any, commercially established beta-galactosidases can match, and it gives process designers an unusual degree of freedom in choosing where and when in the production chain to deploy it.

The broader significance of the work lies in what it says about the untapped enzymatic wealth of extreme environments. Hot springs have long been recognized as reservoirs of thermostable proteins, because the microorganisms adapted to life at elevated temperatures must maintain enzymes that keep their structure and function under conditions that would destroy the proteins of ordinary organisms. Yet the vast majority of these thermophilic communities remain genetically uncharacterized, and functional metagenomics offers a way to access their catalytic repertoire without ever growing a single cell in the laboratory. The As Burgas spring, flowing through a geologically active region of Galicia, proved to be a productive hunting ground, and the researchers suggest that BWbg1’s versatility, spanning lactose hydrolysis, GOS synthesis, pasteurization compatibility, and cold-temperature activity, makes it a strong candidate for industrial application.

For consumers, the practical outcome of such research is tangible. Lactose intolerance affects a substantial share of the global population, and demand for lactose-free dairy continues to climb, while the market for prebiotic ingredients that support gut health grows in parallel. An enzyme that can serve both markets, converting milk into a digestible product for one consumer group and into a GOS-enriched functional food for another, addresses two major trends in food science with a single biocatalyst. As the team’s results demonstrate, the answer to some of the dairy industry’s most persistent processing challenges may have been waiting all along in the steaming waters of a Spanish hot spring, encoded in the genomes of microbes no one has ever cultured.

Subject of Research: A thermostable GH35 beta-galactosidase from a hot spring metagenome for lactose hydrolysis and galacto-oligosaccharide production in milk

Article Title: A novel thermostable beta-galactosidase for low-lactose and galacto-oligosaccharide-rich milk

Article References: A novel thermostable beta-galactosidase for low-lactose and galacto-oligosaccharide-rich milk. (n.d.). https://doi.org/10.1007/s00253-026-14045-z

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14045-z

Keywords: beta-galactosidase, metagenomics, thermostable enzyme, galacto-oligosaccharides, lactose-free milk, hot spring, bioprospecting, dairy biotechnology, prebiotics, pasteurization, GH35, transgalactosylation

Alpha-amylases are the workhorses of the industrial enzyme market, accounting for roughly 30 percent of global enzyme production. These endo-1,4-alpha-D-glucan glucohydrolases randomly hydrolyse the internal bonds of starch chains, depolymerising polysaccharides into reducing sugars in the alpha-configuration. Unlike glucoamylases, which peel glucose units from the non-reducing ends of starch molecules step by step, alpha-amylases act throughout the polymer, making them invaluable for rapid starch liquefaction. Both enzyme classes are calcium-dependent metalloenzymes, with calcium ions playing a critical structural role. Today, they underpin processes ranging from food manufacturing and detergent formulation to wastewater treatment and emerging clinical applications, and demand for them continues to climb.

The commercial enzymes currently used in starch saccharification, which operates at 70 to 110 degrees Celsius, are mesophilic GH-13 amylases that lack sufficient heat tolerance and specificity. This forces manufacturers to make costly pH adjustments and to supplement reactions with calcium. Thermal stability offers real economic rewards: substrates dissolve more readily at high temperature, medium viscosity drops, microbial contamination risk falls, and accompanying non-enzymatic reactions accelerate. For these reasons, the hunt is on for calcium-independent, thermostable, acid-stable amylases that can function near starch’s native pH of approximately 4.5, a combination that would make starch processing cheaper and more energy-efficient. The enzyme from THS-K1, with its broad pH tolerance and robust thermal endurance, is a serious candidate.

Optimising enzyme production from microbial sources is a bioprocess engineering challenge in itself. The researchers began with one-variable-at-a-time screening but quickly moved to statistical designs that capture interactions between factors. A Plackett-Burman design assessed ten medium parameters, including starch concentration, calcium chloride, beef extract, sodium chloride, peptone, pH, yeast extract, and phosphate salts, at two levels each. Regression analysis produced a first-order polynomial model with an R-squared value of 0.79 and a highly significant F-value of 14.39. Three variables emerged as dominant: soluble starch concentration, medium pH, and yeast extract concentration. Starch serves as both inducer and carbon source, while yeast extract supplies a complex blend of nitrogenous compounds and growth factors that inorganic salts cannot replicate.

With the key factors identified, the team applied a Central Composite Design under the Response Surface Methodology framework. Twenty experimental runs, each performed in triplicate, explored combinations of starch from 1.5 to 2.5 grams percent, pH from 6.5 to 7.5, and yeast extract from 0.1 to 1 grams percent. A second-order polynomial model fitted the data with adjusted and experimental R-squared values of 0.99 and 0.98 respectively, indicating an exceptionally strong agreement between predicted and observed responses. Three-dimensional surface plots confirmed meaningful interactions between the variables, with starch and pH jointly shaping the response landscape. Under the optimum conditions, a medium containing 2 percent soluble starch, 0.55 percent yeast extract, calcium chloride, phosphate salts, peptone, magnesium sulphate, and sodium chloride at pH 7 and 50 degrees Celsius for 72 hours, enzyme production rose from 1.1 units per millilitre to 4.94 plus or minus 0.05 units per millilitre, an increase of roughly 4.5-fold.

Purification followed a classical but carefully executed pipeline. The extracellular enzyme was first precipitated with ammonium sulphate, with the active fraction recovered between 50 and 70 percent saturation, then dialysed overnight through a 10 kilodalton cutoff membrane. Size exclusion chromatography on a Bio-Gel P-100 column separated proteins by molecular mass, and a subsequent ion exchange step on DEAE-cellulose, eluted with a sodium chloride gradient, polished the preparation. Together, these steps achieved a 12.8-fold purification with a 25.14 percent yield. SDS-polyacrylamide gel electrophoresis revealed a single band at approximately 25 kilodaltons, a small size for an alpha-amylase, and zymographic analysis, in which a clear band appeared against a dark blue iodine-stained starch background, confirmed that this band was the active enzyme.

Characterisation of the purified enzyme revealed a profile well suited to demanding industrial conditions. Maximum catalytic activity, reaching 3.8 units per millilitre, occurred at 50 plus or minus 1 degree Celsius, although the enzyme remained active across a wide range from 40 to 90 degrees Celsius. The optimum pH was 7, with activity sustained from pH 5 through 10, an unusually broad working window. Thermal stability testing showed half-lives of 13 hours at 50 degrees, 6 hours at 60 degrees, 2 hours at 70 degrees, and an impressive 1 hour and 53 minutes even at 80 degrees. In pH stability trials, the enzyme endured half-lives of 15 to 16 hours between pH 6 and 8 and still retained activity after 8 hours at pH 9. Kinetic analysis using Lineweaver-Burk plots yielded a Michaelis constant of 0.033 milligrams per millilitre, indicating strong substrate affinity, a maximum velocity of 4.18 micromoles per millilitre per minute, a turnover number of 8.71 per second, and a catalytic efficiency of 4.28 times 10 to the fourth per molar per second.

Metal ion profiling added another dimension to the enzyme’s industrial credentials. Divalent cations including copper, iron, calcium, and magnesium significantly enhanced activity, consistent with the metalloenzyme nature of amylases, while monovalent potassium and sodium ions exerted a slight inhibition. To understand the molecular basis of these properties, the team extracted genomic DNA, amplified the amylase gene with Bacillus-specific primers AmyF1 and AmyR1, and recovered a fragment of approximately 1.7 kilobases. Sanger sequencing and BLASTx analysis confirmed that the sequence encodes an alpha-amylase of the conserved cd11315 domain, and translation predicted a polypeptide of roughly 368 amino acids with high sequence identity to the alpha-amylase of Bacillus subtilis. A phylogenetic tree built with the UPGMA method and 1000 bootstrap replicates placed the enzyme firmly within the Bacillus amylase family.

Homology modelling using the SWISS-MODEL server, based on the crystal structure of the Bacillus sp. KR-8104 amylase (PDB entry 3DC0), produced a three-dimensional model showing 61.66 percent sequence identity with a QMEAN quality score of 0.73, indicating reliable local geometry. Ramachandran plotting confirmed that the majority of residues occupy favoured regions, validating the model for structure-function analysis. Comparison with the template revealed multiple amino acid substitutions that the authors suggest may underpin the enzyme’s enhanced thermal stability. Together, the statistical optimisation, biochemical characterisation, kinetic analysis, and in silico modelling represent the first integrated molecular portrait of an amylase from Bacillus stercoris isolated from an Indian hot spring. With its ability to withstand elevated temperatures, function across a broad pH spectrum, and digest starch efficiently, the THS-K1 enzyme and its Amy gene offer a promising foundation for recombinant production and protein engineering aimed at cheaper, greener starch saccharification.

Subject of Research: Bioprocess optimisation and molecular characterisation of a thermostable alpha-amylase from thermotolerant Bacillus stercoris THS-K1

Article Title: Bioprocess optimisation and molecular characterisation of alpha-amylase from thermotolerant Bacillus stercoris THS-K1

Article References: Padia, H. H., Jain, K. S., Goswami, V. C., & Tipre, D. R. (2025). Bioprocess optimisation and molecular characterisation of alpha-amylase from thermotolerant Bacillus stercoris THS-K1. Discover Biotechnology, 2(1), Article 35. https://doi.org/10.1007/s44340-025-00044-9

Image Credits: AI Generated

DOI: 10.1007/s44340-025-00044-9

Keywords: alpha-amylase, Bacillus stercoris, thermostable enzyme, hot spring microbiology, response surface methodology, Plackett-Burman design, central composite design, enzyme purification, starch saccharification, Amy gene, homology modelling, bioprocess optimisation