The successful application of recombinant enzymes in the food industry relies not only on their activity but also on their purity and characterization. For enzymes like beta-galactosidase, ensuring they meet stringent quality and safety standards is critical. This article outlines the essential purification and characterization steps involved in preparing recombinant beta-galactosidase for industrial use, particularly focusing on methods employed for enzymes produced in yeast hosts like Komagataella phaffii.

Following fermentation, the recombinant beta-galactosidase, such as the Paenibacillus wynnii β-gal-Pw, needs to be isolated from the cellular components and other impurities. The initial step typically involves cell disruption, which releases the intracellular enzyme. Various mechanical or chemical methods can be employed for this purpose, aiming to efficiently break open the yeast cells while preserving enzyme activity. Following disruption, a clarification step, often centrifugation, is used to separate the solid cell debris from the liquid enzyme-containing extract.

Purification of beta-galactosidase often involves chromatographic techniques. Hydrophobic interaction chromatography (HIC) is a common and effective method used to separate proteins based on their surface hydrophobicity. In the case of β-gal-Pw, research has demonstrated that HIC can successfully purify the enzyme by employing a step elution strategy. This typically involves binding the enzyme to a hydrophobic resin, washing away unbound impurities, and then eluting the target enzyme using a buffer with a lower salt concentration. This process can significantly increase the specific activity of the enzyme, meaning more enzyme activity per unit of protein mass.

After chromatographic purification, further steps are often taken to concentrate the enzyme and improve its stability for storage and transportation. Adding stabilizers, such as glycerol, is a common practice to protect the enzyme from denaturation. The final enzyme preparation is then thoroughly characterized. This includes determining its specific activity using a substrate like o-nitrophenol-β-D-galactopyranoside (oNPGal) or natural substrates, assessing protein concentration through methods like the Bradford assay, and verifying purity using techniques such as SDS-PAGE. SDS-PAGE is particularly important as it allows for the visualization of protein bands, confirming the presence of the target enzyme at its expected molecular weight and checking for the absence of significant contaminating proteins.

One critical characteristic for enzymes used in the dairy industry is their behavior in the presence of reaction products. As noted in studies, recombinant β-gal-Pw exhibits an advantage over many commercial beta-galactosidases because it is not inhibited by D-galactose; in fact, it can be activated. This unique property ensures consistent and efficient lactose hydrolysis, even as galactose levels rise during the process. This detailed characterization is vital for ensuring that the enzyme performs reliably and safely in food applications.

At NINGBO INNO PHARMCHEM CO.,LTD., we adhere to rigorous purification and characterization protocols to ensure our recombinant beta-galactosidase meets the highest industry standards. Our expertise covers everything from cell disruption and chromatographic purification to comprehensive analytical testing. We provide enzyme solutions that are not only highly active but also pure and stable, making them ideal for sensitive food and beverage applications. Partner with us to secure enzyme products that guarantee quality and performance.