Enzyme Loading Research Articles

Photothermally Enhanced Cascaded Nanozyme‐Functionalized Black Phosphorus Nanosheets for Targeted Treatment of Infected Diabetic Wounds

AbstractDue to the limitations of H2O2 under physiological conditions and defective activity, nanozyme‐catalyzed therapy for infected diabetic wound healing is still a huge challenge. Here, this work designs a novel multifunctional hybrid glucose oxidase (GOx)–CeO2@black phosphorus (BP)/Apt nanosheet that features GOx and CeO2 dual enzyme loading with photothermal enhancement effect and targeting ability for the treatment of infected wounds in type II diabetic mice. Combined with the photothermal properties of the BP nanosheets, the cascade nanozyme effect of GOx and CeO2 is extremely enhanced. The synergistic effect of peroxidase activity and photothermal therapy with targeting aptamer allows for overcoming the catalytic defects of nanozyme and significantly improving in vitro bacterial inhibition rate with 99.9% and 97.8% for Staphylococcus aureus and Escherichia coli, respectively, as well as enhancing in vivo antibacterial performance with the lowest wound remained (0.05%), reduction of infiltration inflammatory cells, and excellent biocompatibility. Overall, this work builds a nanodelivery system with a powerful therapeutic approach, incorporating self‐supplying H2O2 synergistic photothermal and real‐time wound monitoring effect, which holds profound potential as a clinical treatment for infected diabetic wounds.

Statistical Optimization of Tween-80-Assisted Potassium Hydroxide Pretreatment and Enzymatic Hydrolysis for Enhancing Sugar Yields from Corn Cob

With the addition of Tween 80, potassium hydroxide pretreatment and enzymatic hydrolysis were statistically optimized to maximize sugar yields from corn cob (CC). The results indicated that the sugar yields from CC could be influenced significantly by the potassium hydroxide concentration, temperature and time during pretreatment. The optimized pretreatment conditions were as follows: potassium hydroxide, 46 g·L−1; Tween 80, 3.0 g·L−1; solid dose, 200 g·L−1; temperature, 78 °C; and time, 50 min. After optimization, the lignin reduction and recoveries of cellulose and hemicellulose were 89.7%, 97.8% and 68.0%, respectively. In addition, sugar production could also be influenced by the biomass loading, enzyme loading and reaction time. A maximal glucose production (518.48 mg·gds−1, milligrams per gram of dry substrate) and xylose production (351.14 mg·gds−1), 97.2% cellulose conversion and 82.9% hemicellulose conversion from CC could be obtained when the biomass loading was 195 g·L−1 and the enzyme loading was 8.9 FPU·gds−1 (filter paper activity units per gram of dry substrate) and when the Tween 80 concentration was 3.0 g·L−1 at 50 °C for 30.4 h during hydrolysis. This is the first systematic study of combined Tween 80 pretreatment of CC by potassium hydroxide and hydrolysis of CC by cellulase preparation to increase sugar production.

Enzyme Cascade Electrode Reactions with Nanomaterials and Their Applicability towards Biosensor and Biofuel Cells.

Nanomaterials, including carbon nanotubes, graphene oxide, metal-organic frameworks, metal nanoparticles, and porous carbon, play a crucial role as efficient carriers to enhance enzyme activity through substrate channeling while improving enzyme stability and reusability. However, there are significant debates surrounding aspects such as enzyme orientation, enzyme loading, retention of enzyme activity, and immobilization techniques. Consequently, these subjects have become the focus of intensive research in the realm of multi-enzyme cascade reactions. Researchers have undertaken the challenge of creating functional in vitro multi-enzyme systems, drawing inspiration from natural multi-enzyme processes within living organisms. Substantial progress has been achieved in designing multi-step reactions that harness the synthetic capabilities of various enzymes, particularly in applications such as biomarker detection (e.g., biosensors) and the development of biofuel cells. This review provides an overview of recent developments in concurrent and sequential approaches involving two or more enzymes in sequence. It delves into the intricacies of multi-enzyme cascade reactions conducted on nanostructured electrodes, addressing both the challenges encountered and the innovative solutions devised in this field.

Enzyme immobilization on a novel NH2-MOF-5@PEI composite support for efficient kinetic resolution of MOPE enantiomers

The immobilization of enzyme on solid supports as the heterogeneous catalyst is an efficient strategy to enhance the catalytic performance and reusability. MOFs supports show huge potential for enzyme immobilization, however, suffer from poor stability thus reducing the effective loading of enzyme and resulting in low catalytic performance. In this work, NH2-MOF-5 was synthesized and linked with the cross-linked PEI by chemical bond to form a novel composite support, the lipase PS was then immobilized onto the composite support through physical adsorption to synthesized PS@NH2-MOF-5@PEI in hexane. Benefitting from the stable nano-flower structure and strong adsorption for lipase PS of the NH2-MOF-5@PEI, the synthesized PS@NH2-MOF-5@PEI showed huge specific surface area (only decreased 5.90 % after immobilization) and enhanced α-helix and β-turn (increased 29.80 % and 52.88 %, respectively), thus exhibiting excellent catalytic activity which was about 2 times higher than that of free lipase PS, as well as outstanding recyclability after 8 cycles. We believe the strategy of immobilization of enzymes on a novel NH2-MOF-5@PEI support will exhibit huge potential of biocatalyst for further application.

Enzymatic production process of capric acid-rich structured lipids: Development of formulation as a new therapeutic approach

The present work reports an optimization of the synthesis of MLM-type (medium, long, medium) structured lipids (SL) through an acidolysis reaction of grape seed oil with capric acid catalyzed by Rhizopus oryzae lipase immobilized. At first, tests were carried out by preparing the biocatalysts using enzyme loadings (0.15 to 1 g of enzymatic powder) for each gram of support. Enzyme loading was used 0.3 g of enzymatic powder, and hydrolytic activity of 1860 ± 23.4 IU/g was reached. Optimized conditions determined by the Central Composite Rotatable Design (CCRD) revealed that the acidolysis reaction reached approximately 59 % incorporation degree (%ID) after 24 h, in addition to the fact that the biocatalyst could maintain the incorporation degree in five consecutive cycles. From this high incorporation degree, cell viability assays were performed with murine fibroblast cell lines and human cervical adenocarcinoma cell lines. Concerning the cytotoxicity assays, the concentration of MLM-SL to 1.75 and 2 % v/v were able to induce cell death in 56 % and 64 % of adenocarcinoma cells, respectively. Human cervical adenocarcinoma cells showed greater sensitivity to the induction of cell death when using emulsions with MLM-SL > 1.75 % v/v compared to emulsions with lower content indicating a potential for combating carcinogenic cells.

Immobilization of enzymes on functionalized cellulose nanofibrils for bioremediation of antibiotics: Degradation mechanism, kinetics, and thermodynamic study

The deteriorating environmental conditions due to increasing emerging recalcitrant pollutants raised a severe concern for its remediation. In this study, we have reported antibiotic degradation using free and immobilized HRP. The functionalized cellulose support was utilized for efficient immobilization of HRP. Approximately 13.32 ± 0.52 mg/g enzyme loading was achieved with >99% immobilization efficiency. The higher percentage of immobilization is attributed to the higher surface area and carboxylic groups on the support. The kinetic parameter of immobilized enzymes was Km = 2.99 mM/L for CNF-CA@HRP, which is 3.5-fold more than the Michaelis constant (Km = 0.84794 mM/L) for free HRP. The Vmax of CNF-CA@HRP bioconjugate was 2.36072 mM/min and 0.558254 mM/min for free HRP. The highest degradation of 50, 54.3, and 97% were achieved with enzymatic, sonolysis, and sono-enzymatic with CNF-CA@HRP bioconjugate, respectively. The reaction kinetics analysis revealed that applying ultrasound with an enzymatic process could enhance the reaction rate by 2.7–8.4 times compared to the conventional enzymatic process. Also, ultrasound changes the reaction from diffusion mode to the kinetic regime with a more oriented and fruitful collision between the molecules. The thermodynamic analysis suggested that the system was endothermic and spontaneous. While LC-MS analysis and OTC's degradation mechanism suggest, it mainly involves hydroxylation, secondary alcohol oxidation, dehydration, and decarbonylation. Additionally, the toxicity test confirmed that the sono-enzymatic process helps toward achieving complete mineralization. Further, the reusability of bioconjugate shows that immobilized enzymes are more efficient than the free enzyme.

Valorization of spent coffee grounds through integrated bioprocess of fermentable sugars, volatile fatty acids, yeast-based single-cell protein and biofuels production

Recovering nutrients from waste for biological processes aligns with sustainability principles. This study aimed to convert spent coffee grounds (SCG) into valuable products, including fermentable sugars, volatile fatty acids (VFAs), yeast-based single-cell protein and biofuels. Alkaline pretreatment was conducted before enzymatic hydrolysis, in which the pretreated SCG was hydrolyzed with varying enzyme loadings (20–60 filter paper units (FPU)/g-solid) and solid loadings (3–15 % w/v). The hydrolyzed slurry was utilized for VFAs and hydrogen production, yielding high values of 0.66 g/g-volatile solids (VS) and 109 mL/g-VS, respectively, using an enzyme loading of 50 FPU/g-solid and a solid loading of 3 % (w/v). The derived VFAs were used to cultivate a newly isolated yeast, Candida maltosa KKU-ARY2, resulting in an accumulated protein content of 43.7 % and a biomass concentration of 4.6 g/L. This study highlights the conversion of SCG into essential components, emphasizing the benefits of waste utilization through cascade bioprocesses.

Magnetic macroporous chitin microsphere as a support for covalent enzyme immobilization

In this study, a novel magnetic macroporous chitin microsphere (MMCM) was developed for enzyme immobilization. Chitin nanofibers were prepared and subsequently subjected to self-assembly with magnetic nanoparticles and PMMA (polymethyl methacrylate). Following this, microspheres were formed through spray drying, achieving a porous structure through etching. The MMCM serves as an effective support for immobilizing enzymes, allowing for their covalent immobilization both on the microsphere's surface and within its pores. The substantial surface area resulting from the porous structure leads to a 2.1-fold increase in enzyme loading capacity compared to non-porous microspheres. The MMCM enhances stability of the immobilized enzymes under various pH and temperature conditions. Furthermore, after 20 days of storage at 4 °C, the residual activity of the immobilized enzyme was 2.93 times that of the free enzyme. Even after being recycled 10 times, the immobilized enzyme retained 56.7 % of its initial activity. It's noteworthy that the active sites of the enzymes remained unchanged after immobilization using the MMCM, and kinetic analysis revealed that the affinity of the immobilized enzymes rivals that of the free enzymes.

Tween 80 reversed adverse effects of combined autohydrolysis and p-toluenesulfonic acid pretreatment on enzymatic hydrolysis of poplar

In this study, a combined pretreatment involving autohydrolysis and p-toluenesulfonic acid (p-TsOH) was performed on poplar to coproduce xylooligosaccharides (XOSs) and monosaccharides. The autohydrolysis (180 °C, 30 min) yielded 53.2 % XOS and enhanced the delignification efficiency in the subsequent p-TsOH treatment. Furthermore, considerably high glucan contents (64.1 %∼83.1 %) were achieved in the combined pretreated substrates. However, their enzymatic digestibilities were found to be extremely poor (9.6 %∼14.2 %), which were even lower than the single p-TsOH pretreated substrates (10.2 %∼35.8 %). The underlying reasons were revealed by systematically investigating the effects of the single and combined pretreatment strategies on substrate properties. Moreover, the Tween 80 addition successfully reversed the adverse effects of combined pretreatment on the enzymatic hydrolysis, achieving a high glucose yield of 99.3 % at an enzyme loading of 10 filter paper units/g (FPU/g) glucan. These results deepen the understanding of the synergy of combined pretreatment on biomass fractionation and enzymatic saccharification.

Optimising microalgae-derived butanol yield

Rapid global urbanisation emphasises the necessity to explore sustainable energy resources to fulfill escalating energy demands. Biobutanol emerges as a promising biofuel due to heightened energy density and molecular similarity to petrol. Microalgae, rich in carbohydrates, offer a potential glucose source for biobutanol fermentation. The current study shows that varying CO2 concentrations affect carbohydrate content in the biomass of P. kessleri bh-2 and Scenedesmus sp.k-7. The introduction of 1% CO2 significantly improved carbohydrate productivity, reaching 74.5% for P. kessleribh-2 and 71.0% for Scenedesmus sp.k-7. Enzymatic hydrolysis was found to be most effective for ABE fermentation, with optimal results obtained at enzyme loads of 8 mg g−1 cellulase and 2 mg g−1 amylase for both strains. When C. acetobutylicum cells were cultivated with pretreated P. kessleribh-2 biomass, the biobutanol yield amounted to 4.8 gL-1 at a glucose concentration of 5.30 gL-1, demonstrating strain's superior performance compared to Scenedesmus sp.k-7.

Synergistic Hydrolysis of Soy Proteins Using Immobilized Proteases: Assessing Peptide Profiles.

Because of the health benefits and economic opportunities, extracting bioactive peptides from plant proteins, often food processing by-products, garners significant interest. However, the high enzyme costs and the emergence of bitter peptides have posed significant challenges in production. This study achieved the immobilization of Alcalase and Flavorzyme using cost-effective SiO2 microparticles. Mussel-inspired chemistry and biocompatible polymers were employed, with genipin replacing glutaraldehyde for safer crosslinking. This approach yielded an enzyme loading capacity of approximately 25 mg/g support, with specific activity levels reaching around 180 U/mg for immobilized Alcalase (IA) and 35 U/mg for immobilized Flavorzyme (IF). These immobilized proteases exhibited improved activity and stability across a broader pH and temperature range. During the hydrolysis of soy proteins, the use of immobilized proteases avoided the thermal inactivation step, resulting in fewer peptide aggregates. Moreover, this study applied peptidomics and bioinformatics to profile peptides in each hydrolysate and identify bioactive ones. Cascade hydrolysis with IA and IF reduced the presence of bitter peptides by approximately 20%. Additionally, 50% of the identified peptides were predicted to have bioactive properties after in silico digestion simulation. This work offers a cost-effective way of generating bioactive peptides from soy proteins with reducing potential bitterness.

Amorphous-crystalline phase transition and intrinsic magnetic property of nickel organic framework for easy immobilization and recycling of β-Galactosidase

This work describes the synthesis of fibrous nickel-based metal organic framework (Ni-ZIF) via simple solvothermal method. The material formed was calcinated at 400, 600, 800 °C to improve its surface area, porosity and enzyme binding capacity. Changes in X-ray diffraction pattern after calcination revealed the Ni-ZIF transitioned from amorphous to crystalline structure. The surface area, pore volume and pore size for Ni-ZIF@600 were found to be 312.15 m2/g, 0.88 cm3/g and 10.28 nm, with an enzyme loading capacity of 593.85 mg/g after 30 h The free (β-Gal-LEH) and immobilized β-Galactosidase were stable at pH 7.5, temperature 50 °C, and yielded 70.70 and 63.95 mM glucose after milk lactose hydrolysis, respectively. The Ni-ZIF@600@β-Gal-LEH exhibited high enzyme retention capacity, maintaining 59.44 % of its original activity after 6-cycles. The enhanced magnetic property, enzyme binding capacity and easy recoverability of the calcinated Ni-ZIF could guarantee its industrial significance as immobilization module for enzyme-mediated catalysis.

Enterobacter aerogenes Lipase immobilized on ion exchange resin D152 as a supportive carrier

Enterobacter aerogenes Lipase used in this study was heterologously expressed by Pichia pastoris. D152, D152H, D151H, D113, 724), three types of anion resins ( D380, D 301R, D311) and two types of chelating r activities of the immobilized ( EAL). According to the results, D152 was selected for haydrolysis activity as the immobilized enzyme (EAL) . The D152 was selected for hydrolysis activity since the immobilized lipase exhibited the highest specific hydrolysis activity. Immobilization conditions (enzyme loading, immobilization time, temperature, and pH value) . The best results were enzyme loading 4mg/g, time 80min, temperature 30°C, and buffer pH 8; and under the optimized conditions the immobilization efficiency was 95% and the specific activity was 532841.34 U/g.esins (D401, D418) were sieved as support matrix. Hydrolysis assay was employed to evaluate the specific.

Disposable biosensor based on ionic liquid, carbon nanofiber and poly(glutamic acid) for tyramine determination

In this study, an electrochemical biosensor based on carbon nanofibers (CNF), ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (IL), poly(glutamic acid) (PGA) and tyrosinase (Tyr) modified screen printed carbon electrode (SPE) was constructed for tyramine determination. Optimum experimental parameters such as CNF and IL amount, polymerization conditions of glutamic acid, enzyme loading, pH of test solution and operating potential were explored. The construction steps of the Tyr/PGA/CNF−IL/SPE were pursued by scanning electron microscopy and cyclic voltammetry. The Tyr/PGA/CNF−IL/SPE biosensor exhibited linear response to tyramine in the range of 2.0 × 10−7 – 4.8 × 10−5 M with a low detection limit of 9.1 × 10−8 M and sensitivity of 302.6 μA mM−1. The other advantages of Tyr/PGA/CNF−IL/SPE include its high reproducibility, good stability and anti-interference ability. The presented biosensor was also applied for tyramine determination in malt drink and pickle juice samples and mean analytical recoveries of spiked tyramine were calculated as 100.6% and 100.4% respectively.

Model-based optimization of cell-free enzyme cascades exemplified for the production of GDP-fucose

Cell-free production systems are increasingly used for the synthesis of industrially relevant chemicals and biopharmaceuticals. Cell-free systems often utilize cell lysates, but biocatalytic cascades based on recombinant enzymes have emerged as a promising alternative strategy. However, implementing efficient enzyme cascades is a non-trivial task and mathematical modeling and optimization has become a key tool to improve their performance. In this work, we introduce a generic framework for the model-based optimization of cell-free enzyme cascades based on a given kinetic model of the system. We first formulate and systematize seven optimization problems relevant in the context of cell-free production processes including, for example, the maximization of productivity or product yield and the minimization of overall costs. We then present an approach that accounts for parameter uncertainties, not only during model calibration and model analysis but also when performing the actual optimization. After constructing a kinetic model of the enzyme cascade, experimental data are used to generate an ensemble of kinetic parameter sets reflecting their variabilities. For every parameter set, systems optimization is then performed and the resulting solution subsequently cross-validated for all other parameterizations to identify the solution with the highest overall performance under parameter uncertainty. We exemplify our approach for the cell-free synthesis of GDP-fucose, an important sugar nucleotide with various applications. We selected and solved three optimization problems based on a constructed dynamic model and validated two of them experimentally leading to significant improvements of the process (e.g., 50% increase of titer under identical total enzyme load).Overall, our results demonstrate the potential of model-driven optimization for the rational design and improvement of cell-free production systems. The developed approach for systems optimization under parameter uncertainty could also be relevant for the metabolic design of cell factories.

The Impact of Sea Urchin as an Ingredient on the Physicochemical, Microbiological, and Sensory Properties of Fish Sauce Fermentation.

The consequences of using 25% whole or shelled sea urchin as an ingredient in anchovy sauce on its fermentation and development of its physicochemical properties after 20 days fermentation was studied. Two varieties of fish and sea urchin sauce were made with or without shell at 1:2:1 ratio (salt:fish:sea urchin) plus a control fish sauce at 1:3 ratio (salt:fish). All sauces were fermented at 40-50 °C for 20 days, where for the first 7 days the preparation remained in a static phase. During their fermentation, pH, salt concentration, aw, TVB-N, TMA, total nitrogen, formaldehyde nitrogen, amino nitrogen, and ammonium nitrogen, as well as aerobic mesophiles and lactic acid bacteria were monitored. The fermentation of the experimental sauces proved to follow an evolution rather similar to the control sauce. The whole and shelled sea urchins provided the necessary microbial and enzymatic load to trigger an adequate hydrolysis of the fish and the production of total nitrogen (16.0-17.6 g/L), formaldehyde nitrogen (15.1-16.0 g/L), and amino nitrogen (0.7-0.8 g/L) of the same order as the control sauce, despite the lower fish content. According to TMA (9.2-13.1 mg N/100 g), VBT (40.0-47.2 mg N/100 g) contents, and pH levels (5.41-5.46), no deviation of the fermentation process was observed under the experimental conditions (salt content, temperature, and agitation after the static phase). Quantitative descriptive analysis (QDA) sensory revealed that the use of sea urchin results in high quality products characterized by their aromas of crustaceans and mollusks. The present study investigates the potential use of shelled and even whole sea urchin as an ingredient for the preparation of high quality fish sauces.

Process optimization for saccharification and fermentation of the Organic Fraction of Municipal Solid Waste (OFMSW) to maximize ethanol production performance

The Organic Fraction of Municipal Solid Waste (OFMSW) contains paper and cardboard, an attractive cellulosic feedstock for bioethanol production. Waste paper/cardboard (WPC) was subjected to mild acid treatment in solids loadings from 20 to 27.5 %. Different doses of commercial enzyme cocktail and various adjuvants facilitating enzymatic saccharification were tested. Enzyme dosing of 3.75 FPU/g fiber with very slow fed-batch saccharification of 27.5 % (w/v) solids from model lignin-free paper pulp resulted in 12.6 % (w/v) total sugar after 74 h. Use of cheap non-catalytic waste protein adjuvants including Soymeal, Protisyr, Feather meal strongly improved enzymatic saccharification and reduced the overall process cost by reducing commercial enzyme dosing. After fermentation with the industrial yeast strain BMD44 (Cellusec® 1.0) an ethanol titer of 5.77 % (v/v) was obtained using a reduced enzyme loading of 2.5 FPU/g of actual industrial WPC fiber. These results show the potential of economically profitable second-generation bioethanol production with WPC fiber.

In-situ immobilization of lipase on α-alumina membrane for oil fouling control and cleaning

Enzyme immobilization on inorganic membranes is typically a difficult, time-consuming, multi-step process carried out ex-situ (outside the filtration system). To enable process scaling, an in-situ immobilization technique is essential. Therefore, an in-situ method to immobilize the lipase Eversa Transform 2.0 (ET2) on a ceramic membrane was developed using polydopamine as a bonding agent. The in-situ immobilization was successful, and optimum dopamine hydrochloride and ET2 concentrations (0.3 mg·mL−1 and 4 mg·mL−1, respectively) resulted in an enzyme loading of 10 g·m−2 that increased the membrane water affinity and hydrolytic activity towards soybean oil (38 mmol·min−1·m−2). The enzymatic membrane demonstrated great self-cleaning capability (pure water permeance recovery of 97 % after cleaning with deionized water at 40 °C for 6 h) and strong fouling resistance (pure water permeance decline limited to 43 % following oil emulsion filtration). To reuse the membrane after it lost its hydrolytic activity, calcination or in-situ chemical cleaning were tested. There were no morphological or chemical alterations on the membrane surface after five cycles of modification-regeneration. When compared to ex-situ immobilization techniques, in-situ enzyme immobilization and chemical regeneration can save costs, improve the sustainability of the process, and ease scale-up.

The effects of the chemical modification on immobilized lipase features are affected by the enzyme crowding in the support.

In this article, we have analyzed the interactions between enzyme crowding on a given support and its chemical modification (ethylenediamine modification via the carbodiimide route and picryl sulfonic (TNBS) modification of the primary amino groups) on the enzyme activity and stability. Lipase from Thermomyces lanuginosus (TLL) and lipase B from Candida antarctica (CALB) were immobilized on octyl-agarose beads at two very different enzyme loadings, one of them exceeding the capacity of the support, one well under this capacity. Chemical modifications of the highly loaded and lowly loaded biocatalysts gave very different results in terms of activity and stability, which could increase or decrease enzyme activity depending on the enzyme support loading. For example, both lowly loaded biocatalysts increased their activity after modification while the effect was the opposite for the highly loaded biocatalysts. Additionally, the modification with TNBS of highly loaded CALB biocatalyst increased its stability while decrease the activity.

Production of Prebiotic Galacto-Oligosaccharides from Acid Whey Catalyzed by a Novel β-Galactosidase from Thermothielavioides terrestris and Commercial Lactases: A Comparative Study

The steadily increasing global popularity of Greek strained yoghurt has necessitated alternative valorization approaches for acid whey, the major straining process effluent. In this context, prebiotic galacto-oligosaccharides can be enzymatically synthesized from acid whey lactose, via either commercial or novel β-galactosidases. A comparative study of galacto-oligosaccharide production from acid whey was carried out, employing two commercial β-galactosidases (from Kluyveromyces lactis and Aspergillus oryzae) and one novel, in-house produced (from Thermothielavioides terrestris), as a function of the initial lactose content and enzyme load. Selected reaction conditions for β-galactosidases from K. lactis, A. oryzae, and T. terrestris were 35 °C at pH 7.2, 45 °C at pH 4.5, and 50 °C at pH 4.0, respectively. Maximum galacto-oligosaccharide yields equal to 23.7, 23.4, and 25.7% were achieved with, respectively, 0.13 U/mL of K. lactis β-galactosidase in non-concentrated acid whey, 4 U/mL of A. oryzae β-galactosidase, and 8 U/mL of T. terrestris β-galactosidase in acid whey concentrated to 20% w/v initial lactose content. The increased galacto-oligosaccharide productivity of the thermophilic β-galactosidase from T. terrestris can be a determining asset in a combined concentration and oligomerization industrial process. This will allow for high galacto-oligosaccharide yields for efficient, cost-effective production of valuable prebiotics from acid whey.