Determination of the transgalactosylation activity of Aspergillus oryzae β-galactosidase: effect of pH, temperature, and galactose and glucose concentrations

The catalytic potential of β-galactosidase is usually determined by its hydrolytic activity over natural or synthetic substrates. However, this method poorly predicts enzyme behavior when transglycosylation instead of hydrolysis is being performed. A system for determining the transgalactosylation activity of β-galactosidase from Aspergillus oryzae was developed, and its activity was determined under conditions for the synthesis of galacto-oligosaccharides and lactulose. Transgalactosylation activity increased with temperature up to 55 °C while the effect of pH was mild in the range from pH 2.5 to 5.5, decreasing at higher values. The effect of glucose and galactose on transgalactosylation activity was also assessed both in the reactions for the synthesis of galacto-oligosaccharides and lactulose and also in the reaction of hydrolysis of o-nitrophenyl β-d-galactopiranoside. Galactose was a competitive inhibitor and its effect was stronger in the reactions of transgalactosylation than in the reaction of hydrolysis. Glucose was a mild activator of β-galactosidase in the reaction of hydrolysis, but its mechanism of action was more complex in the reactions of transgalactosylation, having this positive effect only at low concentrations while acting as an inhibitor at high concentrations. This information is relevant to properly assess the effect of monosaccharides during the reactions of the synthesis of lactose-derived oligosaccharides, such as galacto-oligosaccharides and lactulose.     

Influence of reaction conditions on the selectivity of the synthesis of lactulose with microbial β-galactosidases

Commercial β-galactosidase preparations from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae were evaluated as catalysts for the synthesis of lactulose. Among them, the enzyme from A. oryzae was selected for further studies. The effect of reaction conditions was then studied on product composition during the kinetically controlled synthesis of lactulose by transgalactosylation with A. oryzae β-galactosidase. Product composition was not affected by pH, temperature, total initial concentration of sugar (lactose plus fructose) and enzyme to substrate ratio within the ranges studied. However, lactose to fructose ratio strongly influenced product composition being then possible to control the lactulose to galacto-oligosaccharide ratio within ample margins. Maximum lactulose yield (0.282 g of lactulose per g initial lactose) was obtained using 1/8 lactose to fructose molar ratio, 50% (w/w) total initial sugars, 40 °C, pH 4.5 and enzyme to initial lactose ratio equivalent to 200 IU/g.     

Cost effective surface functionalization of silver nanoparticles for high yield immobilization of Aspergillus oryzae β-galactosidase and its application in lactose hydrolysis

The present study demonstrates synthesis, characterization and surface functionalization of silver nanoparticles (AgNPs) via glutaraldehyde for high yield immobilization of Aspergillus oryzae β-galactosidase. Soluble β-galactosidase (SβG), enzyme adsorbed on unmodified AgNPs (UβG) and surface modified AgNPs (MβG) showed same pH-optima at pH 4.5. However, it was observed that MβG exhibited enhanced pH stability toward acidic and alkaline sides, and increased temperature resistance as compared to SβG and UβG. Michaelis constant, K_m was increased nearly three-folds for MβG while V_max for soluble and MβG was 0.515 mM/min and 0.495 mM/min, respectively. Furthermore, MβG showed greater resistance to product inhibition mediated by galactose as compared to it soluble counterpart and exhibited excellent catalytic activity even after its fourth successive reuse. The remarkable bioconversion rates of lactose from milk in batch reactors further revealed an attractive catalytic efficiency of β-galactosidase adsorbed on surface functionalized AgNPs thereby promoting its use in the production of lactose free dairy products.     

First enzymatic galactosylation of acyclic nucleoside drugs by β-galactosidase: Synthesis of water-soluble β-D-galactosidic prodrugs

Acyclic nucleoside analogs constitute an important group of antiviral agents. However, these nucleoside drugs suffer from poor water solubility and low oral bioavailability in the clinic use. In the present work, the enzymatic synthesis of the water-soluble galactosidic prodrugs of acyclic nucleosides by using bovine liver β-galactosidase was described. In the enzymatic transgalactosylation between acyclovir (ACV) and o-nitrophenyl β-galactopyranoside (oNPGal), the optimum enzyme dosage, buffer pH, temperature and molar ratio of ACV to oNPGal were 0.225 U/mL, 7.0, 40°C and 2.5, respectively, under which the initial reaction rate and the yield reached 0.40 mM/h and 29%, respectively. In addition, this enzyme could accept ganciclovir (GCV) and penciclovir (PCV) as substrates, affording the corresponding 4’-β-galactosylated derivatives with the yields of 26% and 71%, respectively.     

Characterization and optimization of β-galactosidase immobilization process on a mixed-matrix membrane

β-Galactosidase is an important enzyme catalyzing not only the hydrolysis of lactose to the monosaccharides glucose and galactose but also the transgalactosylation reaction to produce galacto-oligosaccharides (GOS). In this study, β-galactosidase was immobilized by adsorption on a mixed-matrix membrane containing zirconium dioxide. The maximum β-galactosidase adsorbed on these membranes was 1.6 g/m², however, maximal activity was achieved at an enzyme concentration of around 0.5 g/m². The tests conducted to investigate the optimal immobilization parameters suggested that higher immobilization can be achieved under extreme parameters (pH and temperature) but the activity was not retained at such extreme operational parameters. The investigations on immobilized enzymes indicated that no real shift occurred in its optimal temperature after immobilization though the activity in case of immobilized enzyme was better retained at lower temperature (5 °C). A shift of 0.5 unit was observed in optimal pH after immobilization (pH 6.5 to 7). Perhaps the most striking results are the kinetic parameters of the immobilized enzyme; while the Michaelis constant (K_m) value increased almost eight times compared to the free enzyme, the maximum enzyme velocity (V_max) remained almost constant.     

Semicontinual synthesis of alkyl galactosides using β-galactosidase entrapped in polyvinylalcohol hydrogel

Fungal β-galactosidase from Aspergillus oryzae was immobilized into polyvinylalcohol (PVA) hydrogel by LentiKats® technology and used for the production of short-chain alkyl glycosides. Ethyl- and propyl-β-d-galactopyranosides were prepared from lactose (100?g/L) and varying initial amounts of alcohol (10–30% v/v) at 40?°C and pH 4.5. The entrapped β-galactosidase preserved 50% of the initial transgalactosylation activity after 25 repeated cycles in the production of ethyl β-d-galactopyranoside. When 5% (v/v) propanol was used as an acceptor, the enzyme activity (30–32?U/g immobilized enzyme) remained constant for 25 repeated batch runs. These findings suggest that entrapped β-galactosidase into LentiKats® has a great potential to be one effective, reusable and easy producible biocatalyst for the production of alkyl glycosides in a large scale.     

Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis

β-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of β-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8–7.0 with more than 97% activity. Purified β-galactosidase was optimally active at 50 °C. Kinetic parameters K_m and V_max for purified enzyme were 400 µM and 1.22 × 10⁻¹ U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca²⁺, Mg²⁺ and Mn²⁺ slightly activated the enzyme whereas NH₄⁺, Co²⁺ and Fe³⁺ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that β-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min⁻¹ and t_1/2 of 14.74 min. This is better than other studied β-galactosidases. Both sonicated Pediococcus acidilactici cells and purified β-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of β-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca²⁺ shows that it is suitable for milk processing.     

Transglycosylation of a β-galactosyl radical, in the course of enzymic hydrolysis of lactose, in the presence of selected polyhydroxyalcohols

Sorbitol, xylitol, erythritol and lactitol were used as the acceptors of galactosyl radicals, in the process of transgalactosylation accompanying the hydrolysis of lactose, conducted with -galactosidase (4.0 ml Lactozym 3000 was added to 430 ml 1.45 M lactose with 0.95 M polyhydroxyalcohols). The following concentrations of galactosyl derivatives of polyols were obtained after hydrolysis for 4 h at 40°C: 0.31 M Gal-erythritol, 0.22 M Gal-xylitol, 0.18 M Gal-sorbitol and 0.14 M Gal-lactitol. A quadruple increase of xylitol content in dry matter of a solution (from 11.5% (w/w) to 44.5% (w/w) brought about a 2.3-fold increase of the product content in the solution (15.2% (w/w) of dry matter).     

Antarctic,cold-adapted β-galactosidase of Pseudoalteromonas sp. 22b as an effective tool for alkyl galactopyranosides synthesis

The antarctic Pseudoalteromonas sp. 22b β-galactosidase was found to catalyze synthesis of alkyl galactopyranosides. The number of carbon atoms in C3–C6 alcohol molecules only slightly affected the yield of products. Reactions of transgalactosylation were conducted for 80 h and the maximum accumulation of their products was observed between 40 and 50 h. The highest and almost the same yields of alkyl galactosides were achieved at pH 6–9 and 10–30% water concentration. Like known mesophilic β-galactosidases, the antarctic enzyme more efficiently synthesized alkyl galactosides when reactions were carried out in mixtures of buffers and organic solvents (below 50%, v/v).     

Expression,characterization and structural profile of a heterodimeric β-galactosidase from the novel strain Lactobacillus curieae M2011381

A heterodimeric β-galactosidase was discovered in the novel strain Lactobacillus curieae M2011381. The gene encoding the enzyme was expressed in Escherichia coli BL21 (DE3). The specific enzyme activities of the recombinant holoenzyme (LacLM) and large subunit (LacL) measured 11.4 U/mg and 3.8 U/mg, respectively. The k_cat/K_m values of LacLM and LacL were 740 mM⁻¹ s⁻¹ and 1.40 mM⁻¹ s⁻¹, respectively. LacLM showed maximum activity at pH 8.0 and 55 °C, and it could maintain its activity at a neutral pH and below 45 °C. LacLM displayed both hydrolysis and transgalactosylation activity on 200 g/L lactose. When LacLM was added to milk, the lactose was hydrolyzed after 6 h without galactooligosaccharide generation. The sequence alignment and homology modeling of the structures of the holoenzyme and subunits revealed that LacL has a catalytic domain with a catalytic dyad, Glu470 and Glu538, and small subunit LacM is a β-sheet domain with a conserved Trp294. The molecular docking of LacLM helped to illustrate the roles of both subunits in the reaction with lactose.     

Synthesis of benzyl β-d-galactopyranoside by transgalactosylation using a β-galactosidase produced by the over expression of the Kluyveromyces lactis LAC4 gene in Arxula adeninivorans

The LAC4 gene of Kluyveromyces lactis encoding for β-galactosidase was overexpressed in the yeast Arxula adeninivorans to produce the enzyme, which can be used for the synthesis of β-d-galactosides. These compounds play a major role as precursors for the synthesis of glycolipids and glycoproteins in medicine or for the production of tensides.The Xplor^®2 transformation/expression platform was used because it enabled stable integration of the gene in the Arxula genome and the production of high levels of the enzyme. The recombinant β-galactosidase, fused with C-terminal His-tag region (Lac4-6hp), was purified by precipitation with ammonium sulphate and FPLC using hydroxylapatite. The enzyme exhibited optimal activity at 37 to 40 °C, pH 6.5 in 50 mM sodium acetate buffer. Activity was measured by the formation of p-nitrophenol at 405 nm from the hydrolyzed chromogenic substrate, p-nitrophenyl-β-d-gal. Biochemical characterization included the calculation of K_M and apparent k_cat values of the enzyme. The formation of benzyl β-d-gal by 0.1 U enzyme from A. adeninivorans with transgalactosylation was six times higher than that for the prokaryotic enzyme from E. coli. Moreover, the partially purified enzyme was used for the selective hydrolysis of allyl β-d-gal in a mixture of allyl β- and allyl α-d-gal, with 4 g l⁻¹ being hydrolysed within one day by 1 U ml⁻¹. Thus, the recombinant β-galactosidase produced in A. adeninivorans is of potential interest for the enzymatic synthesis of benzyl β-d-gal and other galactosides as well as the selective hydrolysis of anomeric mixtures and could be used to replace difficult chemical procedures.     

Immobilization of Bacillus circulans β-galactosidase and its application in the synthesis of galacto-oligosaccharides under repeated-batch operation

A highly active and stable derivate of immobilized Bacillus circulans β-galactosidase was prepared for the synthesis of galacto-oligosaccharides (GOS) under repeated-batch operation. B. circulans β-galactosidase was immobilized on monofunctional glyoxyl agarose and three heterofunctional supports: amino-, carboxy-, and chelate-glyoxyl agarose. Glyoxyl agarose was the support with highest immobilization yield and stability being selected for the optimization of immobilization conditions and application in GOS synthesis. A central composite rotatable design was conducted to optimize contacted protein and immobilization time, using maximum catalytic potential as the objective function. Optimal conditions of immobilization were 28.9 mg/g and 36.4 h of contact, resulting in a biocatalyst with 595 IU/g and a half-life 89-fold higher than soluble enzyme. Immobilization process did not alter the synthetic capacity of β-galactosidase, obtaining the same GOS yield and product profile than the free enzyme. GOS yield and productivity remained unchanged along 10 repeated batches, with values of 39% (w/w) and 5.7 g GOS/g of biocatalyst·batch. Total product obtained after 10 batches of reaction was 56.5 g GOS/g of biocatalyst (1956 g GOS/g protein). Cumulative productivity in terms of mass of contacted protein was higher for the immobilized enzyme than for its soluble counterpart from the second batch of synthesis onwards.     

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Penicillium notatum

A new low-cost β-galactosidase (lactase) preparation for whey permeate saccharification was developed and characterized. A biocatalyst with a lactase activity of 10 U/mg, a low transgalactosylase activity and a protein content of 0.22 mg protein/mg was obtained from a fermenter culture of the fungus Penicillium notatum. Factors influencing the enzymatic hydrolysis of lactose, such as reaction time, pH, temperature and enzyme and substrate concentration were standardized to maximize sugar yield from whey permeate. Thus, a 98.1% conversion of 5% lactose in whey permeate to sweet (glucose-galactose) syrup was reached in 48 h using 650 β-galactosidase units/g hydrolyzed substrate. After the immobilization of the acid β-galactosidase from Penicillium notatum on silanized porous glass modified by glutaraldehyde binding, more than 90% of the activity was retained. The marked shifts in the pH value (from 4.0 to 5.0) and optimum temperatures (from 50°C to 60°C) of the solid-phase enzyme were observed and discussed. The immobilized preparation showed high catalytic activity and stability at wider pH and temperature ranges than those of the free enzyme, and under the best operating conditions (lactose, 5%; β-galactosidase, 610–650 U/g lactose; pH 5.0; temperature 55°C), a high efficiency of lactose saccharification (84–88%) in whey permeate was achieved when lactolysis was performed both in a batch process and in a recycling packed-bed bioreactor. It seems that the promising results obtained during the assays performed on a laboratory scale make this immobilizate a new and very viable preparation of β-galactosidase for application in the processing of whey and whey permeates.     

β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The K_m, k_cat and k_cat/K_m values for oNPG and lactose are 0.9 mM, 92 s⁻¹, 130 mM⁻¹ s⁻¹ and 29 mM, 98 s⁻¹, 3.3 mM⁻¹ s⁻¹, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising ∼80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-d-Galp-(1→6)-d-Lac, accounting for 34% of total GOS, and β-d-Galp-(1→6)-d-Glc, making up 29% of total GOS.     

Enzymatic synthesis of 3-aminopropyl-1-O-β-D-galactopyranoside catalyzed by Aspergillus oryzae β-galactosidase

Glycosidases represent excellent green chemistry alternatives as catalysts for the synthesis of glycosides, and in particular their stereoselectivity allows the production of anomerically pure glycosides, in only one reaction step using mild reaction conditions. Here, we report the enzymatic synthesis and structural characterization of 3-aminopropyl-1-O-β-D-galactopyranoside. Optimal reaction conditions for the transgalactosylation reaction were 100?mM lactose, 500?mM 3-amino-1-propanol and 24 h of incubation at 50?°C with 6 U/mL of β-galactosidase from Aspergillus oryzae. The fact that the synthesis of 1-propyl-2-O-β-D-galactopyranoside using 1-amino-2-propanol as acceptor was not achieved, and that N-glycoside formation was not observed, confirms the selectivity of β-galactosidase for the synthesis of O-glycosides, and particularly for primary alcohols. The synthesized galactosides were evaluated for their ability to interact with bovine spleen galectin-1 (Gal-1) by using the hemagglutination inhibition assay; results demonstrated that 3-aminopropyl-1-O-β-D-galactopyranoside may be considered as a functionalized galactose moiety more than an efficient Gal-1 inhibitor. The proposed approach constitutes a promising tool for the generation of glycopolymers and glyconanoparticles with potential applications in the development of biosensors as well as construction blocks in chemical synthesis.     

Transgalactosylation and hydrolytic activities of commercial preparations of β-galactosidase for the synthesis of prebiotic carbohydrates

β-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: β-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of β-galactosidases could be tuned by changing the concentration of galactose donor.     

The characteristics of encapsulated whole cell β-galactosidase

We prepared encapsulated whole cell β-galactosidase using E. coli. The cell culture was divided into two steps for the cell accumulation inside the capsule and enzyme production in the cell. Growth and production media were used individually for this purpose. The dry cell weight of the free cell culture was increased 2.8 times by controlling the pH of the growth medium during cultivation. However, the weight of cells accumulated in the capsule reduced 40% with pH control. The dry cell weight increased with lactose concentration of the production medium for both cases of free and capsule cultures. The dry cell weights were 1.5?g/l for free culture and 100?g/l in the capsule when the lactose concentration of the production medium was 10?g/l. The dry cell weight increased about 60% for both cases as the lactose concentration increased from 10 to 50?g/l. The specific activity of whole cell enzyme decreased with lactose concentration from 5 to 1.4?unit/g dry cell for free culture and from 1.1 to 0.65?unit/g dry cell in the capsule. The value of Michaelis constant, K_m, of whole cell enzyme increased 3 times because of the resistance of mass transfer through the capsule membrane. The constants of Michaelis-Menten equation for the whole cell enzyme in the capsule were V_m: 0.0479?mM/min and K_m: 44.86?mM. These constants of the membrane-free cells were V_m: 0.0464?mM/min and K_m: 15.64?mM. To increase the whole cell enzyme activity, we treated encapsulated cells with organic solvents. The activity of encapsulated whole cell enzyme was increased 3.5 times with the treatment of chloroform and ethanol. The activity of the encapsulated whole cell enzymes was reserved after repeating the process 30 times.     

Galactooligosaccharide synthesis by active β-galactosidase inclusion bodies-containing Escherichia coli cells

In this study, galactooligosaccharide (GOS) was synthesized using active β-galactosidase (beta-gal) inclusion bodies (IBs)- containing Escherichia coli (E. coli) cells. Analysis by MALDI-TOF (matrix-assisted laser desorption/ionizationtime of flight) mass spectrometry revealed that a trisaccharide was the major constituent of the synthesized GOS mixture. Additionally, the optimal pH, lactose concentration, amounts of E. coli β-gal IBs, and temperature for GOS synthesis were 7.5, 500 g/l, 3.2 U/ml, and 37 °C, respectively. The total GOS yield from 500 g/l of lactose under these optimal conditions was about 32%, which corresponded to 160.4 g/l of GOS. Western blot analyses revealed that β-gal IBs were gradually destroyed during the reaction. In addition, when both the reaction mixture and E. coli β-gal hydrolysate were analyzed by high-performance thin-layer chromatography (HP-TLC), the trisaccharide was determined to be galactosyl lactose, indicating that a galactose moiety was most likely transferred to a lactose molecule during GOS synthesis. This GOS synthesis system might be useful for the synthesis of galactosylated drugs, which have recently received significant attention owing to the ability of the galactose molecules to improve the drugs solubility while decreasing their toxicity. β-Gal IB utilization is potentially a more convenient and economic approach to enzymatic GOS synthesis, since no enzyme purification steps after the transgalactosylation reaction would be required.     

Improved octyl glucoside synthesis using immobilized β-glucosidase on PA-M with reduced glucose surplus inhibition

A β-glucosidase extracted from bitter almond (Prunus dulcis var. amara) was immobilized on polyamine microspheres (PA-M) for catalytic octyl glucoside (OG) synthesis from glucose and octanol through reversed hydrolysis. The immobilization increased the activity of enzyme at pH 6.0–7.0, and the optimal reaction temperature for immobilized enzyme was identical to the free enzyme. The thermal stability and solvent tolerance of enzyme were increased by its immobilization. In the co-solvent system using 10% t-butyl alcohol and 10% (v/v) water, the yield of OG was increased by 1.7-fold compared to the yield from the system without co-solvent. Based on dynamic and Dixon plot analyses, the initial reaction velocity (V₀) increased approximately three-fold on immobilization and the OG synthesis was inhibited by surplus glucose. The inhibition dissociation constants for free and immobilized enzyme were 219?mM and 116?mM, respectively. A fed-batch mode was applied in the OG synthesis to minimize substrate inhibition. After 336?h of reaction, the OG yield and the conversion rate of glucose reached 134?mM and 59.6%, respectively. Compared to the batch operation, the fed-bath operation increased the OG yield and the conversion rate of glucose by 340% and 381%, respectively.     

β-Galactosidase production using glycerol and byproducts: Whey and residual glycerin

The present study aimed to evaluate β-galactosidase production by liquid-state fermentation using an experimental design and response surface methodology. A culture medium containing lactose and analytical grade glycerol was formulated to maximize β-galactosidase production. The effects of the pH, lactose, and glycerol concentration on the enzyme production were studied using a Central Composite Design (CCD; 2³ plus central points), followed by a Central Composite Rotatable Design (CCRD; 2³ plus axial and central points). The conditions that maximized β-galactosidase production were: lactose concentration of 20?g?L^?1, glycerol concentration of 60?g?L^?1, and pH 5.0. Under these conditions, the highest enzymatic activity was 40.7?U?mL^?1. Glycerol and lactose were replaced by residual glycerin and whey respectively, according to the best condition obtained in CCRD, reaching enzymatic values of 31.8?U?mL^?1, and thus demonstrating to be great alternative sources for β-galactosidase production.