Immobilization of recombinant thermostable β-galactosidase from Bacillus stearothermophilus for lactose hydrolysis in milk

A recombinant thermostable β-galactosidase from Bacillus stearothermophilus was immobilized onto chitosan using Tris(hydroxymethyl)phosphine (THP) and glutaraldehyde, and a packed bed reactor was utilized to hydrolyze lactose in milk. The thermostability and enzyme activity of THP-immobilized β-galactosidase during storage was superior to that of free and glutaraldehyde-immobilized enzymes. The THP-immobilized β-galactosidase showed greater relative activity in the presence of Ca²⁺ than the free enzyme and was stable during the storage at 4°C for 6 wk, whereas the free enzyme lost 31% of the initial activity under the same storage conditions. More than 80% of lactose hydrolysis in milk was achieved after 2 h of operation in the reactor. Therefore, THP-immobilized recombinant thermostable β-galactosidase from Bacillus stearothermophilus has the potential for application in the production of lactose-hydrolyzed milk.     

Prebiotic effects of structurally identified galacto-oligosaccharides produced by β-galactosidase from Aspergillus oryzae

Novel galacto-oligosaccharides were produced by β-galactosidase from Aspergillus oryzae using lactose, and their structural characteristics and prebiotic effects were examined. Highly purified oligosaccharide fraction (HP) was prepared from a crude one (low purified, LP) by gel-filtration on Biogel P-2 column, which was further purified into S1 and S2 fractions by prep-HPLC. S1 and S2 were comprised of galactose (Gal) and glucose (Glc) in the ratio of 2 to 1. ESI-MS-MS and methylation analysis indicated that S1 and S2 were trisaccharides with structures of β-d-Galp-(1,6)-β-d-Galp-(1,4)-β-d-Glcp and β-d-Galp-(1,3)-β-d-Galp-(1,4)-β-d-Glcp, respectively. Herein, LP and HP were used as the carbon sources for determining the prebiotic activity score of probiotics including Lactobacillus and Bifidobacterium species. LP and HP at 1% and 2% (w/v) were observed at all positive scores on several probiotics, especially, B. infantis ATCC 15697 at the 2% level of HP (p<0.05). Consequently, structurally identified trisaccharides of HP can significantly enhance the growth of B. infantis.     

A novel thermostable β-galactosidase from Bacillus coagulans with excellent hydrolysis ability for lactose in whey

β-Galactosidase is one of the most important enzymes used in dairy industry. Here, a novel thermostable β-galactosidase was cloned and overexpressed from Bacillus coagulans NL01 in Escherichia coli. The phylogenetic trees were constructed using neighbor-joining methods. Phylogeny and amino acid analysis indicated that this enzyme belonged to family 42 of glycoside hydrolases. The optimal pH and temperature were, respectively, 6.0 and 55 to 60°C. The purified enzyme had a 3.5-h half-life at 60°C. Enzyme activity was enhanced by Mn²⁺. Compared with other β-galactosidases from glycoside hydrolase family 42, B. coagulans β-galactosidase exhibited excellent hydrolysis activity. The Michaelis constant (K_m) and maximum rate of enzymatic reaction (V_max) values for p-nitrophenyl-β-d-galactopyranoside and o-nitrophenyl-β-d-galactopyranoside were 1.06 mM, 19,383.60 U/mg, and 2.73 mM, 5,978.00 U/mg, respectively. More importantly, the enzyme showed lactose hydrolysis ability superior to that of the commercial enzyme. The specific enzyme activity for lactose was 27.18 U/mg. A total of 104.02 g/L lactose in whey was completely hydrolyzed in 3 h with addition of 2.38 mg of pure enzyme per gram of lactose. In view of the high price of commercial β-galactosidase, B. coagulans β-galactosidase could be a promising prototype for development of commercial enzymes aimed at lactose treatment in the dairy industry.     

Characterisation of a thermostable family 42 β-galactosidase (BgalC) family from Thermotoga maritima showing efficient lactose hydrolysis

A β-galactosidase gene (TM_1195) of Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant β-galactosidase (BgalC), belonging to glycosyl hydrolase (GH) family 42, was purified to homogeneity with 23.4-fold purification and a recovery of 36.6%. Its molecular mass was estimated to be 78 kDa by SDS–PAGE. BgalC exhibited maximum activity at an optimal pH of 5.5 and an optimum temperature of 80 °C. The enzyme displayed important properties, such as stability over a broad pH range of 5.0–9.0 and thermostability up to 75 °C. K_m values of BgalC for p-nitrophenyl-β-galactopyranoside (pNPGal), o-nitrophenyl-β-galactopyranoside (oNPGal) and lactose were 1.21, 7.31 and 6.5 mM, respectively. BgalC was efficient in complete removal of lactose from milk. BgalC is significantly one of the few β-galactosidases from family 42 displaying significant hydrolysis of lactose. These properties make BgalC an ideal candidate for commercial use, in the production of lactose-free milk.     

Immobilization of Aspergillus oryzae with κ-carrageenan for soybean oligosaccharide hydrolysis

The immobilized Aspergillus oryzae spores in κ-carrageenan were used for production of α-galactosidase. The immobilized cells could be used up to 5 repeated cycles for enzyme production. They were employed for raffinose family oligosaccharides (RFO) hydrolysis in batch, repeated batch, and continuous operations after 5 days of fermentation. In batch mode, 65, 73, and 80% of RFOs were hydrolyzed after 3, 6, and 12 h, respectively; in repeated batch; 70, 63, 52, and 45% were hydrolyzed in 1, 2, 3, and 4 repeated cycles. In the fluidized bed reactor, 65, 58, 53, 48, and 44% RFOs were hydrolyzed at flow rates of 25, 50, 75, 100, and 125 mL/h respectively. The κ-carrageenan beads maintain good mechanical strength up to 4 repeated uses for RFO hydrolysis in soymilk, and their use in the hydrolysis of RFOs of soybean is a promising solution to overcome flatulence and to increase consumption of soy products.     

Enhancement of the hydrolysis activity of β-galactosidase from Geobacillus stearothermophilus by saturation mutagenesis

Thermostable β-galactosidase (BgaB) from Geobacillus stearothermophilus is characterized by its thermoactivity in the hydrolysis of lactose to produce lactose-free milk products. However, BgaB has limited activity toward lactose. We established a method for screening evolved mutants with high hydrolysis activity based on prediction of substrate binding sites. Seven amino acid residues were identified as candidates for substrate binding to galactose. To study the hydrolysis activity of these residues, we constructed mutants by site-saturation mutagenesis of these residue sites, and each variant was screened for its hydrolysis activity. The first round of mutagenesis showed that changes in amino acid residues of Arg109, Tyr272, and Glu351 resulted in altered hydrolysis activity, including greater activity toward ortho-nitrophenyl-β-d-galactopyranoside (oNPG). The mutants R109V and R109L displayed changes in the optimum pH from 7.0 to 6.5, and the mutant R109V/L displayed different substrate affinity and catalytic efficiency (k_cat/K_m). Mutant R109G showed complete loss of BgaB enzymatic activity, suggesting that Arg109 plays a significant role in maintaining hydrolysis activity. The optimum pH of mutant E351R increased from 7.0 to 7.5 and this mutant showed a prominent increase in catalytic efficiency with oNPG and lactose as substrates.     

Production of β-galactosidase for lactose hydrolysis in milk and dairy products using thermophilic lactic acid bacteria

The aim of this work was to establish optimal conditions for the maximum production of β-galactosidase using an industrially suitable medium. Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was cultivated in skim milk, whey and whey permeate basal media, supplemented with whey protein products, yeast extract or De Man–Rogosa–Sharpe (MRS) broth, at pH 5.6 and 43°C. All supplementations of the whey and whey permeate basal media resulted in the enhancement of the specific growth rates, rate of lactic acid production and β-galactosidase activity. However, unsupplemented skim milk gave the greatest rate of lactic acid production (3.50±0.269 mg lactic acid ml⁻¹ media h⁻¹) and the highest β-galactosidase activity (5.491±0.116 U activity ml⁻¹ media); far superior to the best whey-based medium supplemented with MRS (2.71±0.176 mg lactic acid ml⁻¹ media h⁻¹ and 3.091±0.089 U activity ml⁻¹ media, respectively). A technologically feasible approach for the reprocessing of the spent skim milk was tested and a conceptual process scheme is proposed.     

Engineering of a β-galactosidase from Bacillus coagulans to relieve product inhibition and improve hydrolysis performance

Most β-galactosidases reported are sensitive to the end product (galactose), making it the rate-limiting component for the efficient degradation of lactose through the enzymatic route. Therefore, there is ongoing interest in searching for galactose-tolerant β-galactosidases. In the present study, the predicted galactose-binding residues of β-galactosidase from Bacillus coagulans, which were determined by molecular docking, were selected for alanine substitution. The asparagine residue at position 148 (N148) is correlated with the reduction of galactose inhibition. Saturation mutations revealed that the N148C, N148D, N148S, and N148G mutants exhibited weaker galactose inhibition effects. The N148D mutant was used for lactose hydrolysis and exhibited a higher hydrolytic rate. Molecular dynamics revealed that the root mean square deviation and gyration radius of the N148D-galactose complex were higher than those of wild-type enzyme-galactose complex. In addition, the N148D mutant had a higher absolute binding free-energy value. All these factors may lead to a lower affinity between galactose and the mutant enzyme. The use of mutant enzyme may have potential value in lactose hydrolysis.     

Galacto-oligosaccharide synthesis using chemically modified β-galactosidase from Aspergillus oryzae immobilised onto macroporous amino resin

The goal of this study was to establish an efficient immobilisation protocol for β-galactosidase from Aspergillus oryzae onto the polystyrenic macroporous resin Purolite^® A-109 for better utilisation of its transglactosylation activity and application in galacto-oligosaccharide (GOS) synthesis. This was achieved by improving simple ionic adsorption by carboxyl group activation on the enzyme surface with carbodiimide, enabling covalent immobilisation. This yielded significantly increased operational stability, assayed as GOS synthesis, in a batch reactor, and even more prominently, in a fluidised bed reactor (73% activity retained after 10 cycles). The immobilised enzyme showed two very beneficial advantages over the free enzyme for future applications: higher affinity towards catalysing transgalactosylation than towards hydrolysis and shift of pH optimum towards more acidic conditions. GOS synthesis performed under the optimum conditions obtained (400 g L⁻¹ lactose, pH 4.5, 50 °C) yielded 87 g L⁻¹ and 100 g L⁻¹ for batch and fluidised bed reactors, respectively.     

Optimisation of the synthesis of high galacto-oligosaccharides (GOS) from lactose with β-galactosidase from Kluyveromyces lactis

A rational optimisation for the synthesis of galacto-oligosaccharides (GOS) catalysed by a commercial β-galactosidase from Kluyveromyces lactis, Lactozym Pure 6500 L, is shown. The study of the main reaction parameters – temperature, enzyme concentration, pH, initial lactose concentration and reaction time – using surface response methodology, was demonstrated to be an accurate tool to optimise empirical production of the most desired galacto-oligosaccharides (tri-GOS and tetra-GOS) with a higher presumed prebiotic effect. The optimal value for the yield towards these high-GOS predicted by the model was 12.18% at 40 °C, 5 U mL⁻¹ enzyme concentration, pH 7.0, 250 g L⁻¹ initial lactose concentration and 3 h of reaction. The pH was found to be a critical parameter affecting the transgalactosylation/hydrolysis enzyme activity ratio, and was used to tune the relative production of tri- or tetra-GOS.     

Batch and continuous synthesis of lactulose from whey lactose by immobilized β-galactosidase

In this study, lactulose synthesis from whey lactose was investigated in batch and continuous systems using immobilized β-galactosidase. In the batch system, the optimal concentration of fructose for lactulose synthesis was 20%, and the effect of galactose, glucose and fructose on β-galactosidase activity was determined for hydrolysis of whey lactose and the transgalactosylation reaction for lactulose synthesis. Galactose and fructose were competitive inhibitors, and glucose acted as a noncompetitive inhibitor. The inhibitory effects of galactose and glucose were stronger in the transgalactosylation reaction than they were in the hydrolysis reaction. In addition, when immobilized β-galactosidase was reused for lactulose synthesis, its catalytic activity was retained to the extent of 52.9% after 10 reuses. Lactulose was synthesized continuously in a packed-bed reactor. We synthesized 19.1 g/l lactulose during the continuous flow reaction at a flow rate of 0.5 ml/min.     

Optimization of conditions for galactooligosaccharide synthesis during lactose hydrolysis by β-galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP G)

A study on optimisation of the conditions for galactooligosaccharide (GOS) formation during lactose hydrolysis, produced by Lactozym 3000 L HP G, was carried out. The synthesis was performed during times up to 300 min at 40, 50 and 60 °C, pH 5.5, 6.5 and 7.5, lactose concentration 150, 250 and 350 mg/mL and enzyme concentration 3, 6 and 9 U/mL. The product mixtures were analysed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). During the hydrolysis of lactose, besides glucose and galactose, galactobiose, allolactose and 6′ galactosyl lactose were also formed as a result of transgalactosylation catalysed by the enzyme. The effect of the reaction conditions was different in the formation of di- and the trisaccharide. Thus, the optimal conditions for galactobiose and allolactose synthesis were 50 °C, pH 6.5, 250 mg/mL of lactose, 3 U/mL of enzyme and 300 min, whereas the best reaction conditions for 6′ galactosyl lactose production were 40 °C, pH 7.5, 250 mg/mL of lactose, 3 U/mL of enzyme and 120 min. These results show the possibility to obtain reaction mixtures with Lactozym 3000 L HP G, with different composition, depending on the assayed conditions.     

Assessment of repetitive batch-wise synthesis of galacto-oligosaccharides from lactose slurry using immobilised β-galactosidase from Bacillus circulans

The synthesis of galacto-oligosaccharides (GOS) using covalently immobilised β-galactosidase from Bacillus circulans was carried out in a lactose slurry rather than in solution. Repeated batch-wise synthesis at 58 °C and 55% (w/w) lactose could be carried out for at least 15 successive runs. All 15 runs were completed within 4 h of reaction time. The reduction of heat exposure due to these short incubation times contributed to the retention of 60% of the initial activity. The product properties of the GOS mixture obtained with the immobilised biocatalyst were compared with the free enzyme product. Oligosaccharide yields and product pattern were highly similar as was the degree of polymerisation. The productivity of the immobilised enzyme system was compared to the free enzyme. The enzymatic productivity (i.e., g GOS g⁻¹ enzyme) after 15 consecutive runs was 165% higher for the immobilised enzyme than for the free enzyme.     

Lactose hydrolysis in milk as affected by neutralizers used for the preparation of crude β-galactosidase extracts from Lactobacillus bulgaricus 11842

Three different neutralizers (NaOH, KOH, NH₄OH) were employed for pH maintenance during the growth of Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, used as a source of β-galactosidase extracts. The crude enzymatic extract (CEE) was obtained by bead milling of the cell paste, collected from the cultivation of the source microorganism in skim milk at 43 °C and constant pH. Lactose hydrolysis kinetics in skim milk and proteolytic activity during the hydrolysis were evaluated. The use of NH₄OH as a neutralizer resulted in significantly (P<0.05) higher enzyme activity of the CEE than that obtained using NaOH or KOH. The kinetic parameters, k_cat and K_m, of the Michaelis–Menten model were determined for lactose hydrolysis in skim milk using 1% (v/v) addition of a CEE. There was no significant (P>0.05) difference in k_cat among the different extracts, with a clear temperature dependence following Arrhenius kinetics. The rate of lactose hydrolysis was dependent on the initial enzyme activity and temperature. The highest initial rate was observed at 65 °C; however, the enzyme deactivation occurred within 1–1.5 h. The proteolytic activity determined by HPLC peptide mapping was significantly (P<0.05) higher in the moderate temperature range (20 and 37 °C) than at 7 or 55 °C. Industrial relevance: Since lactose intolerance affects a large proportion of the world's population, an economically feasible and effective process with a cheap source of β-galactosidase may have a substantial potential. The use of crude β-galactosidase extracts from Lactobacillus bulgaricus 11842 appears to be a promising approach for development of a technologically feasible process of lactose hydrolysis for food or non-food uses.     

Enzymatic synthesis and identification of oligosaccharides obtained by transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis

The enzymatic transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis (KlβGal) leading to the formation of oligosaccharides was investigated in detail. The reaction mixture was analyzed by high performance liquid chromatography with differential refraction detector (HPLC-RI) and two main transgalactosylation products were discovered. To elucidate their overall structures, the products were isolated and purified using preparative liquid chromatography and analyzed by LC/MS, one-dimensional (1D) and two-dimensional (2D) NMR studies. Allo-lactulose(β-d-galactopyranosyl-(1→1)-d-fructose) with two main isomers in D(2)O was identified to be the major transgalactosylation product while lactulose(β-d-galactopyranosyl-(1→4)-d-fructose) turned out to be the minor one, indicating that KlβGal was regioselective with respect to the primary C-1 hydroxyl group of fructose. The maximum yields of allo-lactulose and lactulose were 47.5 and 15.4g/l, respectively, at 66.5% lactose conversion (200g/l initial lactose concentration).     

Kinetics and design relation for enzymatic conversion of lactose into galacto-oligosaccharides using commercial grade β-galactosidase

The enzymatic synthesis of galacto-oligosaccharides (GOS) from lactose was studied using commercial grade β-galactosidase (Biolacta FN5) from Bacillus circulans. The reaction was carried out under free enzyme condition varying initial lactose concentration (ILC: 55-525?g/L), enzyme concentration (0.05-1.575?g/L), temperature (30-50°C) and pH (5.0-6.0). Reaction mixture compositions were analyzed utilizing high performance liquid chromatography (HPLC). A?maximum GOS formation of 39% (dry basis) was achieved at an ILC of 525?g/L converting 60% of the lactose fed. Tri-saccharides were the major types of GOS formed, accounting approximately 24%; whereas, tetra-saccharides and penta-saccharides account approximately 12% and 3%, respectively. Design correlation was developed in order to observe the quantitative effect of operating parameters on GOS yield. Further, based on Michaelis-Menten model, four-step reaction pathways were considered for simplistic understanding of the kinetics. Apart from predicting the reaction mixture composition, the approach also provided kinetic parameters though simulation using COPASI 4.7(?). Excellent agreements were observed between simulated and experimental results.     

Site-directed mutation of β-galactosidase from Streptococcus thermophilus for galactooligosaccharide-enriched yogurt making

β-Galactosidase is one of the most important enzymes used in dairy processing. It converts lactose into glucose and galactose, and also catalyzes galactose to form galactooligosaccharides (GOS), so-called prebiotics. However, most of the β-galactosidases from the starter cultures have low transgalactosylation activities, the process that results in galactose accumulation in yogurt. Here, a site-directed mutation strategy was attempted, to genetically modify β-galactosidase from Streptococcus thermophilus. Out of 28 Strep. thermophilus strains, a β-galactosidase gene named bgaQ, encoded for high β-galactosidase hydrolysis activity (BgaQ), was cloned from the strain Strep. thermophilus SDMCC050237. It was 3,081 bp in size, with 1,027 deduced amino acid residuals, which belonged to the GH2 family. After replacing the Tyr⁸⁰¹ and Pro⁸⁰² around the active sites of BgaQ with His⁸⁰¹ and Gly⁸⁰², the GOS synthesis of the generated mutant protein BgaQ-8012 increased from 20.5% to 26.7% at 5% lactose, and no hydrolysis activity altered obviously. Subsequently, the purified BgaQ or BgaQ-8012 was added to sterilized milk inoculated with 2 starters from Strep. thermophilus SDMCC050237 and Lactobacillus delbrueckii ssp. bulgaricus ATCC11842. The GOS yields with added BgaQ or BgaQ-8012 increased to 5.8 and 8.3 g/L, respectively, compared with a yield of 3.7 g/L without enzymes added. Meanwhile, the addition of the BgaQ or BgaQ-8012 reduced the lactose content by 49.3% and 54.4% in the fermented yogurt and shortened the curd time. Therefore, this study provided a site-directed mutation strategy for improvement of the transgalactosylation activity of β-galactosidase from Strep. thermophilus for GOS-enriched yogurt making.     

Performance of β-galactosidase pretreated with lactose to prevent activity loss during the enzyme immobilisation process

In this study, Kluyveromyces lactis β-galactosidase was pretreated with lactose to prevent loss of activity during the immobilisation process, and glutaraldehyde was used as a linker to immobilise β-galactosidase on the surface of a silica gel. The pretreatment of β-galactosidase strongly improved its activity after immobilisation. Specifically, the activity of pretreated immobilised β-galactosidase was 2.6 times greater than that of non-pretreated immobilised β-galactosidase. The optimal temperature, pH and ionic strength of buffer for pretreated immobilised β-galactosidase were 37 °C, pH 7.5 and 20 mM potassium phosphate buffer, respectively. These values were shifted by 5 °C and pH by 0.5 when compared to the soluble β-galactosidase. Moreover, the pretreated immobilised β-galactosidase showed a better reusability than did non-pretreated immobilised β-galactosidase, with 63.9% of its original activity being retained after 10 reuses.