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).     

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.     

Improved incorporation and stabilisation of β-carotene in hydrocolloids using glycerol

In this paper, a novel method for the incorporation of the antioxidant β-carotene in hydrocolloid matrices is presented, which consists on the use of glycerol as the vehicle for incorporation. This alcohol has been observed to greatly photostabilise the carotenoid molecule within the hydrocolloid matrices without greatly affecting the mechanical properties of the materials. The UV stability of β-carotene in the different hydrocolloid films developed (including starch, soy protein, whey protein concentrate, gelatin and zein) has been studied using UV/visible spectrophotometry, colorimetry and Raman spectroscopy. Raman images of the materials were also generated in order to ascertain the dispersion of the antioxidant within the hydrocolloid materials and it was observed that β-carotene was partially agglomerated in certain areas of the film, fact that could also contribute to enhance the stability of the bioactive molecule.     

Cloning and expression of β-glucosidases from Bifidobacterium lactis AD011

For the conversion of β-glycosides, 3 genes (BLA_0039, BLA_0141, and BLA_0893) annotated as β-glucosidase (E.C 3.2.1.21) from the genome of Bifidobacterium lactis AD011 were cloned and expressed both in Escherichia coli DH5α and Bifidobacterium bifidum BGN4 using previously established bifidobacterial expression system under the control of 16S rRNA promoter. Deduced amino acid sequence analysis revealed that BLA_0141 is highly similar to β-d-glucosidase from Bifidobacterium breve clb and is included in glycosyl hydrolase family 1 (GHF1) while BLA_0893 belongs to glycosyl hydrolase family 3 (GHF3). Recombinant BLA_0893 showed β-glucosidase activity and converted ginsenoside Rb1 to Rd. Recombinant BLA_0141 showed broad range of activities including β-glucosidase, β-galactosidase, β-xylosidase, cellobiosidase, and α-arabinopyranosidase and converted ginsenoside Rb1 and Rb2 to Rd. They also hydrolized cellobiose into glucose. The recombinant enzyme activities of both BLA_0141 and BLA_0893 were stable around pH 4.5–7.5 and below 50°C.     

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.     

Whey valorization using transgalactosylation activity of immobilized β-galactosidase

Whey represents a significant dairy industry by-product that has recently received due attention based on the rich nutrient composition and significant transformation potential. Hereby, we investigated a possibility of whey lactose bioconversion into prebiotic compounds, galacto-oligosaccharides (GOS) using β-galactosidase transgalactosylation activity. The results showed that whey could be successfully used for GOS synthesis, since the highest GOS concentration (around 62 g L⁻¹) was obtained batchwise using 40% (w/w) sweet whey powder solution under optimum conditions (50°C, pH 4.5). Nevertheless, an efficient immobilized preparation using methacrylic Lifetech ECR8409 immobilization carrier was developed, enabling additional process improvement and ensuring at least 4 reaction cycles with unchanged yields and 2.5- fold enhanced productivity in comparison to the soluble enzyme. Therefore, this study provides a valuable contribution to the efficient and economical valorization of whey, which can be further on utilized as functional food and feed constituent.     

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.     

Cloning,expression, and bioinformatics analysis and characterization of a β-galactosidase from Bacillus coagulans T242

The activities of β-galactosidases from bacteria and molds are affected by temperature, pH, and other factors in the processing of dairy products, limiting their application, so it is necessary to find alternative lactases. In this study, the β-galactosidase gene from Bacillus coagulans T242 was cloned, co-expressed with a molecular chaperone in Escherichia coli BL21, and subjected to bioinformatic and kinetic analyses and lactase characterization. The results show that the enzyme is a novel thermostable neutral lactase with optimum hydrolytic activity at pH 6.8 and 50°C. The thermal stability and increased lactose hydrolysis activity of β-galactosidase in the presence of Ca²⁺ indicated its potential application in the dairy industry.     

Heterologous expression and stability of the Escherichia coli β-galactosidase gene in Streptococcus lactis by translation fusion

An Escherichia coli/Streptococcus lactis shuttle vector for creating translation fusions to the E. coli galactosidase (lacZ) has been constructed. Random S. lactis chromosomal DNA fragments inserted upstream of the lacZ gene and in frame promote the expression of active galactosidase in both E. coli and S. lactis. One fusion pSK216 has been characterized by Western blotting to reveal a fusion protein of 116 kd. S. lactis LM0230, a plasmidless lac⁻ derivative of S. lactis C2 when carrying pSK216, exhibits a Lac⁺ phenotype suggesting the presence of a chromosomally encoded lactose permease. Further evidence for this lactose permease system is provided by assay of lactose uptake by S. lactis LM0230. In the absence of selection pSK216 was less stable in S. lactis LM0230 than its derivative pSK41. The growth rate of S. lactis LM0230 carrying pSK216 is slower in comparison to the plasmid-free strain. Two genetic events were observed, the deletion of the lacZ gene and the loss of the entire plasmid. These results indicate that recombinant plasmids are unstable in S. lactis and imply that there is an obstacle in the genetic engineering of lactic acid bacteria.     

Characterisation of a thermostable family 42 β-galactosidase from Thermotoga maritima

A β-galactosidase gene (TM_0310) of Thermotoga maritima MSB8 was expressed in Escherichia coli. The recombinant β-galactosidase (designated BgalB) was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. BgalB belongs to the glycoside hydrolase family 42. Its molecular mass was estimated to be 78 kDa and 76 kDa by SDS–PAGE and gel filtration, respectively. The enzyme was optimum at pH 5.5, and it was quite stable over the pH range 5.0–11.4 at 70 °C. It was optimally active at 80 °C and was stable up to 75 °C. Besides, BgalB exhibited broad substrate specificity with a preference for p-nitrophenyl-β-galactopyranoside (pNPGal). K_m values of the purified enzyme for pNPGal, o-nitrophenyl-β-galactopyranoside (oNPGal) and pNP-β-fucopyranoside were 2.7 mM, 12.5 mM and 1.4 mM, respectively. These properties make this enzyme an interesting candidate for biotechnological applications. This is the first report of the family 42 β-galactosidases from T. maritima.     

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.     

Isolation and some properties of β-galactosidase from the thermophilic bacterium Thermus thermophilus

The highest β-galactosidase activity in Thermus thermophilus cells was achieved after 40 h of cultivation at 70°C in a medium containing 0.8% peptone, 0.4% yeast extract and 0.2% NaCl. After the addition to the medium of 2% of lactose or galactose, enzyme synthesis in the cells increased by about 33% and 61% of its constitutive value. The thermostable β-galactosidase isolation and partial purification involved extraction in 0.01 m phosphate buffer at pH 7.2, ammonium sulphate precipitation, dialysis and gel filtration on Sephadex G-200 or lyophilization. The highest recovery yield of activity during the isolation process was about 72% of its total value in the extract. The gel filtration step resulted in an increase of β-galactosidase specific activity of two-fold more than the ammonium sulphate precipitate. The crude enzyme obtained exhibited highest activity at pH 6.6 and 87°C. Decimal reduction times of activity at 75, 80, 85, 90 and 95°C were 174, 50, 22, 5.5 and 0.9 h, respectively..     

Production of galactooligosaccharides using a hyperthermophilic β-galactosidase in permeabilized whole cells of Lactococcus lactis

Galactooligosaccharides (GOS) are novel prebiotic food ingredients that can be produced from lactose using β-galactosidase, but the process is more efficient at higher temperatures. To efficiently express the lacS gene from the hyperthermophile Sulfolobus solfataricus, in Lactococcus lactis a synthetic gene (lacSt) with optimized codon usage for Lc. lactis was designed and synthesized. This hyperthermostable β-galactosidase enzyme was successfully overexpressed in Lc. lactis LM0230 using a nisin-controlled gene expression system. Enzyme-containing cells were then killed and permeabilized using 50% ethanol and were used to determine both hydrolysis and transgalactosylation activity. The optimum conditions for GOS synthesis was found to be at pH 6.0 and 85°C. A maximum production of 197 g/L of GOS tri- and tetrasaccharides was obtained from 40% initial lactose, after 55 h of incubation. The total GOS yield increased with the initial lactose concentration, whereas the highest lactose conversion rate (72%) was achieved from a low lactose solution (5%). Given that a significant proportion of the remaining lactose would be expected to be converted into disaccharide GOS, this should enable the future development of a cost-effective approach for the conversion of whey-based substrates into GOS-enriched food ingredients using this cell-based technology.     

Overexpression and characterization of a novel GH4 galactosidase with β-galactosidase activity from Bacillus velezensis SW5

A novel galactosidase gene (gal3149) was identified from Bacillus velezensis SW5 and heterologously expressed in Escherichia coli BL21 (DE3). The novel galactosidase, Gal3149, encoded by gal3149 in an open reading frame of 1,299 bp, was 433 amino acids in length. Protein sequence analysis showed that Gal3149 belonged to family 4 of glycoside hydrolases (GH4). Gal3149 displayed higher enzyme activity for the substrate 2-nitrophenyl-β-d-galactopyranoside (oNPG) than for 4-nitrophenyl-α-d-galactopyranoside (pNPαG). This is the first time that an enzyme belonging to GH4 has been shown to exhibit β-galactosidase activity. Gal3149 showed optimal activity at pH 8.0 and 50°C, and exhibited excellent thermal stability, with retention of 50% relative activity after incubation at a temperature range of 0 to 50°C for 48 h. Gal3149 activity was significantly improved by K⁺ and Na⁺, and was strongly or completely inhibited by Ag⁺, Zn²⁺, Tween-80, Cu²⁺, carboxymethyl cellulose, and oleic acid. The rate of hydrolyzed lactose in 1 mL of milk by 1 U of Gal3149 reached about 50% after incubation for 4 h. These properties lay a solid foundation for Gal3149 in application of the lactose-reduced dairy industry.     

Some characteristics and isolation of novel thermostable β-galactosidase from Thermus oshimai DSM 12092

The β-galactosidases belong to the class of hydrolytic enzymes and have been used as lactose hyrolysis. The enzyme is used in reducing lactose milk production, an outstanding industrial product used by a large lactoseintolerant population. This is the first detailed report of some characteristics of β-galactosidase and the gene encoding β-galactosidase in Thermus oshimai DSM 12092. The growth rate (μ, 1/h), and the doubling time (t_D, h) for T. oshimai grown both in shaking flasks and in a bioreactor were determined. The optimal temperature and pH for β-galactosidase were determined as 75°C and 7.4, respectively. This enzyme was thermostable and was retained by more than 70% at 90°C for 3 h. The β-galactosidase from T. oshimai DSM 12092 was more stable in basic pH and Zn²⁺ was the most effective divalent cation. Also, 2 steps of purification consisting of ammonium sulfate precipitation and gel filtration were employed and purified 32-fold.     

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.     

Extraction of phenolic compounds from grapes and their pomace using β-cyclodextrin

Use of aqueous cyclodextrins (CD) for recovery of selected bioactive phenolic compounds from grapes and their pomace was evaluated. When α, β and γ forms of CD were compared, β-CD was the most effective in recovering stilbenes, flavonols, and flavan-3-ols from grape pomace. The maximum quantified phenolics were obtained from the powder and the slurry of grape pomace when extracted with 2.5% (w/v) aqueous β-CD solutions at 60 °C for 12–24 h. With β-CD, total quantified phenolics obtained from the dry powder were 123 mg/100 g (DW) while from the slurry, they were 35.8 mg/100 g (FW). Incorporation of β-CD to grape mash prior to pressing for juice enhanced the recovery of phenolics in juice. Incorporation of β-CD was more effective in recovering flavan-3-ols than flavonols. Aqueous CD can effectively be used in recovering phenolics from by-products of fruit processing and therefore for functional foods and nutraceutical applications.     

Drying and storage of crude β-galactosidase extracts from Lactobacillus delbrueckii ssp. bulgaricus 11842

The retention of β-galactosidase activity in crude cellular extract (CCE) preparations from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was investigated after spray drying at three different outlet air temperatures (40, 50 or 60 °C), freeze drying, and after 30 days storage. Lactose, skim milk and whey protein preparations in concentrations ranging from 5 to 30% (w/w) were used as drying adjuncts. To further investigate the protective role of sugars in the enzyme activity preservation, cellobiose and sucrose were also employed in 5 and 10% concentrations during spray-drying at 60 °C or freeze-drying. The addition of lactose or skim milk in all examined concentrations resulted in significantly (P<0.05) higher β-galactosidase activity retention in comparison to all other CCE spray dried at 60°C. The effect was less pronounced at lower spray drying temperatures and increased whey protein concentrations, especially during freeze drying, when almost complete recovery of the enzyme activity upon reconstitution was achieved. Cellobiose provided less β-gal protection in comparison to lactose or sucrose. Lactose was more effective than sucrose at 5% concentration, but both sugars were equal at 10%. The β-gal activity retention in dry CCE preparations during storage at 7 °C over 30 day period was related to the initial water activity; higher initial a_w of powders obtained at lower spray drying temperature was correlated with significant (P<0.05) β-gal activity loss. Freeze dried and spray dried (60 °C) preparations were more stable in comparison to all other samples, retaining high β-gal activity during storage up to 30 days.