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College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo 315211, People's Republic of ChinaInstitute of Quality Standards and Testing Technology for Agro-Products, Fujian Academy of Agricultural Sciences, Fuzhou 350003, People's Republic of China
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.
Galactosidases, including α-galactosidase (EC 3.2.1.22) and β-galactosidase (EC 3.2.1.23), are complex carbohydrate-active enzymes with extensive applications in food, pharma, and biotechnology industries (
). The former can hydrolyze lactose by attacking the o-glucosyl bond; the latter can transfer the cleaved galactosyl from one lactose onto another molecule, such as lactose or galactose (
β-Galactosidase can reduce the lactose content in milk and produce low-sugar products that are more likely to be consumed by lactose-intolerant people (
), which show multiple beneficial effects such as enhancing the growth of bifidobacteria and lactobacilli in the intestinal tract, reducing the growth of pathogenic microorganisms, and improving mineral absorption (
A multifunctional thermophilic glycoside hydrolase from Caldicellulosiruptor owensensis with potential applications in production of biofuels and biochemicals.
Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions.
). Previous studies showed that β-galactosidase derived from microorganisms exhibits the capacity of transglycosylation and hydrolysis of lactose in milk and fresh whey (
Heterologous expression is an important approach to investigate gene function and interactions. Most reported β-galactosidases are recombinant enzymes from different expression systems. In recent years, β-galactosidases from Bacillus licheniformis (
A novel cold-active β-d-galactosidase with transglycosylation activity from the Antarctic Arthrobacter sp. 32cB—Gene cloning, purification and characterization.
) have been successfully expressed in Escherichia coli. Bacillus velezensis SW5 with the ability to produce galactosidase was isolated from fish sauce in our laboratory. As reported previously, a cold-adapted phospho-β-galactosidase from B. velezensis SW5 was expressed in E. coli and characterized (
). In the current study, a novel galactosidase belonging to GH4 with the capacity of lactose hydrolysis and generating GOS was found. To explore enzymatic and functional properties, it was cloned and expressed in E. coli BL21(DE3) using the pET-his vector. The effects of different factors such as temperature, pH, metal ions, and reagents on recombinant Gal3149 activity and its kinetic parameters [half-saturation coefficient (Km), maximum velocity (Vmax), and turnover number (kcat)] were investigated. Moreover, the ability of Gal3149 for lactose hydrolysis in milk and the composition of hydrolysis products were also analyzed. These properties lay a solid foundation for Gal3149 in application of the lactose-reduced dairy industry.
MATERIALS AND METHODS
Materials
We purchased 2-nitrophenyl-β-d-galactopyranoside (oNPG), 4-nitrophenyl-β-d-galactopyranoside (pNPG), 4-nitrophenyl-α-d-galactopyranoside (pNPαG), 2-nitrophenyl-β-d-glucopyranoside (oNPGlu), 4-nitrophenyl-β-d-glucopyranoside (pNPGlu), 4-nitrophenyl-α-d-glucopyranoside (pNPαGlu), and 4-nitrophenyl-β-d-xylopyranoside (pNPXyl) from Sigma-Aldrich. Restriction endonucleases, T4 DNA ligase, and corresponding buffers were purchased from New England Biolabs. Bacterial DNA Extraction Kit, Plasmid Mini Extraction Kit, and Gel Extraction and Purification Kit were purchased from TaKaRa Bio Inc. All other reagents were of analytical grade unless otherwise stated.
Bacterial Strains, Plasmids, and Culture Conditions
The strain of B. velezensis SW5 was isolated from fish sauce in our previous experiment and deposited in the China General Microbiological Culture Collection Center (Accession No. 1.16723). Escherichia coli DH5α and E. coli BL21 (DE3) were purchased from TransGen Biotech and were used as the cloning strain and the expression strain, respectively. The pET-his vector with T7 promoter was purchased from Biofeng Company. The strains used in this study were cultivated on Luria-Bertani (LB) medium (15 g/L agar for solid) at 37°C. Strains harboring the vector were grown in LB medium with ampicillin at the final concentration of 100 μg/mL.
Molecular Cloning and Plasmid Construction
The genomic DNA of B. velezensis SW5 was extracted using the Bacterial DNA Extraction Kit according to the manufacturer's instructions. A pair of specific primers, the forward primer F: 5′-CGCGCGGATCCTTGAAGAAAATTACGTTTA-3′ and the reverse primer R: 5′-CTAGCTAGCTTAGTGATAAGCCGGAAGC-3′ with BamHI and NheI restriction sites (underlined), were designed according to DNAMAN software 7.0 (Lynnon Biosoft) to amplify the complete open reading frame of the gal3149 gene. After initial denaturation at 95°C for 5 min, the mixture was subjected to 30 cycles, each consisting of 30-s denaturation at 95°C, 30-s annealing at 55°C, and 90-s extension at 72°C, followed by 10 min of final extension at 72°C to complete the elongation. The PCR products were resolved by 1% agarose nucleic acid electrophoresis, and the target fragment was recovered using the Gel Extraction and Purification Kit and subjected to DNA sequencing by the Shanghai Sangon Biotech Company. After sequencing, the recovered fragment was digested by BamHI and NheI and ligated into pET-his vector digested by the same restriction enzymes. They were connected at 16°C overnight and transformed into E. coli DH5α, yielding the recombinant plasmid pET-Gal3149. After conformation by DNA sequencing, pET-Gal3149 was transformed into E. coli BL21 (DE3).
Expression and Purification of Recombinant Gal3149
Escherichia coli BL21 (DE3) harboring pET-Gal3149 was cultivated in LB medium supplemented with ampicillin (final concentration of 100 μg/mL) at 37°C overnight and then inoculated (2%, vol/vol) into 20 mL of LB broth medium in a 250-mL conical flask at 37°C with shaking at 180 rpm. When the optical density at 600 nm reached 0.6 to 0.8, isopropyl-β-d-thiogalactopyranoside was added at a final concentration of 0.2 mM, and incubation continued for 8 h at 16°C with shaking at 180 rpm. Escherichia coli BL21 (DE3) bacteria harboring the empty plasmid pET-his were used as control.
The cells were harvested by centrifugation at 7,100 × g for 15 min at 4°C and then resuspended using 50 mM PBS, pH 8.0. The resuspended cells were subsequently lysed by ultrasonication (300 W, 3-s strokes and 5-s intervals for 10 min) in an ice bath. The cell suspension containing the recombinant Gal3149 was obtained after centrifugation at 12,800 × g for 20 min at 4°C, and then subjected to purification by Ni2+-chelating affinity column (His-GraviTrap, GE Healthcare; performed by Shanghai Sangon Biotech Company;
). The linear gradient eluent (a mixture of 20–250 mM imidazole and equilibration buffer) was prepared in accordance with the kit instructions and then was added to the Ni2+-chelating affinity column from least to greatest concentration. The crude Gal3149 was eluted in a linear gradient eluent. Finally, the purified Gal3149 was collected and dialyzed by filtration (3,000 Da) to remove imidazole and then stored at 4°C for further analysis of enzymatic properties. The protein concentration was determined using a Modified BCA Protein Assay Kit (Sangon Biotech Company) with BSA as a standard. The molecular weight of Gal3149 was evaluated using 12% SDS-PAGE. The standard protein marker (10–180 kDa, Sangon Biotech Company) was used to calculate the molecular weight of Gal3149.
Enzyme Activity Assay
The β-galactosidase activity was assayed using substrate oNPG as described by
, with some modifications. The reaction mixture consisted of 100 μL of enzymes (appropriately diluted) and 100 μL of 8 mM oNPG in 50 mM PBS, and was incubated at 50°C for 10 min. The reaction was terminated by adding 800 μL of 0.5 M Na2CO3. The absorbance of the released o-nitrophenol (oNP) was measured at 420 nm and quantified using an oNP standard curve. One unit (U) of enzyme activity was defined as the amount of enzyme required to liberate 1 µmol of oNP per minute under the assay conditions. The specific activity of enzyme referred to the number of units of enzyme activity per milligram of protein.
The α-galactosidase activity assay was measured using substrate pNPαG, as described by
, with slight modification. All operating conditions and steps were the same as in the β-galactosidase activity assay. The absorbance of the released p-nitrophenol (pNP) was measured at 405 nm and quantified using a pNP standard curve. One unit (U) of enzyme activity was defined as the amount of enzyme required for the liberation of 1 µmol of pNP per minute under the assay conditions. The specific activity of enzyme referred to the number of units of enzyme activity per milligram of protein.
Effects of Temperature and pH on Gal3149 Activity
The optimal temperature and pH of Gal3149 activity were measured at different temperatures (0–80°C) and various pH (3.0–11.0) using oNPG as substrate. The different pH buffers were 0.02 mol/L citrate (pH 3.0–4.0), 0.02 mol/L sodium acetate (pH 4.0–6.0), 0.02 mol/L sodium phosphate (pH 6.0–8.0), 0.02 mol/L Tris-HCl (pH 8.0–9.0), and 0.02 mol/L glycine-NaOH (pH 9.0–11.0), respectively.
To assess thermal stability, Gal3149 was previously incubated at different temperature ranging from 0°C to 80°C for 48 h, and then the residual enzyme activity was measured under standard conditions. The pH stability of Gal3149 was investigated by incubating the enzyme in different pH buffers for 180 min. The residual activity was then determined by the method previously described and expressed as a percentage relative to the initial activity.
Effects of Metal Ions and Reagents on Gal3149 Activity
The effects of different metal ions (FeCl2, FeCl3, MgSO4, KCl, NaCl, BaCl2, CaCl2, MnSO4, CuSO4, CoCl2, ZnSO4, and AgCl) on Gal3149 activity were examined at final concentrations of 1 mM and 10 mM at 50°C for 30 min, respectively. The effects of different reagents on Gal3149 activity were also investigated, such as Triton X-100, Tween-80, Tween-40, SDS, EDTA, urea, ethanol, isopropanol, ethanediol, butanol, glycerin, oleic acid, and carboxymethyl cellulose, at final concentrations of 1% and 10% under the previously described conditions. Relative activity (%) in the presence of metal ions and reagents was calculated based on the activity measured without these additives under standard conditions.
Effect of NaCl on Gal3149 Activity
The influence of NaCl on Gal3149 activity also investigated in the range of 0–3 M (final concentration) under optimal conditions. The relative activity (%) was defined as the residual activity relative to that of the control, without NaCl.
Substrate Specificity and Kinetic Analysis
The substrate specificity of the purified Gal3149 was measured under optimal conditions using various substrates: oNPG (β-galactosidase activity), pNPG (β-galactosidase activity), pNPαG (α-galactosidase activity), oNPGlu (β-glucosidase activity), pNPGlu (β-glucosidase activity), pNPαGlu (α-glucosidase activity), and pNPXyl (β-xylanase activity).
The kinetic parameters of Gal3149 toward oNPG (0.1–5 mM) and lactose (0.2–40 mM) were determined under standard conditions. For lactose substrate, after incubation at 50°C for 10 min, the reaction was terminated by placing the sample in boiling water (not less than 95°C) for 10 min. The released glucose in the lactose hydrolysis reaction was determined using a commercial Glucose Oxidase-Peroxidase Assay Kit (Shanghai Rongsheng Biotech Co. Ltd.). One unit (U) of enzyme activity was defined as 1 µmol of glucose released per minute. Values for Vmax, kcat, Km, and catalytic efficiency (kcat/Km) were calculated based on the Lineweaver-Burk method.
Bioinformatics Analysis
Bioinformatics analysis of Gal3149 was performed as previously described by
. Briefly, the nucleotide and amino acid sequences of Gal3149 were analyzed using different websites and software. Two websites, BlastP at the NCBI server (http://www.ncbi.nlm.nih.gov/blast) and the CAZy website (http://www.cazy.org/Glycoside-Hydrolases.html), were used to analyze sequence identity and similarity, conserved domains, and glycoside hydrolase (GH) families of Gal3149, respectively. Physicochemical properties were calculated using the ExPASy ProtParam site (https://web.expasy.org/protparam/). The prediction of an amino-terminal signal peptide and its cleavage site were performed using the signal 5.0 server (https://www.cbs.dtu.dk/services/SignalP-5.0/). The phylogenetic tree of Gal3149 was constructed by MEGA 7.0 software (
To evaluate the efficiency of Gal3149 in hydrolyzing lactose in milk, the reaction, containing 1 U of Gal3149 and 1 mL of commercial skim milk, was performed at 4°C, 10°C, or 40°C for 4 h. The reaction was then terminated by incubating the samples in boiling water for 10 min. The lactose hydrolysis rate was calculated as follows:
where A is the content of initial lactose and B is the content of remaining lactose.
To analyze the hydrolysis products and observe the formation of GOS in a low-temperature milk environment, the mixture (1 U of Gal3149 and 1 mL of commercial skim milk) was incubated for 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 12, and 24 h at 4°C, respectively. After various time intervals, reaction mixtures were collected and boiled to stop the reaction, and then stored at 4°C. All reaction supernatant (properly diluted) were spotted on silica gel 60 thin-layer chromatography (TLC) aluminum sheets (Merck) as described by
. The components were separated in a solvent system containing n-butanol, isopropyl alcohol, and water (3:12:4, vol/vol/vol) as mobile phase. Detection was achieved by spraying with aniline-diphenylamine phosphate reagent, and the spot was visualized by baking at 85°C for 10 min. In addition, HPLC was used as described by
, with some modifications. The lactose hydrolysis products were monitored by HPLC analysis using a Shim-Pack GIST NH2 column (5 μm, 4.6 × 250 mm, Waters), with 75% acetonitrile used as a mobile phase at a flow rate of 1 mL/min and a 2414 Refractive Index Detector (Waters).
Statistical Analysis
All data were analyzed using SPSS 23.0 software (IBM Corp.). The mean ± standard deviation (SD) was determined for each group in the individual experiments. All experiments were performed at least in triplicate.
RESULTS AND DISCUSSION
Bioinformatic Analysis
The genomic DNA of B. velezensis SW5 was extracted, and sequence analysis revealed that gal3149 was a galactosidase gene (Accession No. MW282921). The gal3149 gene contained an open reading frame of 1,299 bp that encoded 433 amino acids residues. ExPASy online analysis determined that the formula of Gal3149 is C2165H3428N588O648S23 and its total number of negatively charged residues (Asp + Glu) and positively charged residues (Arg + Lys) were 58 and 48, respectively. Additionally, the theoretical molecular weight and putative isoelectric point of Gal3149 were 48.8 kDa and 5.76, respectively. The results from the Signal IP 5.0 server showed no signal peptide of Gal3149.
The BlastP search against the NCBI database revealed that Gal3149 shared high identity with the α-galactosidas gene predicted from the complete genome sequence of Bacillus (Accession No. WP_060674854.1), B. velezensis (Accession No. WP_064107373.1), and B. amyloliquefaciens (Accession No. WP_033574232.1), with identities of 99.7%, 99%, and 99%, respectively. However, these enzymes were deduced from whole-genome sequencing, and their enzymatic characteristics were not investigated. Among the characterized amino acid sequence, Gal3149 exhibited sequence similarity with the GH4 α-galactosidase-encoding gene melA (85%; Accession No. O34645) from Bacillus subtilis strain 168 (
The chitin disaccharide, N,N'-diacetylchitobiose, is catabolized by Escherichia coli and is transported/phosphorylated by the phosphoenolpyruvate:glycose phosphotransferase system.
). The phylogenetic tree was constructed using the neighbor-joining method to reveal the relationship between Gal3149 from B. velezensis SW5 and other characterized galactosidases. Gal3149 showed close relatedness and clustering with the α-galactosidase from B. subtilis (Accession No. AAC00383.1), which belonged to GH4 (Figure 1). Similarly, the result from the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/cdd) analysis also identified Gal3149 as a member of the GH4 galactosidase and classified galactosidase into the Ce1F superfamily. Based on the information from phylogenetic analysis and the Conserved Domain Database in NCBI, we consider Gal3149 to represent a novel member of the GH4 family.
Figure 1Phylogenetic tree of Gal3149 from Bacillus velezensis SW5 and other galactosidase amino acid sequences, based on the neighbor-joining method.
In addition, a salient feature of α-galactosidase is that its sequence contains GAGS, which can bind to NAD+ (Figure 2A; ▲ represented GAGS). The results from sequence alignment also showed that Gal3149 exhibited a certain similarity (46%) with other reported β-galactosidases. Some amino acids of Gal3149 were found, as shown in Figure 2A and Figure 2B (red boxes). Therefore, we speculate that Gal3149 belonging to GH4 has properties of both α-galactosidase and β-galactosidase. The enzymatic characteristics of Gal3149 with β-galactosidase activity will be further explored in our next work.
Figure 2Amino acid sequence alignment of Gal3149 from Bacillus velezensis SW5 with other α-galactosidases and other reported β-galactosidases. (A) Amino acid sequence alignment of Gal3149 with other α-galactosidases from GH4: Gal3149 from Bacillus velezensis SW5 (Accession No. MW282921), α-galactosidase from Bacillus subtilis (GenBank AAC00383.1), Bacillus megaterium (GenBank AGG11047.1), Bacillus halodurans C-125 (Accession No. NP_243094.1), Enterococcus faecium E980 (GenBank EFF38781.1). ▲ represents predicted NAD+ binding amino acid residue. (B) Amino acid sequence alignment of Gal3149 with other reported β-galactosidases from GH 42. Tnap 1577 from Thermotoga naphthophila RKU-10 (GenBank ADA67652.1), bgaA from Thermus sp. T2 (GenBank CAB07810.1), ef1 from Marinomonas sp. ef1 (Accession No. WP_100635792.1), BgaP from Planococcus sp. L4 (GenBank ABI64125.1). The black background indicates a strictly conserved amino acid; the purple and blue backgrounds indicate similarity of amino acid residues over 75% and 50%, respectively. Red boxes represent some amino acids of Gal3149 that were found in both 2A and 2B.
After connection with pET-his, pET-Gal3149 was subcloned in E. coli DH5α and then expressed into E. coli BL21 (DE3). Significant β-galactosidase activity was detected in the lysate of recombinant cells after induction with 0.2 mM isopropyl-β-d-thiogalactopyranoside for 8 h at 16°C, but no β-galactosidase activity was observed in the lysate of cells of hosts or with the empty vector. These results indicate that the recombinant enzyme was successfully transformed and expressed in E. coli BL21 (DE3), which provided the basis for exploring the biochemical properties of Gal3149.
After purification, Gal3149 exhibited higher activity (147 U/mg) with using oNPG as substrate (β-galactosidase activity) than using pNPαG (44 U/mg) as substrate (α-galactosidase activity). These results further verified that the recombinant Gal3149 had β-galactosidase activity, and its β-galactosidase properties were further explored. Moreover, the capacity of lactose hydrolysis and its hydrolysis products were also studied. The result of SDS-PAGE showed that a single band of the purified Gal3149 was detected, which was similar to the band of crude enzyme (Figure 3), indicating that recombinant Gal3149 was successfully obtained by the purification process.
Figure 3Sodium dodecyl sulfate-PAGE analysis of recombinant Gal3149. Lane M = protein maker 250–180 kDa; lane 1 = crude Gal3149; lane 2 = purified Gal3149.
Effect of Temperature on Gal3149 Activity and its Thermal Stability
The optimal temperature of the purified Gal3149 was determined using substrate oNPG under different temperature conditions (0–80°C). The optimal temperature of Gal3149 was 50°C, which was consistent with the studies of
. The enzyme activity increased with increasing temperature (0°C to 50°C), due to the rise of kinetic energy leading to collision of enzyme and substrate. When the samples were incubated above the optimal temperature (50°C), Gal3149 activity decreased rapidly (Figure 4A) but still retained 40% activity at 80°C. Evidence shows that the optimal temperature of β-galactosidase from most microorganisms ranges from 25 to 60°C (
Figure 4Effects of temperature and pH on Gal3149 activity and its temperature and pH stability. (A) Effects of temperature from 0 to 80°C on Gal3149 activity using substrate oNPG. (B) Thermal stability of Gal3149 incubated at 0–80°C for 48 h. (C) Effects of pH from 3 to 11 on Gal3149 activity using substrate oNPG. (D) pH stability of Gal3149 at pH 3.0–11.0 for 3 h. Data are shown as the mean ± SD.
The thermal stability of Gal3149 was carried out between 0°C and 80°C using oNPG substrate for 48 h (Figure 4B). The results revealed that Gal3149 was stable at temperatures no more than 50°C, with 55% remaining enzymatic activity after 48-h incubation. As shown in Figure 4B, the activity of Gal3149 significantly decreased at 60°C, 70°C, and 80°C after 0.5-h incubation, but the residual activity was still higher than that of β-galactosidase from Bacillus megaterium 2-37-4-1 (
). With the temperature and time increasing, Gal3149 activity gradually reduced. When incubated at 80°C for 20 h, no enzyme activity was observed. These results indicated that Gal3149 was relatively stable and maintained a high activity at low temperature.
Effect of pH on Gal3149 Activity and its pH Stability
The pH-dependency of Gal3149 activity was investigated in the range of pH 3 to 11, using substrate oNPG. As shown in Figure 4C, the optimal pH of Gal3149 was 8.0. With an increase of pH from 3.0 to 8.0, the enzyme activity of Gal3149 also increased. In contrast, the enzyme activity of Gal3149 decreased as the pH further increased (from 8.0 to 11.0). The activity of Gal3149 sharply increased from pH 5.0 to 8.0, which could be due to the use of buffers containing Na+. According to our analysis of the effects of metal ions on enzyme activity (Table 1), Na+ had an obvious activation effect on Gal3149 activity. As shown in Figure 4C, Gal3149 exhibited high relative activity at the pH of 6.0 to 9.0, indicating it had a wide pH value. The pH stability results showed that Gal3149 was stable at pH 6.0 to 8.0, retaining more than 68% of the initial activity after 180-min incubation (Figure 4D). Compared with previous study (
), the pH stability of β-galactosidase from B. megaterium ranged from 6.0 to 9.0, which was consistent with that of Gal3149 in this study. These properties of Gal3149 provide a theoretical foundation for its application in the dairy industry and may greatly enhance its development in the future.
Table 1Effects of different metal ions on activity of the novel galactosidase Gal3149
Effects of Metal Ions and Reagents on Gal3149 Activity
The effects of metal ions at different concentrations on Gal3149 activity were investigated and are shown in Table 1. It was found that Na+ and K+ enhanced the activity of Gal3149 at the final concentration of 1 mM and 10 mM; this may be related to the source of B. velezensis SW5 (from fish sauce with high salt concentration). The stimulating effects of Na+ and K+ were also observed in β-galactosidase from Lactobacillus reuteri (
Comparison between discontinuous and continuous lactose conversion processes for the production of prebiotic galacto-oligosaccharides using β-galactosidase from Lactobacillus reuteri.
J. Agric. Food Chem.2007; 55 (17630761): 6772-6777
). The change of Gal3149 activity was negligible in the Mg2+ buffer, which retained over 99% relative activity regardless of the concentration (1 mM or 10 mM). The enzyme activity reported in other studies was promoted by Mg2+ at the low concentration but was inhibited at the high concentration (
Purification and characterization of β-galactosidase from newly isolated Aspergillus terreus (KUBCF1306) and evaluating its efficacy on breast cancer cell line (MCF-7).
). The enzyme activity strongly decreased in the presence of Ba2+, Ca2+, Mn2+, and Co2+ at high concentrations (10 mM). In particular, with Ca2+ and Co2+ at 1 mM or 10 mM, the activity of Gal3149 decreased from 73% to 26% and from 58% to 14%, respectively. The activities of β-galactosidase from Bifidobacterium longum CCRC 15708 (
) were also inhibited by Ca2+. Table 1 shows that some metal ions (Fe2+, Fe3+, Ag+, and Zn2+) greatly reduced the enzyme activity, whether in the concentrations of 1 or 10 mM. Moreover, Cu2+ completely inhibited the enzyme activity. Zn2+ and Cu2+ also markedly or completely inhibited the enzyme activity in β-galactosidase from Alteromonas sp. ML52 (
Similarly, the effects of various reagents on Gal3149 activity were also investigated and are shown in Table 2. The relative activity of Gal3149 exceeded 94% in SDS, urea, ethanol, isopropanol, ethanediol, and glycerin, illustrating that they had minor effects on Gal3149 and could be considered negligible. With the concentration rising, the activity of Gal3149 was strongly inhibited by reagents such as Triton X-100, Tween-40, and Tween-80. Moreover, Gal3149 activity was completely inhibited by carboxymethyl cellulose and oleic acid at concentrations of both 1% and 10%. In some cases, the concentration of the reagent was negatively correlated with enzyme activity. For example, the presence of EDTA with low concentration exhibited a stronger inhibitory effect on enzyme activity (19%) than that of high concentration (48%). In another study, the enzyme activity was also inhibited by EDTA (
). Activity of Gal3149 was improved by the presence of Na+ (Table 1). High-concentration EDTA contains a certain amount of Na+ ions, which can reduce the effect of EDTA on enzyme activity. This phenomenon can also be seen in the effect of pH on enzyme activity. Activity of Gal3149 was greatly increased due to the presence of Na+ (pH 4.0–8.0).
Table 2Effects of different reagents on activity of the novel galactosidase Gal3149
Recombinant Gal3149 may have a high tolerance to NaCl, because B. velezensis SW5 was isolated from a fish sauce mash that contained a high concentration of salt (
). The tolerance test showed that Gal3149 activity was markedly promoted by NaCl at concentrations ranging from 0 mM to 0.1 M, and was slightly influenced at concentrations from 0.05 M to 3 M (Figure 5). In addition, the residual activity was more than 85% at 3 M NaCl, indicating that Gal3149 was tolerant to high salt concentrations.
Figure 5Effects of NaCl on Gal3149 activity; concentration of NaCl from 0 to 3 M. Data are shown as the mean ± SD.
The substrate specificity of recombinant Gal3149 was investigated with 7 chromogenic substrates: oNPG, pNPG, pNPαG, oNPGlu, pNPGlu, pNPαGlu, and pNPXyl (Table 3). When using pNPG and pNPαGlu as substrates, Gal3149 showed about 340% and 83% of its activity compared with oNPG, respectively. When other substrates were used (oNPGlu, pNPGlu, and pNPXyl), the relative activity was extremely low, and thus we considered their activities to be negligible.
The kinetic parameters of Gal3149 were calculated under optimal conditions with oNPG and lactose as substrates, as shown in Table 4. The half-saturation coefficient, Km, is a characteristic constant of the enzyme, which is related to the character of the enzyme rather than to the concentration of enzyme. The Km value approximately represents the affinity of the enzyme and the substrate; that is, the smaller the Km value, the greater the affinity of the enzyme and the substrate (
). The Km value of lactose was higher (about 15 times) than that of oNPG under the same assay conditions, suggesting that Gal3149 had a higher affinity for oNPG than for lactose. The Km values of Gal3149 and β-galactosidase from Lactobacillus pentosus (
), which may be adjusted by the increase of kcat, or by decrease of Km, or by changing these 2 parameters at the same time. The catalytic efficiency of Gal3149 toward lactose was higher than those of β-galactosidases from Alteromonas sp. ML117 (2.1 s−1 mM−1;
A novel cold-active β-d-galactosidase with transglycosylation activity from the Antarctic Arthrobacter sp. 32cB—Gene cloning, purification and characterization.
β-Galactosidase is a key enzyme in the preparation of lactose-free milk and generation of GOS. The application of β-galactosidase in the dairy industry is limited, owing to the specific pH of milk (6.5–7.0) and its low storage temperature. Gal3149 was relatively stable in the range of pH 6.0–8.0 and at low temperature (0–10°C), so the purified Gal3149 was suitable to hydrolyze lactose in milk. The rate of hydrolyzed lactose reached about 36%, 45%, and 69%, respectively, after incubation at 4°C, 10°C, and 40°C for 4 h. With increasing temperature, the rate of hydrolyzed lactose increased. These results demonstrate that Gal3149 has the capacity of lactose hydrolysis with tremendous potential in the dairy product industry to generate lactose-free milk.
The production of GOS was detected within a certain reaction time. During the reaction, lactose in milk was degraded into monomers. At the same time, the degradation process was observed on TLC plate and via HPLC. After a 30-min reaction, a small quantity of glucose and galactose were generated, indicating that lactose in milk had begun to hydrolyze. After a 60-min reaction, a new spot, representing GOS, was clearly displayed on the TLC plate (Figure 6A). As can be seen from the TLC plate (Figure 6A), the amount of lactose in the milk gradually decreased as the reaction continued, while the amount of galactose and GOS gradually increased. Similarly, a new peak next to the peak of lactose was observed on the HPLC (Figure 6B), which further confirmed the presence of GOS. Relevant studies should be carried out to determine the structure of GOS in the future. Another study also reported that lactose was hydrolyzed by β-galactosidase from Alteromonas sp. QD01 and generated GOS (
). We found, for the first time (to the best of our knowledge), that a galactosidase belonging to the GH4 family exhibited β-galactosidase activity. Therefore, Gal3149 from B. velezensis SW5 displays the capacity of lactose hydrolysis and production of GOS products. These properties demonstrate that Gal3149 has the potential to be used in the food industry for producing lactose-free milk and GOS prebiotics.
Figure 6Analysis of hydrolysis products using thin-layer chromatography (TLC) and HPLC. (A) TLC analysis. Lanes M1–M3 = lactose, galactose, and glucose, respectively; lanes 1–11 = incubation for 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 8, 12, and 24 h, respectively. (B) HPLC analysis. GOS = galactooligosaccharides.
In this study, a novel GH4 galactosidase from B. velezensis SW5 was successfully cloned and expressed in E. coli. The recombinant Gal3149 was purified, and its enzymatic properties were also characterized. Based on sequence analysis and experimental verification, Gal3149 not only demonstrated high similarity to α-galactosidase in GH4 but also exhibited the properties of β-galactosidase. The optimal temperature and pH were 50°C and pH 8.0, respectively. Moreover, Gal3149 was studied for its β-galactosidase properties, and exhibited the capacity of lactose hydrolysis and generation of GOS. This research provides a novel galactosidase for development of lactose-free milk and lays a solid foundation for Gal3149 in application of the lactose-reduced dairy industry.
ACKNOWLEDGMENTS
This work was financially supported by the Natural Science Fund Project of Ningbo (2018A610337) and the Public Welfare Project of Zhejiang (GG19C200003). The authors declare no competing financial interest.
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