Journal of Dairy Science
Volume 91, Issue 5 , Pages 1751-1758, May 2008

Production, Purification, and Characterization of a Potential Thermostable Galactosidase for Milk Lactose Hydrolysis from Bacillus stearothermophilus

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

Received 15 August 2007; accepted 8 January 2008.

Article Outline

Abstract 

β-Galactosidase, commonly named lactase, is one of the most important enzymes used in dairy processing; it catalyzes the hydrolysis of lactose to its constituent monosaccharides glucose and galactose. Here, a thermostable β-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. sub-tilis WB600. The recombinant enzyme was purified by a combination of heat treatment, ammonium sulfate fractionation, ion exchange, and gel filtration chromatography techniques. The purified β-galactosidase appeared as a single protein band in sodium dodecyl sulfate-PAGE gel with a molecular mass of approximately 70 kDa. Its isoelectric point, determined by polyacryl-amide gel isoelectric focusing, was close to 5.1. The optimum temperature and pH for this β-galactosidase activity were 70°C and pH 7.0, respectively. Kinetics of thermal inactivation and half-life times for this thermostable enzyme at 65 and 70°C were 50 and 9h, respectively, and the Km and Vmax values were 2.96mM and 6.62μmol/min per mg. Metal cations and EDTA could not activate this thermostable enzyme, and some divalent metal ions, namely, Fe2+, Zn2+, Cu2+, Pb2+, and Sn2+, inhibited its activity. Thiol reagents had no effect on the enzyme activity, and sulfhydryl group blocking reagents inactivated the enzyme. This enzyme possessed a high level of transgalactosylation activity in hydrolysis of lactose in milk. The results suggest that this recombinant thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galactooligosaccharides in milk processing.

Key words: thermostable β-galactosidase, lactose, purification, hydrolysis

 

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Introduction 

β-Galactosidase (β-d-galactohydrolase; EC 3.2.1.23) has been used to hydrolyze lactose to produce lactose-free milk products and has recently become of interest for the production of galactooligosaccharides from lactose by the transgalactosylation reaction (Prenosil et al., 1987; Panesar et al., 2006). Although the yeast Kluyveromyces lactis is still the major commercial source of β-galactosidase because of its dairy environmental habitat and outstanding lactose hydrolysis activity, K. lactis-produced β-galactosidase has a major drawback in terms of thermostability (Ganeva et al., 2001). In recent years, several types of thermostable β-galactosidases have been reported (Daabrowski et al., 2000; Lebbink et al., 2000; Wanarska et al., 2005; Synowiecki et al., 2006). Thermostable β-galactosidases can work at higher temperatures and prevent microbial contamination to some extent in milk processing, functioning under conditions that normally denature mesophilic enzymes (Zeikus et al., 1998). Thus, these thermostable enzymes have considerable industrial potential because they can give better yields at high temperatures.

Many β-galactosidases from thermophilic microorganisms have been characterized and used for the hydrolysis of lactose and galactooligosaccharide production (Panesar et al., 2006). Nevertheless, the production of these enzymes in thermophilic microorganisms is difficult to achieve on an industrial scale, and therefore the possibility of producing them in a mesophilic host using recombinant techniques has become of interest (Bruins et al., 2001; Demirjian et al., 2001; Kang et al., 2005). One of the hosts for large-scale production of foreign proteins is the gram-positive bacterium Bacillus subtilis. The major advantage of this strain is its classification as GRAS (generally recognized as safe), and is an organism free of any endotoxin, so it is favorable in food industry (van Wely et al., 2001).

In our laboratory, the bgaB gene from Bacillus stearothermophilus had been expressed in Escherichia coli(Chen et al., 2002a) and its enzymatic activity was characterized; however, its enzymatic half-life at 60°C was just 0.5h. In the present study, the B. subtilis expression system was used to evaluate its applicability in thermostable β-galactosidase production, and the characteristics of this recombinant thermostable β-galactosidase were examined after multistep separation for the enzyme purification.

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Materials and Methods 

Reagents, Microorganisms, and Plasmids 

The restriction endonucleases, T4 DNA ligase, and Pfu DNA polymerase were purchased from Takara Bio-tech Co. Ltd. (Dalian, China). The Gel Extraction Mini-Kit and PCR Purification Mini Kit were purchased from Watson Biotechnologies Inc. (Shanghai, China). Other reagents purchased were of analytic grade.

Escherichia coli TG1 was used as host strain for cloning and for the preparation of template plasmids. Bacillus stearothermophilus ATCC8005 was the donor of thermostable β-galactosidase coding sequence, and B. subtilis WB600 was used for expression of β-galactosidase. The plasmid pBSKII KS+ was the clone vector, and pMA5 was used for construction of the thermostable β-galactosidase expression plasmid in B. subtilis.

Construction of Expression Vector 

The bgaB coding sequence was amplified by PCR with primers U-5′-ATACATATGAATGTGTTATCCTC-3′ and d-5′-TATGGATCCCTAAACCTTCCCGGCTTC-3′, and then cloned into the pBSKII KS+ vector in accordance with the manufacturer's (Stratagene, La Jolla, CA) protocol to obtain the cloning vector, pBSK-bgaB. Following the digestion with the restriction enzymes NdeI and BamHI (the restriction sites, underlined above, were introduced through PCR primers), the fragment encoding the bgaB was recovered, and then inserted between the same sites of NdeI and BamHI into pMA5 to obtain the expression vector pMA5-bgaB. The expression vector was then confirmed by restriction en-donuclease digestion and sequencing.

Expression of the Recombinant Thermostable β-Galactosidase 

A fresh clone of B. subtilis WB600, harboring the pMA5-bgaB vector, was grown in Luria broth (LB) medium containing 50μg of kanamycin/mL for 12h. Seed was then taken and inoculated at the ratio of 5% (volume) into LB medium in a flask or fermenter and fermented at 37°C for 18h; cells were harvested and stored at −70°C.

Purification of Thermostable β-Galactosidase 

The frozen B. subtilis WB600/pMA5-bgaB cells were thawed at room temperature and suspended in buffer A, 20mM sodium phosphate (pH 7.0), and then treated with sonication. The supernatant was collected after centrifugation at 4°C and 18,522×g for 20min on Beckman J2-21M centrifuge (Beckman Coulter Inc., Fullerton, CA), and then the heating treatment was performed at 65°C for 50min. The solution was subjected to centrifugation at 18,522×g for 30min and the supernatant was collected for enzymatic assay. The clarified supernatant was brought to 30% ammonium sulfate saturation and centrifuged at 18,522×g for 30min. All subsequent fractionation steps involving ammonium sulfate were carried out at 4°C. After centrifugation, the pellet was discarded and the supernatant was brought to 65% ammonium sulfate saturation and centrifuged at 18,522×g for 30min again, and the pellet was dissolved in buffer B, 20mM sodium phosphate (pH 7.5), and dialyzed at 4°C for 24h; further purification was done by anion exchange followed by gel filtration chromatography.

Anion exchange chromatography was performed on an ÄKTA explorer system (Amersham Pharmacia Bio-tech, Uppsala, Sweden) using a DEAE HiPrep16/10 column and equilibrated with buffer B; the target enzyme was eluted by a linear gradient of 0 to 500mM NaCl in buffer B at 2 mL/min. The effluents and eluents were collected for further evaluation. Gel filtration chromatography was also performed on ÄKTA explorer system using a Sephacryl S-100 HiPrep16/60 column (Amer-sham Pharmacia Biotech, Uppsala, Sweden). The thermostable β-galactosidase was eluted in sodium phosphate buffer (20mM sodium phosphate, 100mM NaCl, pH = 7.0) at 1 mL/min.

PAGE and Protein Quantification 

Electrophoretic analysis of the synthesized products and purified samples was performed in 15% SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue for band visualization. The molecular weight of the purified enzyme was estimated on the basis of migration on 15% SDS-PAGE.

The protein concentration was determined with Bradford protein assay using BSA as the standard. The percentage of target protein in total protein was quantified by Quantity One software (BioRad Laboratories Inc., Hercules, CA) according to protein band visualization.

Isoelectric Point Estimation 

Isoelectric focusing (IEF) gel electrophoresis was performed using Model 111 mini cell (BioRad) for estimation of isoelectric point according to the protocol provided by the manufacturer. The gel was stained with Coomassie Brilliant Blue R-250 to determine the location of the protein bands, and the isoelectric point of the enzyme was estimated by comparing its mobility with the following pI values of IEF protein markers (BioRad): phycocyanin, 4.7; β-LG, 5.1; bovine carbonic anhydrase, 6.0; human carbonic anhydrase, 6.5; equine myoglobin (minor), 6.8: equine myoglobin (major), 7.0; human hemoglobin A, 7.1; human hemoglobin C, 7.5; lentil lectin (three hands), 7.8, 8.0, and 8.2; and cytochrome c, 9.6.

Enzyme Assay 

The thermostable β-galactosidases activity was determined as described by Gist-Brocades (Xia et al., 2005), except that the incubation temperature of the enzyme was adjusted to 55°C. One unit of enzyme activity was defined as the amount of enzyme hydrolyzing 1 micromole of substrate orthonitrophenyl-β-d-galacto-pyranoside (ONPG) per minute. The specific activity of thermostable β-galactosidase was expressed in units per milligram of cellular or enzyme protein.

The enzyme activity on lactose in milk was assayed under the same conditions as described above (Xia et al., 2005), and the hydrolysis rate of lactose in milk (%) was defined as [glucose]×1.9/[lactose]×100. The released glucose was measured by the glucose oxidaseperoxidase method (Zhao, 2002), and the lactose concentration was determined by Fehling method (Zhao, 2002).

Effect of pH, Temperature, and Various Reagents on Enzyme Activity 

The pH dependency of enzyme activity was determined at 55°C in 2 buffer systems: sodium citrate-phosphate buffer (200mM) from pH 5.0 to 7.5 and sodium potassium-phosphate buffer (200mM) from pH 7.5 to 9.0. The effect of temperature on enzyme activity was determined at temperatures ranging from 45 to 80°C. The thermostability of the enzyme was tested by incubation at 60°C for 6h, 65°C for 5h, and 70°C for 4h, and aliquots were withdrawn at 0.5- to 1-h intervals.

The effect of various reagents on enzyme activity were examined by assaying the residual activity after incubating the enzyme with 1 to 10mM of various reagents dissolved in potassium-phosphate buffer (200mM, pH 6.5) for 20min at 25°C. The cations tested were Li+, Na+, K+, Mg2+, Ca2+, Zn2+, Cu2+, Fe2+, Mn2+, Pb2+, and Sn2+; the chelating agent, EDTA, the thiol reagents 2-mercaptoethanol and dithiothreitol, and enzyme inhibitors p-chloromercuribenzoic acid and iodoacetic acid, were also examined.

Kinetic Analysis 

The kinetic parameters (Km and Vmax) were calculated from Lineweaver-Burk plots using a substrate concentration range of 0.83 to 10mM ONPG. All the experiments were carried out at 55°C and pH 7.0.

Amino Acid Composition Analysis 

The AA composition was analyzed by adding 10mL of 6 M HCl and 12.7mg of the further purified thermostable β-galactosidases to a hydrolysis vessel, which was kept at 110°C for 24h. Afterward, the HCl was removed from 1mL of sample, and then 1mL of 20mM HCl was added. Chromatographic analysis was performed on an Agilent 1100 HPLC system (Agilent, Santa Clara, CA) using a C18 column with a flow rate of 1 mL/min. The AA of the target enzyme were detected with a UV wavelength of 338nm.

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Results and Discussion 

Expression Vector Construction of Thermostable β-Galactosidase and Its Expression in B. subtilis 

For expression the thermostable β-galactosidase in B. subtilis, an E. coli-B. subtilis shuttle plasmid pMA5 was exploited as backbone to construct the expression vector. To construct expression vector pMA5-bgaB, PCR was performed to introduce restriction sites NdeI and BamHI to the 5′- and 3′-ends of bgaB gene. The 1% agarose gel electrophoresis analysis of the PCR product showed the length of the above gene was approximately 2,000 bp, in accordance with the calculated length (2,016 bp). The bgaB gene was confirmed for correct orientation using restriction endonuclease digestion and sequencing.

The recombinant strain B. subtilis WB600/pMA5-bgaB was cultured in LB medium at 37°C for 18h, and the cells were harvested and treated with sonication to determine enzymatic activity. Results showed that thermostable β-galactosidase was expressed and the enzymatic activity reached 5.8 U/mg of protein, more than 45 times of that obtained from B. stearothermophilus (0.1 U/mg of protein; Hirata et al., 1986).

Purification of Thermostable β-Galactosidase 

Because the recombinant β-galactosidase is a thermostable enzyme, the crude enzyme solution was first treated with heating at 65°C for 50min. After ammonium sulfate precipitation, the enzyme was further separated by 2-step purification in which the anion exchange chromatography was followed by gel filtration. The final enzyme activity reached 125.2 U/mg of protein (higher than that reported in Hirata et al., 1985) and the purity reached about 95.1%. The purification procedures and results of the thermostable β-galactosidase are summarized in Table 1, which depicts specific activity and the purification fold of every step. An overall recovery of 19.7% and a 19.4-fold enrichment of the recombinant enzyme were obtained after 4 steps. The enzyme samples of every purification step were subjected to 15% SDS-PAGE analysis, and the final purified thermostable β-galactosidase showed a single band of about 70 kDa (Figure 1). Its molecular weight was in accordance with that reported by Hirata et al. (1985), but lower than the molecular weight of 78 kDa deduced from the AA sequence. It is possible that the recombinant enzyme is modified by the host B. subtilis.

Table 1. Summary of the thermostable β-galactosidase purification from recombinant Bacillus subtilis
StepTotal protein (mg)Total activity (U)Specific activity (U/mg)Yield (%)Purification (fold)
Cell-free extract20,580.5133,350.36.5100.01.0
Heat treatment7,100.1133,000.618.799.72.9
30 to 65% ammonium sulfate precipitation1,221.198,100.180.373.612.4
Anion exchange (DEAE)680.972,400.7106.454.316.4
Gel filtration (Sephacryl S-100)209.326,200.3125. 219.719.4
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  • Figure 1. 

    Sodium dodecyl sulfate-PAGE analysis of the purified thermostable β-galactosidase from different stages. M = protein marker; lane 1 = cell-free extract; lane 2 = fraction of heat treatment; lane 3 = fraction of (NH4)2SO4 precipitation; lane 4 = fraction of ÄKTA DEAE column; lane 5 = fraction of ÄKTA Sephacryl S-100 column.

Molecular Properties 

The isoelectric point of the thermostable β-galactosidase, estimated by IEF on a typical analytical IEF poly-acrylamide gel, was in the range of 4.7 to 5.1. It was lower than the calculated value of 5.7, and similar to most of the reported pI for thermostable β-galactosidases (4.1 to 4.5) (Onishi and Tanaka, 1995).

The AA composition is indicated in Table 2. From the 17 kinds of AA, the determined weight percentage of Asp and Glu were greater than the calculated value, whereas the weight percent of the other AA were similar to the calculated values, especially for Ala, Val, Leu, and Ile, the side-acyclic AA. The AA Ala, Val, Leu, and Ile are very helpful for the thermostability of β-galactosidase (He et al., 2000).

Table 2. Amino acid composition of the thermostable β-galactosidase
Amino acidNumber count (calculated)% by frequency (calculated)% by weight (calculated)% by weight (determined)
Ala365.353.554.19
Arg365.356.956.90
Asp405.945.9016.09
Cys101.491.340.84
Gly436.393.585.69
Glu517.588.3113.55
His192.823.272.18
Ile487.136.986.54
Lys385.656.156.04
Leu527.737.568.06
Met152.232.481.83
Phe294.315.315.44
Pro375.504.724.20
Ser294.313.382.37
Thr253.713.303.73
Val497.286.366.28
Tyr355.207.026.08
Asn385.655.56ND1
Gln213.123.40ND
Trp213.124.75ND

1ND = not determined.

Enzyme Properties 

pH and Temperature Profiles of the Enzymes 

The pH profile of the thermostable β-galactosidase activity is shown in Figure 2. The enzyme displayed its maximum activity at pH = 7.0 and retained more than 80% of its activity at a pH range of 6.0 to 7.5. This optimal pH range is a good property with respect to milk processing because it is near to pH = 6.7 of normal bovine milk.

The effect of temperature on the enzyme activity was studied at temperatures ranging from 45 to 80°C at pH = 7.0 (Figure 3). This thermophilic enzyme showed the maximum activity at 70°C, which was double that obtained at 55°C, and retained 80% of its activity even at 75°C. Thermostability was determined by treating the enzyme at 60, 65, and 70°C (Figure 4). Results revealed that the enzymatic half-life at these 3 temperatures were 120, 50, and 9h, respectively, higher than the data reported (Hirata et al., 1985; Wei et al., 2001). The enzymatic half life of recombinant β-galactosidase from B. subtilis is much greater than that from E. coli (Chen et al., 2002b), which may due to the greater homogeneity between B. subtilis and B. stearothermophilus than between E. coli and B. stearothermophilus. The favorable temperature activity of this enzyme and its thermostability are desirable properties for normal lactose-free and lactose-reduced milk processing.

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  • Figure 4. 

    The thermostability of β-galactosidase from Bacillus subtilis WB600/pMA5-bgaB at different temperatures: ■ = 60°C; • = 65°C; ▴ = 70°C. Error bars represent the standard deviation from 3 separate experiments.

Effects of Metal Cations, EDTA, Thiol Reagents, and Enzyme Inhibitors on Enzyme Activity 

The cations Fe2+, Zn2+, Cu2+, Pb2+, and Sn2+ inhibited 52, 76.6, 85.3, 100, and 100% of the enzyme activity, respectively, whereas other metal cations and EDTA did not (Table 3). Therefore, no metal cations are required for enzyme activity according to these results. The sulfhydryl group-blocking reagents p-chloromercuribenzoic acid and iodoacetic acid inhibited 86.2 and 73.0% of the enzyme activity, respectively, indicating that a sulfhydryl group is involved at or near the active domain of this recombinant thermostable β-galactosidase. Furthermore, the thiol reagents 2-mercaptoethanol and dithiothreitol had no distinct promotion effect on enzyme activity, showing that intact disulfide groups are not important for enzyme activity.

Table 3. Effects of metal cations, EDTA, thiol reagents, and enzyme inhibitors on the thermostable β-galactosidase activity (n = 4)
Concentration (mM)Relative activity (%)
Control0100.0a
Cations
Li+1097.5a
Na+10100.0a
K+10103.6a
Mg2+10109.0a
Ca2+1095.0a
Zn2+523.4b
Cu2+514.7b
Fe2+548.0c
Mn2+2102.0a
Pb2+20.0d
Sn2+20.0d
Chelating agents
EDTA197.4a
Thiol reagents
2-mercaptoethanol1117.5a
Dithithreitol1125.0a
Enzyme inhibitors
PCMB1113.8b
Iodoacetic acid127.0b

a-dMeans within the column with different superscripts differ (P<0.05).

1PCMB = p-chloromercuribenzoic acid.

Novel Kinetic Properties with Substrate or Substrate-Like Activation 

The enzyme kinetic parameters for ONPG or other substrate-like inhibitors were calculated from a Lineweaver-Burk plot. The Km and Vmax values for ONPG were 2.96mM and 6.62μmol/min per mg (Figure 5). The substrate-like inhibitors, lactose (10mM), galactose (100mM), melibiose (100mM) and melitriose (100mM), known to be potent substrate inhibitors of many β-galactosidases in an ONPG system (Chen, 2001), were added to obtain the inhibitor constant value Ki according to the inhibitor substrate concentration. The results showed that lactose and galactose had a competitive inhibitor effect against ONPG (Figure 6). The inhibitory activity of lactose was greater than galactose: the Ki was 15.7 and 87.3mM for lactose and galactose, respectively.

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  • Figure 6. 

    Effect of substrate-like inhibitors on the activity of thermostable β-galactosidase: ■ = control; ▴ = lactose (10mM); • = galactose (100mM); ♦= melibiose (100mM); ♦= melitriose (100mM).

Hydrolysis of Lactose in Milk by Thermostable β-Galactosidase 

The ability of thermostable β-galac-tosidase to catalyze lactose for producing d-glucose was tested with hydrolysis of lactose in milk at different temperatures using different amounts of the recombinant enzyme. The determination of the d -glucose/β-galactosidase ratio (hydrolysis rate) as a function of the reaction time provides a good estimate of the extent that transgalactosylation (to lactose or d -galactose receptors) competes with complete hydrolysis during lactose conversion. The maximum value for the hydrolysis rate was100% when using 2 U of thermostable β-galactosidase/mL at 65°C (Figure 7). Note that the hydrolysis rate of lactose in milk went up when the reaction temperature and β-galactosidase amounts increased, and also increased with time. These results showed that the thermostable β-galactosidase from B. subtilis that has been produced using recombinant techniques has a high level of transgalactosylation activity.

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  • Figure 7. 

    Hydrolysis of lactose in milk by thermostable β-galactos-idase: ■ = 55°C, 1 U/mL; • = 55°C, 2 U/mL; ▴ = 60°C, 1 U/mL; ♦ = 65°C, 1 U/mL; ♦ = 65°C, 2 U/mL. Error bars represent the standard deviation from 3 separate experiments.

The thermostable β-galactosidase gene from B. stear-othermophilus, which was expressed in recombinant B. subtilis, was first reported by Hirata et al. (1985). We analyzed enzyme kinetics with substrate-like inhibitors, metal cations, EDTA, thiol reagents, and enzyme inhibitors and showed that this recombinant enzyme is generally effective in hydrolyzing lactose in milk.

One of the general methods for milk pasteurization is the low-temperature, long-time process, which involves heating the milk to a temperature between 62.8 and 65.6°C for 30min (Robinson, 1994). Considering the molecular properties, temperature profiles, and its hydrolytic activity of lactose in milk, the thermostable β-galactosidase has a potential for enzyme application in low-lactose milk production during the low-temperature, long-time process. In addition, B. subtilis is recognized as safe organism free of any endotoxin. Therefore, the thermostable β-galactosidase from B. subtilis is well suited for direct application to lactose hydrolysis during milk pasteurization.

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Conclusions 

Extensive studies on thermostable β-galactosidases and their recombinant production have been carried out previously (Panesar et al., 2006). Almost all the thermostable β-galactosidases have so far been found in thermophilic and thermotolerant microorganisms. This paper reports the purification and characterization of a recombinant thermostable β-galactosidase with a high level of transgalactosylation activity. The enzyme is active at high temperatures up to 75°C and also shows a high level of thermostability. The enzyme has a molecular weight of about 70 kDa and its optimum pH is 7.0. Some divalent cations Fe2+, Zn2+, Cu2+, Pb2+, and Sn2+ can inhibit the enzyme activity from 52 to 100% (inhibition ratio), and EDTA does not activate it. The enzyme includes a sulfhydryl group around the enzyme active site and intact disulfide groups are not essential for enzyme activity. In addition, its isoelectric point is near 5.1 and the Km and Vmax values for ONPG at 55°C are 2.96mM and 6.62μmol/min per mg. The enhanced stability properties of this recombinant thermostable β-galactosidase, combined with its neutral pH activity and favorable temperature activity optima, suggest that the enzyme is an ideal candidate for the hydrolysis of lactose in milk, and would be suitable for application in low-lactose milk production during milk pasteurization.

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Acknowledgments 

This work was financially supported by National Natural Science Foundation of China (No. 30670065) and the National High Technology Research and Development Program of China (No. 2007AA10Z316).

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PII: S0022-0302(08)71211-6

doi:10.3168/jds.2007-617

Journal of Dairy Science
Volume 91, Issue 5 , Pages 1751-1758, May 2008

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