Food Chemistry 125 (2011) 614621

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Characterisation of a thermostable family 42 b-galactosidase (BgalC) family from Thermotoga maritima showing efcient lactose hydrolysis
Priti Katrolia a, Min Zhang a, Qiaojuan Yan b, Zhengqiang Jiang a,, Chunlei Song a, Lite Li a
a b

Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, PR China Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing 100083, PR China

a r t i c l e

i n f o

a b s t r a c t
A b-galactosidase gene (TM_1195) of Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant b-galactosidase (BgalC), belonging to glycosyl hydrolase (GH) family 42, was puried to homogeneity with 23.4-fold purication and a recovery of 36.6%. Its molecular mass was estimated to be 78 kDa by SDSPAGE. 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 5.09.0 and thermostability up to 75 C. Km values of BgalC for p-nitrophenyl-b-galactopyranoside (pNPGal), o-nitrophenyl-b-galactopyranoside (oNPGal) and lactose were 1.21, 7.31 and 6.5 mM, respectively. BgalC was efcient in complete removal of lactose from milk. BgalC is signicantly one of the few b-galactosidases from family 42 displaying signicant hydrolysis of lactose. These properties make BgalC an ideal candidate for commercial use, in the production of lactose-free milk. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 6 March 2010 Received in revised form 30 June 2010 Accepted 24 August 2010

Keywords: Glycosyl hydrolase b-Galactosidase Lactose hydrolysis Thermotoga maritima Thermostability

1. Introduction b-Galactosidases (b-D-galactoside galactohydrolase or lactase; EC 3.2.1.23) are known to catalyse the conversion of lactose to glucose and galactose. This property has given the enzyme widespread use in food industries, particularly in the dairy industry for the production of galacto-oligosaccharides as probiotic foodstuffs (Gaur, Pant, Jain, & Khare, 2006; Martnez-Villaluenga, Cardelle-Cobas, Corzo, Olano, & Villamiel, 2008) and the production of low-lactose milk (Chen et al., 2008; Wang et al., 2009). Due to growing interest, some b-galactosidases from different microbial sources have been investigated (Chen et al., 2008; Hinz, van den Brock, Beldman, Vincken, & Voragen, 2004; Hu et al., 2007; Wang et al., 2009). Although a large number of b-galactosidases have been isolated and characterised, the focus has now been
Abbreviations: CAPS, (cyclohexylamino)-1-propanesulphonic acid; CHES, 2(cyclohexylamino)ethanesulfonic acid; DTT, dithiothreitol; GH, glycosyl hydrolase; IPTG, isopropyl b-D-thiogalactoside; LB, Luria-Bertani broth; MES, 2-(N-morpholino)ethane sulfonic acid; MOPS, 4-(N-morpholino)-propane sulphonic acid; NiIDA, nickeliminodiacetic acid; oNP, o-nitrophenol; oNPGal, oNP-b-galactopyranoside; PDA, potato dextrose-agar; pNP, p-nitrophenol; pNPFuc, pNP-b-fucopyranoside; pNPGal, pNP-b-galactopyranoside; SH, sulhydryl; TLC, thin-layer chromatography; BgalC, the recombinant b-galactosidase C (TM_1195) from Thermotoga maritima MSB8. Corresponding author. Address: China Agricultural University, Post Box 294, No. 17 Qinghua Donglu, Haidian District, Beijing 100083, PR China. Tel.: +86 10 62737689; fax: +86 10 82388508. E-mail address: zhqjiang@cau.edu.cn (Z. Jiang). 0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.08.075

shifted to b-galactosidases from thermophilic organisms. Thermostable b-galactosidases have signicant advantages in the production of low-lactose milk, compared to mesophilic enzymes. The enzyme reaction is more favourable at high temperatures due to several factors, such as higher reaction velocity, longer half-life of the enzyme and reduced product inhibition. Also, the high temperatures tend to inhibit microbial contamination. As pasteurisation of milk occurs at the high temperature of 6585 C, b-galactosidases that can hydrolyse lactose at high temperatures are highly desirable (Chen et al., 2008; Kang et al., 2005; Kim, Ji, & Oh, 2004; Van Laere, Abee, Schols, Beldman, & Voragen, 2000). Hence, identication and characterisation of such thermostable b-galactosidases could greatly benet the dairy industry. On the basis of amino acid similarities, b-galactosidases have been divided into four glycoside hydrolase (GH) families: 1, 2, 35 and 42 (Henrissat & Davies, 1997; Hidaka et al., 2002; Nakkharat & Haltrich, 2006; Wang et al., 2009). GH family 42 is comprised of b-galactosidases from thermophilic, psychrophilic and halophilic organisms (Di Lauro et al., 2008; Gul-Guven, Guven, Poli, & Nicolaus, 2007; Holmes et al., 1997; Hu et al., 2007; Li, Zhang, Jiang, Tang, & Cong, 2009; Ohtsu, Motoshima, Goto, Tsukasaki, & Matsuzawa, 1998). Several GH family 42 b-galactosidases have been studied in great detail. Recently, the crystal structure of b-galactosidase from Thermus thermophilus A4, a GH family 42 b-galactosidase, has been solved (Hidaka et al., 2002). b-Galactosidases are enzymes that catalyse the hydrolysis of the b-D-1,4galactosidic linkage of lactose, forming the products, galactose

P. Katrolia et al. / Food Chemistry 125 (2011) 614621

615

and glucose. However, many GH family 42 b-galactosidases are unable to hydrolyse lactose completely (Di Lauro et al., 2008; Hinz et al., 2004; Holmes et al., 1997; Shaikh, Khire, & Khan, 1999; Shipkowski & Brenchley, 2006; Van Laere et al., 2000). This may be due to inhibition by the products, galactose and glucose (Hildebrandt, Wanarska, & Kur, 2009; Park & Oh, 2010). Most of the biochemical and kinetic studies of b-galactosidase have been reported with the b-linked galactosidic substrates, o-nitrophenolb-D-galactopyranoside or p-nitrophenol-b-D-galactopyranoside, since most of the b-galactosidases show preferential hydrolysis of synthetic substrates as compared to lactose. Hence, it is necessary to identify and characterise thermostable GH family 42 b-galactosidases with respect to lactose hydrolysis in order to fully explore their commercial value in the dairy industry. BgaA from Thermotoga maritima has earlier been reported to possess transglycosylation abilities at high lactose concentrations (30%, w/v) whereas another b-galactosidase, BgalB from T. maritima, does not show lactose hydrolysis (Kim et al., 2004; Li et al., 2009). Hence it was in our interest to study the properties of the third b-galactosidase from T. maritima to gain better understanding of the functional role of enzymes within the same species. The genome of hyper-thermophilic bacteria, T. maritima contains three b-galactosidase genes, namely TM_0310 (Accession No. AAD35398.1, designated as BgalB), TM_1193 (Accession No. 08186, designated as BgaA) and TM_1195 (Accession No.U/ AAD36270.1, designated as BgalC) (Gabelsberger, Liebl, & Schleifer, 1993; Nelson et al., 1999). Previously, BgalB was cloned and expressed in Escherichia coli. Also, BgalB was puried and characterised (Li et al., 2009). In the present study, we have cloned and expressed BgalC in E. coli. BgalC shares 44% homology with BgalB. The recombinant b-galactosidase (BgalC) was puried and characterised. We have also studied the hydrolysis of lactose and explored the role of BgalC in production of lactose-free milk. It was interesting to nd that BgalC, though a GH family 42 enzyme, could hydrolyse lactose completely at high temperatures. 2. Materials and methods 2.1. Materials All the substrates used in the activity assay, namely o-nitrophenyl (oNP)-b-galactopyranoside (oNPGal), p-nitrophenyl (pNP) substrates, pNP-b-galactopyranoside (pNPGal), pNP-b-fucopyranoside (pNPFuc), pNP-a-galactopyranoside, pNP-b-glucopyranoside, pNP-a-glucopyranoside, pNP-b-D-xylopyranoside, pNP-a-arabinofuranoside and pNP-b-mannopyranoside, were purchased from Sigma Chemical Company (St. Louis, MO, USA). Chelating Sepharose (NiIDA) resin, Sephacryl S-100 and Sephacryl S-200 gel ltration matrix, used for purication of recombinant b-galactosidase, were purchased from GE Life sciences (USA). Whole milk was purchased from the local supermarket. All other chemicals used were analytical grade reagents unless otherwise stated. 2.2. Bacterial strains and plasmids The genomic DNA from T. maritima MSB8 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, DSM 3109) was used as the source of the b-galactosidase gene (TM_1195, BgalC). E. coli DH5a strain was used for DNA manipulations and amplication. E. coli BL21 (DE3) was used as host for the recombinant plasmid harbouring pET-28a (+) (Novagen, USA) and for protein expression. Recombinant DNA techniques, including plasmid extraction, restriction endonuclease digestion and DNA ligation, were performed using standard methods (Sambrook & Russell, 2001).

2.3. PCR and cloning the gene The open reading frame (ORF) encoding BgalC was retrieved from GenBank (Accession No. U/AAD36270.1). The gene was amplied by polymerase chain reaction (PCR) using a 50 -forward primer containing a restriction site for NcoI (50 -CCATGGTCGGCGTCTGTTACTATCCA-30 ; the restriction site is underlined) and 30 -reverse primer with a HindIII site (50 -AAGCTTGCGTTCGTTTTCCTTCCATA30 ). The restriction sites were chosen so as to insert a C-terminal 6 His-tag into the construct. PCR conditions were performed as follows: a hot start at 94 C for 5 min, 30 repeated cycles of 94 C for 30 s, 62 C for 30 s and 72 C for 2 min, followed by one cycle of 72 C for 10 min. The PCR products were puried from agarose gels. The puried DNA fragment was ligated to the pMD18-T and the plasmid was transformed into E. coli DH5a cells. The resulting recombinant plasmids (TM-BgalC-pMD18-T) were isolated from a positive clone and DNA sequencing was performed using a BigDye terminator cycle sequencing kit (PerkinElmer, Applied Biosystems, Norwalk, CT, USA). 2.4. Expression of BgalC in E. coli BgalC was excised from the recombinant plasmid TM-BgalCpMD18-T using one pair of restriction enzymes, NcoI and HindIII, and ligated with the pET-28a (+) vector, which had been previously digested with the same pair of restriction enzymes. The ligation of DNA-insert was conducted overnight at 16 C, using the ligation High T4 DNA ligase kit (TOYOBO Co., Osaka, Japan). The host E. coli BL21 (DE3) competent cells were transformed with the ligated recominant plasmid BgalC-pET28. The positive colonies were screened from LB (Luria-Bertani) agar-plates containing kanamycin (50 lg/ml) by the direct colony PCR, using the vectorspecic primers (T7 promoter and T7 terminator primers). One of the positive clones was sequenced to conrm that the DNA sequence was identical to the data base sequence for T. maritima. E. coli BL21 cells, transformed with the recombinant plasmid pET-28a, were grown in LB media containing 50 lg/ml of kanamycin on a rotary shaker at 200 rpm at 37 C. When the absorbance at 600 nm reached 0.6, the enzyme was expressed by inducing with 1% lactose and the culture was grown at 35 C for 24 h in a rotary shaker at 200 rpm. The cells were harvested by centrifugation at 8000 rpm for 15 min at 4 C. 2.5. Purication of b-galactosidase The cell pellet was lysed in 50 mM phosphate buffer (pH 8.0) by sonication, followed by centrifugation at 12,000 rpm for 30 min. The claried crude lysate was subjected to a heat treatment at 70 C for 10 min, followed by rapid cooling on ice. This step facilitated efcient removal of most of the E. coli proteins. The precipitated host proteins were removed by centrifugation at 12,000 rpm for 30 min at 4 C. The heat-treated sample was loaded onto a 7 ml NiIDA column after adding 300 mM NaCl and 20 mM imidazole. After binding, the column was washed with 1 column-volume of buffer A (50 mM phosphate buffer, 300 mM NaCl and 20 mM imidazole), followed by 5 columnvolumes of buffer B (50 mM phosphate buffer, 300 mM NaCl and 50 mM imidazole). The bound enzyme was eluted with buffer C (50 mM phosphate buffer, 300 mM NaCl and 200 mM imidazole) and the elution fractions were collected in the presence of 5 mM EDTA in order to prevent precipitation due to leaching of nickel. The elution was dialysed against 50 mM sodium phosphate buffer (pH 6.8) for 16 h at 4 C. The dialysed protein was concentrated with a 30 kDa membrane, using a Millipore centrifugal device (USA). The concentrated enzyme was further puried on a Sephacryl S-100 gel ltration column. The protein was

616

P. Katrolia et al. / Food Chemistry 125 (2011) 614621

loaded onto the column (100 1 cm) and eluted at a ow-rate of 0.3 ml/min in 50 mM sodium phosphate buffer, pH 6.8. The fractions from the gel ltration column were pooled and analyzed, as described. All the studies were performed using the puried enzyme. 2.6. Enzyme assay and protein determination For determination of b-galactosidase activity, 25 ll of suitably diluted enzyme solution were incubated with 5 mM pNPGal in 225 ll of 50 mM phosphate buffer (pH 7.0) at 70 C for 10 min. The reaction was terminated by adding 0.75 ml of 2.0 M Na2CO3. The enzyme activity was determined by measuring the amount of p-nitrophenol released, which was measured spectrophotometrically at 410 nm. One unit of enzyme activity was dened as 1 lmol of p-nitrophenol released per minute under the conditions described above. The protein concentration was determined by Bradfords method, using bovine serum albumin (BSA) as the standard (Bradford, 1976). Specic activity was expressed as U/mg of protein. 2.7. SDSPAGE and determination of native molecular mass The purity of the enzyme was determined on a 10% SDSPAGE by Laemmlis method (Laemmli, 1970). The molecular mass of the enzyme subunit was determined using a low molecular mass standard (Amersham) comprised of phosphorylase b (97.0 kDa), albumin (66.0 kDa), ovalbumin (45.0 kDa) and carbonic anhydrase (30.0 kDa). The protein bands were visualised by staining with Coomassie brilliant blue R-250. The native molecular mass of the enzyme was determined on a Sephacryl S-200 column by comparison with protein standards of known molecular masses. The molecular mass standards used were fetuin (68.0 kDa), albumin from chicken egg white (45.0 kDa), a-chymotrypsinogen (25.7 kDa) and cytochrome c (13.0 kDa). 2.8. Determination of pH and temperature proles The effect of pH-dependence on b-galactosidase activity was examined by incubating the enzyme (192 U/mg) and 5 mM pNPGal mixture in 50 mM buffers of different pH, namely sodium citrate (pH 3.06.0), sodium acetate (pH 4.05.5), sodium phosphate (pH 6.08.0), MOPS (pH 6.58.5), CHES (pH 8.011.0) and CAPS (pH 9.011.0) at 70 C for 10 min. Stability of the enzyme, at different pH values, was determined by incubating the enzyme at 70 C for 30 min in various buffers, as mentioned above, followed by rapid cooling on ice. The activity of the above-treated samples was determined by the standard assay method, as given above. The effect of temperature on b-galactosidase activity was assayed using 5 mM pNPGal in 50 mM sodium citrate buffer (pH 5.5) at temperatures ranging from 30 to 100 C. All the buffers were preheated at desired temperatures before adding the enzyme solution. Thermal stability of the enzyme was determined by incubating the enzyme solution at temperatures ranging from 30 to 100 C for 30 min, followed by rapid cooling on ice. The activity of the above samples, incubated at different temperatures, was assayed as described earlier. 2.9. Inuence of metal ions and other reagents The activity of b-galactosidase was measured in the presence of 1 mM individual metal ions. To further investigate the dependence of activity on metal ions, the enzyme activity was assayed in the presence of a metal-chelator, EDTA (1 mM). The enzyme was incu-

bated with the above reagents at 70 C for 30 min, cooled on ice, and the activity was determined. 2.10. Substrate specicity and kinetic parameters The substrate specicity of the enzyme was determined by assaying the enzyme with various nitrophenol substrates at a nal concentration of 5 mM in 50 mM sodium citrate buffer (pH 5.5) at 70 C for 10 min. The released o- or p-nitrophenol was measured spectrophotometrically at 410 nm, as described earlier. The kinetic parameters for BgalC were determined at six different concentrations of substrates in 50 mM sodium citrate buffer (pH 5.5) at 70 C for 5 min. For determining the kinetic parameters with lactose, the reaction was terminated by boiling for 15 min, followed by monitoring the glucose release, using a glucose-oxidase kit (Beijing BHKT clinical Reagent Co., Ltd.). Km and kcat and their standard errors were calculated by using the nonlinear regression analysis programme Grat. 2.11. Study of lactose hydrolysis property of BgalC The ability of BgalC to hydrolyse lactose was monitored as given below. Suitably diluted enzyme was incubated with 5.0% lactose (w/v) in 50 mM sodium citrate buffer (pH 5.5) at 70 C for 10 min. The reaction was terminated by boiling for 15 min, after which the amount of glucose released was measured using the glucose-oxidase kit. The absorbance at 505 nm was converted to glucose concentration, using a standard glucose curve. One unit of enzyme activity is dened as the amount of enzyme needed to produce 1 lmol of glucose per minute under the dened conditions. The hydrolysis of lactose was studied by incubating 5.0% of lactose with 2.0 U/ml of enzyme in 50 mM sodium citrate buffer (pH 5.5) at 80 C for 8 h. Samples were withdrawn at various time-intervals, boiled for 15 min to stop the reaction and analyzed by thin-layer chromatography (TLC). The reaction mixtures were deionized and spotted onto a silica gel plate (Merck Silica Gel 60F 254, Germany), and developed twice in a solvent system containing butan-1-olethanolwater (5:3:2, v/v). Saccharides were detected by heating in an oven after spraying the plates with a mixture of methanol: sulphuric acid (95:5, v/v). The concentrations of lactose were analyzed with a Sugar-D Waters column (4.6 250 mm), operated by an HPLC system equipped with a Refractive Index Detector (RID). The column was eluted at 40 C with mobile phase of 75:25 (v/v) acetonitrilewater at a ow rate of 1.0 ml/min. The hydrolysis effect of BgalC was evaluated by the following equations: lactose hydrolysis (%) = 100residual lactose concentration (g/l) 100/initial lactose concentration (g/l). 2.12. Removal of lactose from milk by BgalC Hydrolysis of lactose in milk was further studied. Different concentrations of the enzyme (0.256.0 U/ml of milk) were added (in 7.5 ml milk) and incubated at 80 C for 4 h, the reaction was stopped by adding ethanol to a nal concentration of 75% (v/v). The mixture was shaken gently for 15 min and the supernatant was collected after centrifugation at 10,000 rpm for 10 min. The amount of residual lactose in the supernatant was determined by HPLC. Untreated milk was considered as a control for the calculation of percentage of lactose hydrolysis. The time-course of lactose hydrolysis in milk was studied as follows: After addition of the enzyme (4 U/ml of milk) to the milk, the reaction was allowed to proceed for 3 h, during which aliquots were removed and analyzed for residual lactose by HPLC, as described above.

P. Katrolia et al. / Food Chemistry 125 (2011) 614621

617

3. Results and discussion 3.1. Cloning and expression of a b-galactosidase gene from T. maritima A b-galactosidase gene (TM_1195, BgalC) from genomic DNA of T. maritima (Nelson et al., 1999) was cloned and expressed in E. coli. The amplied BgalC-coding DNA fragment was inserted into the pET-28a (+) plasmid with both NcoI and HindIII restriction sites, resulting in an expression vector designated BgalC-pET28. The recombinant b-galactosidase (BgalC) was a soluble protein that contained a hexa-histidine tag at the carboxyl terminus, and it consisted of 649 amino acid residues. According to the homology search of the deduced amino acid sequence, the enzyme showed the highest degree of similarity (94% identity) to the putative b-galactosidases of Thermotoga petrophila RKU-1 (Accession No. ABQ47564.1) and of Thermotoga sp. RQ2 (Accession No. ACB09959.1) and was next close to the putative b-galactosidase from Thermotoga neapolitana (83% identity, Accession No. AAC24217.1), all belonging to GH family 42. Although the above putative b-galactosidase genes were identied from Thermotoga species, their gene products have not been characterised. The rest of the b-galactosidases showed identities of less than 59%. Particularly, BgalC showed 44% identity with BgalB, another b-galactosidase from T. maritima (Li et al., 2009). Furthermore, comparison of the BgalC protein sequence to other b-galactosidases in the Genbank database revealed that the enzyme was a member of GH family 42 (Henrissat & Davies, 1997).

Fig. 1. SDSPAGE of the recombinant b-galactosidase (BgalC) purication steps. (Lane M) low molecular weight standard; (lane 1) crude lysate; (lane 2) after heat treatment; (lane 3) after Ni-IDA; (lane 4) after Sephacryl S-100 (puried BgalC).

dases are enzymes of high molecular masses and are multimers, usually dimers or trimers, with subunit masses of about 75 kDa (Di Lauro et al., 2008; Gul-Guven et al., 2007; Hu et al., 2007; Lu et al., 2007; Mller et al., 2001; Park & Oh, 2010). 3.3. Characterisation of BgalC The effects of pH and temperature on b-galactosidase activity are examined in Figs. 2 and 3, respectively. The pH curve displayed a maximal activity of b-galactosidase at pH 5.5 in 50 mM sodium citrate buffer (Fig. 2a). Furthermore, the enzyme was stable over a broad pH range of 6.09.5, where it retained around 80% or more of its activity (Fig. 2b). This pH optimum was found to be similar to several thermostable b-galactosidases belonging to the GH family 42 such as b-galactosidase from Pyrococcus wosei (Dabrowski, ska, & Synowiecki, 1998), lactose-hydrolases from Thermus Maciun sp. IB-21 (Kang et al., 2005) and BgalB from T. maritima (Li et al., 2009). The optimum pH value for BgaA from T. maritima was 6.5 (Kim et al., 2004). The enzyme showed an optimum temperature of 80 C and its activity showed a sharp decline above 80 C, showing almost complete inactivation at 95 C (Fig. 3a). The enzyme exhibited thermal stability up to 75 C, retaining more than 90% of enzyme activity (Fig. 3b). Among the b-galactosidases isolated from thermophilic organisms, the enzyme from Thermus aquaticus displayed the highest temperature optimum of 80 C at pH 5.5 (Berger, Lee, & Lacroix, 1997), whereas the enzyme from Thermus sp. had maximum activity at 70 C at pH 6.5 (Ladero et al., 2002). Among the hyper-thermophilic organisms, those from Sulfolobus solfataricus display maximum activity at 75 C at pH 6.5 (Pisani et al., 1990). The other two b-galactosidases from T. maritima exhibit temperature optima at 80 and 85 C (Kim et al., 2004; Li et al., 2009). Thus, the thermostability of BgalC is comparable to those of other thermostable b-galactosidases. The high stability of the enzyme at high temperatures, coupled with the broad pH range makes it suitable for industrial use.

3.2. Purication of the recombinant b-galactosidase The recombinant b-galactosidase (BgalC), being a histidinetagged protein, was expressed in E. coli and could be puried to homogeneity by a simple process, which included heat treatment, afnity chromatography on Ni-IDA and gel ltration. Puried BgalC showed a specic activity of 185 U/mg (Table 1). The specic activity of the puried enzyme was increased 23.4-fold compared to that of the crude extract with a recovery yield of 36.6%. SDSPAGE of the puried enzyme displayed a single homogeneous band having an apparent molecular mass of 78 kDa (Fig. 1). The estimated subunit molecular mass of BgalC is similar to that (7080 kDa) of typical GH family 42 b-galactosidases (Gul-Guven et al., 2007; Hinz et al., 2004; Holmes et al., 1997; Hu et al., 2007; Kang et al., 2005; Li et al., 2009; Lu, Xiao, Xu, Li, & Li, 2007; Mller, Jrgensen, Hansen, Madsen, & Stougaard, 2001; Wanarska, Kur, Pladzyk, & Turkiewicz, 2005). The native molecular mass of the enzyme, BgalC, as estimated by gel ltration on Sephacryl S-200 column, was 286 kDa (data not shown), indicating that the enzyme is a trimer. The molecular mass of BgalC is thus found to be close to that of other GH family 42 b-galactosidases from Thermus sp. A4 (Hidaka et al., 2002) and Caldicellulosiruptor saccharolyticus (Park & Oh, 2010), which also has a trimeric structure. The other two b-galactosidases from T. maritima, namely BgaA and BgalB, exist as dimer (240 kDa) and monomer (78 kDa), respectively (Kim et al., 2004; Li et al., 2009). GH family 42 b-galactosidases exhibit great variations with respect to native molecular masses. Most of the GH family 42 b-galactosiTable 1 Summary of the recombinant b-galactosidase (BgalC) purication. Purication steps Crude lysate Heat treatment NiIDA Sephacryl S-100
a b

Total activity (U)a 2521 2491 1112 924

Total protein (mg)b 319 76.0 7.4 5.0

Specic activity (U/mg) 7.9 32.8 150 185

Yield (%) 100 98.8 44.1 36.6

Purication (fold) 1 4.1 19.0 23.4

Activity was measured in 50 mM phosphate buffer (pH 7.0) at 70 C for 10 min, using pNPGal as substrate. The protein was measured by the Bradford method (Bradford, 1976), using BSA as the standard.

618

P. Katrolia et al. / Food Chemistry 125 (2011) 614621

a 100
90 80 Relative activity (%)

a 100
90 80 Relative activity (%) 70 60 50 40 30 20 10 0 30 40 50 60 70 80 Temperature (C) 90 100

70 60 50 40 30 20 10 0 2 3 4 5 6 7 pH 8 9 10 11 12

b 100
90 80 Relative activity (%) 70 60 50 40 30 20 10 0 30 40 50 60 70 80 Temperature (C) 90 100

b 100

90 80 70 60 50 40 30 20 10 0
2 3 4 5 6 7 pH 8 9 10 11 12

Relative activity (%)

Fig. 2. Optimal pH (a) and pH stability (b) of the recombinant b-galactosidase (BgalC). The inuence of pH on b-galactosidase activity was determined at 70 C using 50 mM concentrations of different buffers. To determine pH stability, the remaining activity was measured after incubation for 30 min at 70 C over various pH ranges. Buffers used: acetate (+), citrate (), phosphate (j), MOPS (N), CHES (h), CAPS (}).

Fig. 3. Optimal temperature (a) and thermal stability (b) of the recombinant bgalactosidase (BgalC). The temperature prole was measured at different temperatures in 50 mM citrate buffer (pH 5.5). For determination of thermostability, the residual activity of the treated enzyme was measured according to the standard assay after a 30 min pre-incubation at different temperatures.

a-glucopyranoside, pNP-b-D-xylopyranoside, pNP-a-arabinofuranoside and pNP-b-mannopyranoside and results indicate that BgalC shows high specicity for oNPG and pNPG as compared to other substrates. Moreover, the catalytic efciency of BgalC for pNPG was found to be ninefold higher than that for oNPG and 60-fold higher than that for lactose. The catalytic efciency of BgalC for pNPG (kcat/Km = 4.23 mM1 s1) was found to be twofold higher than that of BgalB (kcat/Km = 2.13 mM1s1) (Li et al., 2009). For oNPG too, the catalytic efciency of BgalC was fourfold higher (kcat/Km = 0.47 mM1 s1) than that of BgalB (kcat/Km = 0.11 mM1 s1). A b-galactosidase from Alicylcobacillus acidocaldarius also showed a higher catalytic efciency for pNPG (1.3-fold) as compared to oNPG (Di Lauro et al., 2008). Similarly, a b-galactosidase from C. saccharolyticus exhibited higher efciency (2.4-fold) for pNPG hydrolysis than for oNPG and 211-fold than that for lactose (Park & Oh, 2010). GH family 42 b-galactosidases are found to exhibit low kcat/Km values for lactose (Park & Oh, 2010). The kinetic parameters, such as Km and Vmax, for BgalC were investigated (data not shown). Km values for pNPGal, oNPGal, pNPFuc and lactose were 1.21, 7.31, 2.56 and 6.5 mM, respectively at 70 C. The above values suggest that the enzyme displays highest afnity towards pNPGal, which is the preferred substrate. Our results indicate that BgalC has a weak afnity for oNPG, compared to some other GH family 42 b-galactosidases, e.g., Km value of 5.9 mM for the b-galactosidase in Thermus sp. A4 (Ohtsu et al., 1998) and Km of 4.9 mM for the b-galactosidase of Planococcus isolate (Sheridan & Brenchley, 2000). Further, comparison of binding afnities of BgalC with other b-galactosidases from T. maritima

The effects of various metal ions and other reagents on enzyme activity were studied (data not shown). The activity of BgalC was strongly inhibited by SDS (6.6%) and Hg2+ (1.47%). By contrast, it was activated by most metal ions (169% for Mg2+, 163% for Mg2+, 160% for Ca2+, 150% for Na+, 150% for K+, 147% for Sr2+, 146% for Mn2+, 135% for Li+, 124% for Cu2+, 119% for Ni2+ and 115% for EDTA). The enzyme activity was signicantly stimulated by many divalent and monovalent ions, such as Mg2+, Mn2+, Na+ and K+, similar to bgalactosidases from Bacillus licheniformis (Phan Trn et al., 1998) and Enterobacter agglomerans (Lu et al., 2007). Interestingly, in our study, Ca2+, which is known to inhibit many b-galactosidases, also increased the enzyme activity. This activation of enzyme in the presence of Ca2+ is particularly attractive for hydrolysis of lactose in milk, which contains Ca2+ ions. Cu2+ has been previously reported to deactivate the other two b-galactosidases, BgaA and BgalB, from T. maritima (Kim et al., 2004; Li et al., 2009) which is contrary to our ndings on BgalC where there is an increase in enzyme activity in the presence of Cu2+. Similar inhibition of enzyme activity with Cu2+ has also been reported for the b-galactosidase from Thermus sp. A4 (Ohtsu et al., 1998). 3.4. Substrate specicity and kinetic parameters The specicity of BgalC was assayed with various nitrophenyl derivatives (data not shown), such as pNPG, oNPG, pNP-b-fucopyranoside, pNP-a-galactopyranoside, pNP-b-glucopyranoside, pNP-

P. Katrolia et al. / Food Chemistry 125 (2011) 614621

619

shows some interesting results. The Km value (1.21 mM) of BgalC obtained for pNPGal was less than that obtained for BgalB (2.74 mM) from T. maritima and also it had a much higher afnity towards oNPGal (Km of 12.5 mM) in comparison to BgalB (Li et al., 2009). However, the enzyme showed very weak afnity towards oNPGal as compared to another b-galactosidase from T. maritima (Kim et al., 2004) where a Km value of 0.33 mM was obtained. Another major difference was observed in binding afnity towards pNPFuc. BgalC has a lower afnity for pNPFuc than has BgalB (Km = 1.44 mM) (Li et al., 2009). The analysis of kinetic parameters of BgalC for its natural substrate, lactose, yields some interesting results. BgalC shows a relatively low Michaelis constant for lactose at 70 C (Km = 6.5 mM), which indicates that the enzyme exhibits better binding afnity for lactose than that reported for many bgalactosidases of GH family 42, showing Km values of 1942 mM (Berger et al., 1997; Kang et al., 2005; Nakkharat & Haltrich, 2006; Ohtsu et al., 1998). For b-galactosidase from T. maritima (BgaA), the Km value for lactose was dependent on substrate concentrations; 1.6 mM at lower concentrations up to 10 mM lactose and 27.8 mM at higher concentrations of lactose (Kim et al., 2004).

3.5. Study of lactose hydrolysis property of BgalC Since lactose hydrolysis is an important property of b-galactosidase with respect to commercial application in the dairy industry, we have studied this property of the enzyme. Lactose hydrolysis assay of BgalC with 5% lactose (normal concentration of lactose in milk) were performed at 70 C for 10 min in order to assess whether BgalC is suitable for production of low-lactose milk in the dairy industry. The specic activity of BgalC with 5% lactose (138 mM) was 40.1 U/mg. A GH family 42 b-galactosidase, BgaA from Thermus sp. IB-21, was reported to show specic activity of 8.5 U/mg with 5.0% lactose (Kang et al., 2005) at 70 C and pH 7.0. Another b-galactosidase from Thermus sp. T2 was shown to have a specic activity of 13 U/mg (Koyama, Okamoto, & Furukawa, 1990). It was also reported that two GH family 42 b-galactosidases do not cleave lactose in vitro (Holmes et al., 1997; Van Laere et al., 2000). Thus, our ndings suggest that a GH family 42 b-galactosidase that can hydrolyse lactose could have important implications. Furthermore, the ability of BgalC to hydrolyse lactose at a concentration found in milk was assayed by incubating the enzyme with 5% lactose at 80 C and analyzing the hydrolysis products on TLC. As seen in Fig. 4a, even at low enzyme concentration (2.0 U/ml), BgalC is effective in hydrolysing lactose to yield galactose and glucose. The enzyme could completely hydrolyse lactose to galactose and glucose in 4 h. The hydrolysis rate was also monitored by determining decrease in lactose concentrations at increasing time-intervals. The HPLC analysis results (Fig. 4b) also conrmed that, by 4 h, 100% hydrolysis of lactose had occurred.

a 100
Lactose hydrolysis (%) 80 60 40 20 0

b
Lactose concentration (g/l)

50

100

1 2 3 4 5 Enzyme concentration (U/ml milk)

40

80 Lactose hydrolysis (%)

b 100
80 Lactose hydrolysis (%) 60 40 20 0

30

Lactose concentration Lactose hydrolysis

60

20

40

10

20

0 0 1 2 3 4 5 6 Incubation time (h) 7 8

0.5

1 1.5 2 Incubation time (h)

2.5

Fig. 4. TLC analysis (a) and HPLC data (b) of hydrolysis products from 5.0% of lactose by the recombinant b-galactosidase (BgalC). Lane M: a mixture of galactose, lactose and glucose; Samples were analyzed after 15 min, 30 min, 1 h, 2 h, 4 h, 6 h and 8 h time-intervals.

Fig. 5. Lactose hydrolysis in milk by BgalC. (a) Effect of initial enzyme concentration on the lactose hydrolysis in milk. The hydrolysis reaction was carried out at 80 C for 4 h. (b) Time-course of lactose hydrolysis in milk by BgalC. The hydrolysis reaction with 4 U of BgalC/ml of milk was carried out at 80 C for 3 h.

620

P. Katrolia et al. / Food Chemistry 125 (2011) 614621 the thermoacidophilic bacterium Alicyclobacillus acidocaldarius: Identication of the active site residues. Biochimica et Biophysica Acta, 1784(1), 292301. Gabelsberger, J., Liebl, W., & Schleifer, K. H. (1993). Cloning and characterization of b-galactoside and b-glucoside hydrolysing enzymes of Thermotoga maritima. FEMS Microbiology Letters, 109(23), 131137. Gaur, R., Pant, H., Jain, R., & Khare, S. K. (2006). Galacto-oligosaccharide synthesis by immobilized Aspergillus oryzae b-galactosidase. Food Chemistry, 97(3), 426430. Gul-Guven, R., Guven, K., Poli, A., & Nicolaus, B. (2007). Purication and some properties of a -galactosidase from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. Rittmannii isolated from Antarctica. Enzyme and Microbial Technology, 40, 15701577. Henrissat, B., & Davies, G. (1997). Structural and sequence-based classication of glycoside hydrolases. Current Opinion in Structural Biology, 7, 637644. Hidaka, M., Fushinobu, S., Ohtsu, N., Motoshima, H., Matsuzawa, H., Shoun, H., et al. (2002). Trimeric crystal structure of the glycoside hydrolase family 42 bgalactosidase from Thermus thermophilus A4 and the structure of its complex with galactose. Journal of Molecular Biology, 322(1), 7991. Hildebrandt, P., Wanarska, M., & Kur, J. (2009). A new cold-adapted b-Dgalactosidase from the Antarctic Arthrobacter sp. 32c-gene cloning, overexpression, purication and properties. BMC Microbiology, 9, 151. Hinz, S. W., van den Brock, L. A., Beldman, G., Vincken, J. P., & Voragen, A. G. (2004). b-Galactosidase from Bidobacterium adolescentis DSM20083 prefers b(1,4)galactosidase over lactose. Applied Microbiology and Biotechnology, 66, 276284. Holmes, M. L., Scopes, R. K., Moritz, R. L., Simpson, R. J., Englert, C., Pfeifer, F., et al. (1997). Purication and analysis of an extremely halophilic b-galactosidase from Haloferax alicantei. Biochimica et Biophysica Acta, 1337, 276286. Hu, J. M., Li, H., Cao, L. X., Wu, P. C., Zhang, C. T., Sang, S. L., et al. (2007). Molecular cloning and characterization of the gene encoding cold-active b-galactosidase from a psychrotrophic and halotolerant Planococcus sp. L4. Journal of Agricultural and Food Chemistry, 55, 22172224. Kang, S. K., Cho, K. K., Ahn, J. K., Bok, J. D., Kang, S. H., Woo, J. H., et al. (2005). Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: Cloning, expression, and enzyme characterization. Journal of Biotechnology, 116, 337346. Kim, C. S., Ji, E. S., & Oh, D. K. (2004). Characterization of a thermostable recombinant b-galactosidase from Thermotoga maritima. Journal of Applied Microbiology, 97(5), 10061014. Koyama, Y., Okamoto, S., & Furukawa, K. (1990). Cloning of a- and b-galactosidase genes from an extreme thermophile, Thermus strain T2 and their expression in Thermus thermophilus HB27. Applied and Environmental Microbiology, 56, 22512254. Ladero, M., Santos, A., Garcia, J., Carrascosa, A., Pessela, B., & Garcia-Ochoa, F. (2002). Studies on the activity and the stability of b-galactosidases from Thermus sp. strain T2 and from Kluyveromyces fragilis. Enzyme and Microbial Technology, 30, 392405. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685. Li, L. T., Zhang, M., Jiang, Z. Q., Tang, L., & Cong, Q. Q. (2009). Characterisation of a thermostable family 42 b-galactosidase from Thermotoga maritima. Food Chemistry, 112, 844850. Lu, L., Xiao, M., Xu, X., Li, Z., & Li, Y. (2007). A novel b-galactosidase capable of glycosyl transfer from Enterobacter agglomerans B1. Biochemical and Biophysical Research Communications, 356(1), 7884. Martnez-Villaluenga, C., Cardelle-Cobas, A., Corzo, N., Olano, A., & Villamiel, M. (2008). Optimization of conditions for galactooligosaccharide synthesis during lactose hydrolysis by b-galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP G). Food Chemistry, 107, 258264. Mller, P. L., Jrgensen, F., Hansen, O. C., Madsen, S. M., & Stougaard, P. (2001). Intraand extracellular b-galactosidases from Bidobacterium bidum and B. Infantis: Molecular cloning, heterologous expression, and comparative characterization. Applied and Environmental Microbiology, 67, 22762283. Nakkharat, P., & Haltrich, D. (2006). Purication and characterisation of an intracellular enzyme with b-glucosidase and b-galactosidase activity from the thermophilic fungus Talaromyces thermophilus CBS 236.58. Journal of Biotechnology, 123, 304313. Nelson, K. E., Clyton, R. A., Gill, S. R., Gwinn, M. L., Dodson, R. J., Haft, D. H., et al. (1999). Evidence for lateral gene transfer between archaea and bacteria from genome sequence of Thermotoga maritima. Nature, 399, 323329. Ohtsu, N., Motoshima, H., Goto, K., Tsukasaki, F., & Matsuzawa, H. (1998). Thermostable b-galactosidase from an extreme thermophile, Thermus sp. A4: Enzyme purication and characterization, and gene cloning and sequencing. Bioscience, Biotechnology, and Biochemistry, 62, 15391545. Park, A. R., & Oh, D. K. (2010). Effects of galactose and glucose on the hydrolysis reaction of a thermostable b-galactosidase from Caldicellulosiruptor saccharolyticus. Applied Microbiology and Biotechnology, 85(5), 14271435. Phan Trn, L. S., Szabo, L., Fulop, L., Orosz, L., Sik, T., & Holczinger, A. (1998). Isolation of a b-galactosidase-encoding gene from Bacillus licheniformis: Purication and characterization of the recombinant enzyme expressed in Escherichia coli. Current Microbiology, 37, 3943. Pisani, F. M., Rella, R., Raia, C. A., Rozzo, C., Nucci, R., Gambacorta, A., et al. (1990). Thermostable b-galactosidase from the archaebacterium Sulfolobus solfataricus: Purication and properties. European Journal of Biochemistry, 187(2), 321328. Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual (3rd ed.). New York: Cold Spring Harbor Laboratory Press. Shaikh, S. A., Khire, J. M., & Khan, M. I. (1999). Characterization of a thermostable extracellular b-galactosidase from a thermophilic fungus Rhizomucor sp. Biochimica et Biophysica Acta, 1472(12), 314322.

3.6. Removal of lactose from milk by BgalC We further studied the hydrolysis of lactose in milk in order to assess whether BgalC is suitable for its removal from milk. The effect of different enzyme concentrations on lactose hydrolysis was studied at 80 C for 4 h. As shown in Fig. 5a, lactose was hydrolysed, reaching up to 99% and 100% lactose hydrolysis with 2 and 4 U of BgalC/ml of milk, respectively. The time course of lactose hydrolysis in milk with 4 U of BgalC /ml of milk was monitored at 80 C for 3 h. Results indicate that BgalC was capable of completely hydrolysing lactose after 1.5 h. As shown in Fig. 5b, 56.7% lactose was hydrolysed in the rst 15 min. However, as time proceeded, the rate of lactose hydrolysis slowed down. One explanation could be a possible decrease in the concentration of lactose and increase in the concentration of the reaction products, glucose and galactose. The complete hydrolysis of lactose in milk by BgalC indicates the suitability of the enzyme for production of lactose-free milk. Although there are a few b-galactosidases of GH family 42 that can hydrolyse lactose weakly (Di Lauro et al., 2008; Hu et al., 2007; Li et al., 2009; Mller et al., 2001; Phan Trn et al., 1998; Shipkowski & Brenchley, 2006), most of them prefer to hydrolyse chromogenic substrates instead. Particularly, for thermostable bgalactosidases, complete hydrolysis of lactose is difcult to achieve since the products (galactose and glucose) tend to inhibit the enzyme activity (Kim et al., 2004; Park & Oh, 2010). This is also conrmed by our previous results which showed that BGalB, a b-galactosidase from T. maritima, could not effectively hydrolyse lactose (Li et al., 2009). Surprisingly, this study indicates that BgalC, a GH family 42 b-galactosidase was more efcient in hydrolysing lactose. 4. Conclusions The recombinant b-galactosidase (BgalC) from T. maritima belongs to GH family 42 and displays characteristics similar to those of other b-galactosidases from the same strain, such as a high temperature optimum of 80 C, broad pH stability and high thermostability. BgalC was found to be an efcient lactose-hydrolysing enzyme, having a high specic activity on lactose and a low Km value. Moreover, the enzyme was capable of complete lactose hydrolysis in milk at high temperatures. Considering the properties, temperature proles and hydrolytic activity of lactose in milk, BgalC has potential for enzyme application in lactose-free milk production. Acknowledgements This work was nancially supported by the Program for New Century Excellent Talents in University (NCET-08-0534) and the National Natural Science Foundation of China (Project No. 20776152). References
Berger, J. L., Lee, B. H., & Lacroix, C. (1997). Purication, properties and characterization of a high-molecular-mass b-galactosidase isoenzyme from Thermus aquaticus YT1. Biotechnology and Applied Biochemistry, 25, 2941. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Chemistry, 72, 248254. Chen, W., Chen, H., Xia, Y., Zhao, J., Tian, F., & Zhang, H. (2008). Production, purication, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus. Journal of Dairy Science, 91, 17511758. ska, J., & Synowiecki, J. (1998). Cloning and nucleotide Dabrowski, S., Maciun sequence of the thermostable b-galactosidase gene from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme. Molecular Biotechnology, 10(3), 217222. Di Lauro, B., Strazzulli, A., Perugino, G., La Cara, F., Bedini, E., Corsaro, M., et al. (2008). Isolation and characterization of a new family 42 b-galactosidase from

P. Katrolia et al. / Food Chemistry 125 (2011) 614621 Sheridan, P. P., & Brenchley, J. E. (2000). Characterization of a salt-tolerant family 42 b-galactosidase from a psychrophilic antarctic Planococcus isolate. Applied and Environmental Microbiology, 66(6), 24382444. Shipkowski, S., & Brenchley, J. E. (2006). Bioinformatic, genetic, and biochemical evidence that some glycoside hydrolase family 42 b-galactosidases are arabinogalactan type I oligomer hydrolases. Applied and Environmental Microbiology, 72(12), 77307738. Van Laere, K. M., Abee, T., Schols, H. A., Beldman, G., & Voragen, A. G. (2000). Characterization of a novel b-galactosidase from Bidobacterium adolescentis

621

DSM 20083 active towards transgalactooligosaccharides. Applied and Environmental Microbiology, 66, 13791384. Wanarska, M., Kur, J., Pladzyk, R., & Turkiewicz, M. (2005). Thermostable Pyrococcus woesei b-D-galactosidasehigh level expression, purication and biochemical properties. Acta Biochimica Polonica, 52, 781787. Wang, H., Luo, H. Y., Bai, Y. G., Wang, Y. R., Yang, P. L., Shi, P. J., et al. (2009). An acidophilic b-galactosidase from Bispora sp. MEY-1 with high lactose hydrolytic activity under simulated gastric conditions. Journal of Agricultural and Food Chemistry, 57(12), 55355541.