Cloning, purification and characterization of a thermostable β-galactosidase from Thermotoga naphthophila RUK-10

Process Biochemistry 49 (2014) 775–782

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Cloning, purification and characterization of a thermostable ␤-galactosidase from Thermotoga naphthophila RUK-10 Fansi Kong a , Yeqing Wang a , Shugui Cao a , Renjun Gao a,∗ , Guiqiu Xie b,∗∗ a b

Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, College of Life Science, Jilin University, Changchun 130012, China College of Pharmacy, Jilin University, Changchun 130021, China

a r t i c l e

i n f o

Article history: Received 16 December 2013 Received in revised form 12 February 2014 Accepted 13 February 2014 Available online 5 March 2014 Keywords: ␤-Galactosidase Thermostable enzyme Thermotoga naphthophila Transglycosylation

a b s t r a c t A novel ␤-galactosidase gene (Tnap1577) from the hyperthermophilic bacterium Thermotoga naphthophila RUK-10 was cloned and expressed in Escherichia coli BL21 (DE3) cells to produce ␤-galactosidase. The recombinant ␤-galactosidase was purified in three steps: heat treatment to deactivate E. coli proteins, Ni-NTA affinity chromatography and Q-sepharose chromatography. The optimum temperatures for the hydrolysis of o-nitrophenyl-␤-d-galactoside (o-NPG) and lactose with the recombinant ␤-galactosidase were found to be 90 ◦ C and 70 ◦ C, respectively. The corresponding optimum pH values were 6.8 and 5.8, respectively. The molecular mass of the enzyme was estimated to be 70 kDa by SDS-PAGE analysis. Thermostability studies showed that the half-lives of the recombinant enzyme at 75 ◦ C, 80 ◦ C, 85 ◦ C and 90 ◦ C were 10.5, 4, 1, and 0.3 h, respectively. Kinetic studies on the recombinant ␤-galactosidase revealed Km values for the hydrolysis of o-NPG and lactose of 1.31 mM and 1.43 mM, respectively. These values are considerably lower than those reported for other hyperthermophilic ␤-galactosidases, indicating high intrinsic affinity for these substrates. The recombinant ␤-galactosidase from Thermotoga naphthophila RUK-10 also showed transglycosylation activity in the synthesis of alkyl galactopyranoside. This additional activity suggests the enzyme has potential for broader biotechnological applications beyond the degradation of lactose. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction ␤-Galactosidases (EC 3.2.1.23), hydrolases that catalyze the hydrolysis of ␤-galactosides into monosaccharides, have been isolated and characterized from many different sources, including animals, plants and microorganisms [1,2]. This class of enzyme has drawn increased attention in recent years because of its hydrolysis and transglycosylation activities [3]. More specifically, as ␤-galactosidase catalyzes the hydrolysis of lactose into glucose and galactose in nature, this enzyme could have applications in the dairy industry. Approximately 80% of Asian people are lactose intolerant due to an insufficient quantity or decreased activity of intestinal lactase. In addition, many dairy products have an unpleasant sandy texture because lactose has a low solubility that gives it a tendency to crystallize at high concentrations. The catalytic activity of ␤-galactosidase could be applied to the in situ conversion of lactose to enhance the quality and

∗ Corresponding author. Tel.: +86 43185155212; fax: +86 43185155200. ∗∗ Corresponding author. Tel.: +86 13180890093. E-mail addresses: [email protected] (R. Gao), [email protected] (G. Xie). http://dx.doi.org/10.1016/j.procbio.2014.02.008 1359-5113/© 2014 Elsevier Ltd. All rights reserved.

digestibility of dairy products. Many ␤-galactosidases can also transfer galactose residues to other sugars or alcohols to form galacto-oligosaccharides (GOS) [3] or alkyl glycosides [4,5]. GOS are probiotic agents that can promote the growth of bifidobacteria in the intestine. In addition, alkyl glycosides produced by ␤-galactosidases constitute a group of non-ionic surfactants that may have a variety of applications in the pharmaceutical, detergent and food industries. Based on similarities between their functions and their amino acid sequences, ␤-galactosidases can be divided into four different glycoside hydrolase (GH) families, named 1, 2, 35 and 42. ␤-Galactosidases from GH family 2 have been the subject of many research studies (e.g., ␤-galactosidase from E. coli). However, less is known about the function of ␤-galactosidases belonging to GH family 42. ␤-Galactosidases of GH family 42 are derived mainly from extremophiles such as thermophilic and halophilic microbes [6,7]. Microbial ␤-galactosidases have attracted particular attention due to their high yields, high activity and abundance. Many of these enzymes have been cloned and expressed [8,9]. However, most of the ␤-galactosidases reported to date are derived from mesophilic organisms; consequently, the main drawback of these enzymes is low thermostability [3]. It is generally acknowledged that reactions

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catalyzed at elevated temperature may have two benefits. The first is improved reaction kinetics, which gives a higher initial reaction rate. The second benefit is that increased substrate solubility results in high volumetric productivity [10]. Therefore, in addition to improved thermostability and longer half-lives, thermophilic ␤galactosidases have additional advantages over their mesophilic counterparts in terms of reaction kinetics and utility. In the present study, the gene Tnap1577 encoding ␤galactosidase was isolated from Thermotoga naphthophila RUK-10, a hyperthermophilic bacterium with optimum cell growth at 80 ◦ C. This gene was cloned and expressed in E. coli in a soluble form with a His-tag at the N-terminus. The recombinant enzyme was purified using column chromatography. The molecular mass, biochemical properties and transglycosylation activity of this enzyme were evaluated. 2. Materials and methods 2.1. Materials o-Nitrophenyl-␤-d-galactopyranoside (o-NPG) was purchased from Sigma (St. Louis, MO, USA). All restriction enzymes were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). T4 DNA ligase, DNA polymerase and protein molecular weight standards (Blue Plus Protein Marker and Blue Plus II Protein Marker) were supplied by Beijing TransGen Biotech Co., Ltd. (Beijing, China). The glucose oxidase kit was purchased from Changchun HuiLi Biotech Co., Ltd. (Changchun, China). Silica gel plates were supplied by Branch of Qingdao Haiyang Chemical Co., Ltd. (Qingdao, China). All other chemicals were of reagent grade and were purchased from Beijing DingGuo Changsheng Biotechnology Co., Ltd. (Beijing, China). 2.2. Bacterial strains and plasmids The genomic DNA of Thermotoga naphthophila RUK-10 (Japan Collection of Microorganisms, JCM10882) was used as the PCR template, and E. coli JM109 cells were used for DNA manipulation and amplification. E. coli BL21 (DE3) cells were used for ␤-galactosidase expression. The plasmid pET-15b was used as the DNA cloning and expression vector. 2.3. Cloning and expression of ˇ-galactosidase from Thermotoga naphthophila RUK-10 The gene Tnap1577 encoding ␤-galactosidase (1.95 kb) from Thermotoga naphthophila RUK-10 was amplified by polymerase chain reaction (PCR) with EasyTaq DNA polymerase. Based on the DNA sequence of the ␤-galactosidase gene from Thermotoga naphthophila RUK-10 reported in GenBank (accession number ADA67652.1), two oligonucleotides, a 5 -forward primer (5 TGACGTACCATATGCCGGA GTCTGCTA-3 ) and a 3 reverse primer (5 -GCCATCGGATCCTTAACGTCCAGTTTCT-3 ), were designed to introduce the underlined NdeI and BamHI restriction sites, respectively. The amplified DNA fragment was purified on an agarose gel and digested with both NdeI and BamHI endonucleases. The digested DNA fragment was purified and inserted into the pET-15b plasmid digested with the same restriction enzymes. E. coli JM109 cells were transformed with the ligation mixture and plated on Luria–Bertani (LB) agar containing 1 mg/ml ampicillin. The resulting recombinant plasmid was isolated from a positive clone and confirmed by digestion with both NdeI and BamHI endonucleases. To express the enzyme, E. coli BL21 (DE3) cells transformed with the recombinant plasmid were grown with agitation in one liter of LB medium containing 1 mg/ml ampicillin at 37 ◦ C. Isopropyl-␤d-thiogalactopyranoside (IPTG, 0.7 mM) was added to the culture

when it reached an optical density at 600 nm (O.D.600 ) of 1.0. The medium was further incubated with agitation at 150 rev min−1 and 25 ◦ C for 12 h. 2.4. Enzyme purification The induced cells were harvested with centrifugation and washed once with 50 mM sodium phosphate buffer, pH 7.0. A 5 g dispersion of cells in 50 ml of 50 mM sodium phosphate buffer, pH 7.0 containing 0.4 M NaCl was disrupted by sonication. Cellular debris was removed by centrifugation (15,000 × g for 15 min at 4 ◦ C) to obtain the crude lysate. The crude extract was incubated in a water bath for 10 min at 80 ◦ C to denature E. coli proteins. The extract was then centrifuged to separate the crude enzyme from heat-denatured cellular components and heat-denatured proteins. The supernatant was loaded onto a 5 ml Ni-NTA agarose resin column equilibrated with 20 mM sodium phosphate buffer (pH 7.0) containing 0.4 M NaCl. The enzyme was eluted on a linear gradient of 0–200 mM imidazole in 20 mM sodium phosphate buffer (pH 7.0) containing 0.4 M NaCl. The protein solution was further purified by Q-sepharose chromatography after dialysis with 20 mM sodium phosphate buffer (pH 7.5). The protein solution was loaded onto a 5 ml Q-sepharose column equilibrated with 20 mM sodium phosphate buffer (pH 7.5). The enzyme was then eluted on a linear gradient of 0–1 M NaCl sodium phosphate buffer (pH 7.5). The molecular mass and purity were determined on a 12% SDS-PAGE gel, and the protein concentration was determined using the Bradford (1976) assay with bovine serum albumin (BSA) as the standard. 2.5. ˇ-Galactosidase activity assay ␤-Galactosidase activity was measured using o-NPG as an artificial substrate. Kinetic parameters were determined using a UV–Visible Spectrophotometer 2550 (SHIMADZU, Tokyo, Japan) combined with UVProbe 2.33 software. The change in absorbance at 420 nm (A420 ) over 1 min was observed using 1 ml of the reaction mixture containing 0.5 mM o-NPG, 50 mM sodium phosphate buffer (pH 6.8) and an appropriate amount of ␤-galactosidase at 80 ◦ C. One unit of enzyme activity was defined as the hydrolysis of 1 ␮mol of o-NP per minute under the defined conditions. To determine the lactose hydrolysis activity of the enzyme, 1 mg/ml of enzyme was added to a reaction mixture containing 5% lactose and 50 mM acetate-acetic acid buffer (pH 5.8) to achieve a final volume of 1 ml. The mixture was incubated at 70 ◦ C for 30 min, which is the standard assay method. The amount of glucose produced during incubation was assessed using a glucose oxidase kit and observing changes in absorbance at 510 nm (A510 ). One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 ␮mol of glucose per minute under the defined conditions. 2.6. Effects of pH and temperature on enzyme activity The temperature dependence of enzyme activity was determined by measuring the hydrolysis of o-NPG and lactose over the temperature ranges of 50–93 ◦ C and 50–95 ◦ C, respectively. Reactions were performed in 50 mM sodium phosphate (pH 6.8). To estimate its thermostability, the enzyme (0.2 mg/ml) was preincubated in 50 mM sodium phosphate (pH 6.8) at 75 ◦ C, 80 ◦ C, 85 ◦ C and 90 ◦ C. Samples were withdrawn at specific time intervals, and residual activity was measured using the standard assay method with o-NPG as the substrate. The optimum pH for ␤-galactosidase activity was determined at 80 ◦ C and 70 ◦ C for the hydrolysis of o-NPG and lactose, respectively. Various 50 mM buffer systems were examined: sodium acetate-acetic acid buffer (pH 4.0–5.8), sodium phosphate buffer

F. Kong et al. / Process Biochemistry 49 (2014) 775–782

(pH 5.8–8.0) and Tris–HCl buffer (pH 7.5–8.9). To determine pH stability, the enzyme (0.2 mg/ml) was incubated at 70 ◦ C in buffer at various pH values. Residual enzyme activity was determined at various time intervals under the standard assay conditions using o-NPG as the substrate.

2.7. Effects of various cations The catalytic activity of ␤-galactosidase toward the hydrolysis of o-NPG was measured in the presence of EDTA and various metal cations with a final concentration of 1 mM at 80 ◦ C in 50 mM Tris–HCl buffer (pH 8.0). The activity of the enzyme solution under the same conditions with no added cations was used as a comparison.

2.8. Kinetic analysis Various concentrations of o-NPG (0.08 to 0.5 mM) were used to determine the kinetic parameters of the enzyme. All experiments were performed in 50 mM phosphate buffer (pH 6.8) for 5 min at 80 ◦ C. The kinetic parameters of the enzyme for lactose hydrolysis were determined at 70 ◦ C in acetate-acetic acid buffer (pH 5.8) for 30 min by varying the concentration of lactose (0.33–3 mM). Values for Km (mM) and Vmax (␮mol min−1 mg−1 ) were calculated via Lineweaver–Burk Plot.

2.9. Transglycosylation Transglycosylation tests were performed at 70 ◦ C with lactose as the sugar donor and n-butanol as the acceptor. The reaction mixture contained 180 ␮l of lactose (36 mg) in 20 mM sodium phosphate buffer (pH 6.0), 20 ␮l of the enzyme (0.26 U) and 1.8 ml of acceptor (n-butanol, n-hexanol or n-octanol) in a total volume of 2 ml. Samples were withdrawn at defined time intervals. The transglycosylation product was analyzed by thin-layer chromatography (TLC). Reaction mixtures were loaded onto silica gel plates, separated in a solvent system containing methanol–chloroform–acetic acid–water (30:60:5:5, v/v) and dried. Plates were stained with ␣-naphthol (2.56 g/l) in an ethanol:sulfuric acid mixture (90:10, v/v). Carbohydrates were detected by heating for a few minutes at 110 ◦ C.

2.10. Purification of alkyl galactopyranosides Organic phases were separated from aqueous phases by centrifugation. The products were then concentrated using rotary evaporation. The residues of the organic phases were redissolved in 2 ml of mobile phase described above, and the alkyl galactopyranosides in the mixture were separated from other components with silica gel chromatography. The products were monitored by TLC, and the collected fractions were evaporated to remove the mobile phase. Purified butyl galactopyranoside, hexyl galactopyranoside and octyl galactopyranoside were obtained.

2.11. Mass spectrometry and nuclear magnetic resonance The purified alkyl galactopyranosides were analyzed with high performance liquid chromatography-electrospray tandem mass spectrometry (Agilent 1290-microTOF Q II, Bruker) equipped with a C18 column (4.6 × 50 mm, 3.5 ␮m particle size). NMR spectra (1 H, 500 MHz) were acquired on a Bruker AVANCE III500.

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3. Results and discussion 3.1. Gene cloning and enzyme expression A 1,956 bp gene encoding a putative ␤-galactosidase sequence from Thermotoga naphthophila RUK-10, with the same sequence as that reported in GenBank, was cloned and expressed in E. coli. A protein with a calculated molecular mass of approximately 70,000 Da was expressed as a soluble protein containing a hexa-histidine tag at the N-terminus. Expression conditions were optimized by varying IPTG concentration (0.1–1 mM) and induction temperature (25 ◦ C, 30 ◦ C and 37 ◦ C). A maximum expression level was obtained after incubation at 25 ◦ C for 12 h with 0.7 mM IPTG. Based on the Carbohydrate-Active Enzymes databank (http://www.cazy.org) and amino acid sequence alignment, ␤-galactosidase from Thermotoga naphthophila RUK-10 can be classified as a member of the GH-42 family. Comparison of the amino acid sequences of Thermotoga naphthophila RUK-10 ␤galactosidase with those from other bacterial GH-42 members is shown in Fig. 1. Inspection of the amino acid sequence alignment reveals that the catalytic residues of ␤-galactosidase from Thermotoga naphthophila RUK-10 are Glu141 and Glu314, which correspond to Glu141 and Glu312 of the ␤-galactosidase from Thermus sp. A4 [11]. As expected, these two amino acid residues are highly conserved in all GH42 members. 3.2. Purification and molecular mass determination of Thermotoga naphthophila RUK-10 ˇ-galactosidase The recombinant ␤-galactosidase was purified in a three-step process. Heat treatment was applied at 80 ◦ C to deactivate other non-thermostable proteins, followed by purification using Ni-NTA agarose affinity chromatography and Q-sepharose chromatography. The ␤-galactosidase was purified 12.88-fold, based on specific activity, with total activity recovery of 16.7%. Approximately 6185.88 U of purified recombinant enzyme was obtained per liter of culture, giving a final specific activity of 1294.12 U/mg (Table 1). The molecular mass of purified ␤-galactosidase was 70 kDa, as determined by SDS-PAGE (Fig. 2A). This finding was consistent with the calculated value of 71,610 Da based on the 651-residue amino acid sequence. Non-denaturing PAGE shows that the molecular mass of the native enzyme was approximately 130–140 kDa (Fig. 2B), indicating that the recombinant enzyme is a homodimer. Most reported recombinant ␤-galactosidases are multimeric enzymes. Mesophilic ␤-galactosidases from Haloferax alicantei [9] and thermostable ␤-galactosidases from Rhizomucor sp. [12] are homodimers, whereas ␤-galactosidase from Sulfolobus solfataricus [13] is a homotrimer. Most of the reported microbial ␤-galactosidases from GH family 42 have a molecular mass of 70–80 kDa, such as ␤-galactosidases from Thermus sp. A4 [7] and Thermotoga maritima (BgalB) [14]; these enzymes are monomeric proteins with molecular masses of 75 and 78 kDa, respectively. However, ␤-galactosidase from Bacillus licheniformis is observed as a homodimer with a subunit mass of 75 kDa [15], and Thermotoga maritima (BgalC) produces a homotrimeric ␤-galactosidase with a subunit mass of 78 kDa. [16] The specific activity of purified ␤-galactosidase from Thermotoga naphthophila RUK-10 is higher than that shown by enzymes from Thermus sp. A4 [7] and Alicyclobacillus acidocaldarius subsp. rittmannii [17] but lower than that of ␤-galactosidase from Bacillus sp. [18]. 3.3. Effects of temperature and pH The optimum temperature and pH for o-NPG hydrolysis with the purified ␤-galactosidase were found to be 90 ◦ C and pH 6.8 (Fig. 3A and B). The pH effect was measured at 80 ◦ C, and the

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Fig. 1. Multiple alignments of the amino acid sequence of ␤-galactosidase from T. naphthophila (Tnap1577; GenBank accession no. ADA67652.1), ␤-galactosidases from Thermus sp. A4 (A4-bGal; BAA28362), Thermus sp. IB-21 (IB-bGal; AAN05443) and Haloferax lucentense SB1 (Hal-bGal; AAB40123). The gray down arrows indicate the conserved catalytic glutamic acid residues. The black arrows domain structure (A, B, and C) defined as Hidaka et al. (2002). Domain A contains the catalytic residues; domain B is involved in trimer formation; and the function of domain C is unknown.

enzyme achieved greater than 80% of the maximum activity over the pH range from 6.5 to 6.9 (Fig. 3B). However, even a small variation in pH (0.1 pH unit) led to a marked decrease in activity. This finding indicates that at elevated temperature (80 ◦ C), the ␤-galactosidase is very sensitive to changes in pH. When lactose was used as the substrate, the optimum conditions were different for the same enzyme: 70 ◦ C and pH 5.8 (Fig. 3A and C). The

thermostability of ␤-galactosidase was examined by measuring the time course of its residual activity at different temperatures (Fig. 4A). The half-lives of the enzyme at 75 ◦ C, 80 ◦ C, 85 ◦ C and 90 ◦ C were found to be 10.5, 4, 1 and 0.3 h, respectively. The enzyme showed obvious thermal activation at 75 ◦ C and 80 ◦ C, likely due to conformational changes in the enzyme at elevated temperature that enhanced its catalytic activity toward hydrolysis

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Table 1 Purification of recombinant ␤-galactosidase from Thermotoga naphthophila RUK-10. Total protein (mg) Crude enzyme Heat treatment Ni-NTA chromatography Q-sepharose chromatography a

368.6 25.1 6.14 4.78

Total activity (U) 37,050 16,011.9 7003.5 6185.9

Specific activity (U/mg)a

Purification fold

Recovery (%)

100.5 638.4 1140.6 1294.1

1 6.4 11.4 12.9

100 43.2 18.9 16.7

Activity was measured in 50 mM phosphate buffer (pH 6.8) at 80 ◦ C using 0.5 mM o-NPG as substrate.

of the substrate. The recombinant enzyme was very stable at pH 6.8 and 70 ◦ C, with no obvious decline in activity observed over 12 h. The enzyme retained 57–67% of its original activity after incubation at 70 ◦ C in buffers between pH 6.0 and 7.0 (Fig. 4B). The properties of ␤-galactosidase may differ significantly depending on its source, as listed in Table 2. The ␤-galactosidases from hyperthermophiles such as Thermotoga maritima (BgalC) [16], Pyrococcus woesei [19], and Thermus sp. IB-21 (BgaA) [20] are among the group of highly thermostable galactosidases that typically exhibit optimal activity between 80 and 95 ◦ C and from pH 5.5–6.5 using o-NPG or p-NPG as a substrate. The ␤-galactosidase from Thermotoga naphthophila RUK-10 studied in this work can be placed in the same category, with its optimal activity at 90 ◦ C and pH 6.8. The half-lives of the ␤-galactosidase from Thermotoga naphthophila RUK-10 were 10.5, 4 and 1 h at 75 ◦ C, 80 ◦ C and 85 ◦ C, respectively. In comparison, the half-lives of the hyperthermophilic ␤-galactosidase from Sulfolobus solfataricus [13] were 24, 10 and 3 h at 75 ◦ C, 80 ◦ C and 85 ◦ C, respectively, and the half-life of ␤-galactosidase from Rhizomucor sp. [12] was 150 min at 70 ◦ C. ␤-Galactosidase from Thermus sp. A4 [7] retained 75% of its activity after incubation at 85 ◦ C for 2 h. Although thermal inactivation was

Fig. 2. SDS-PAGE (A) and Native-gradient PAGE (B) analysis of the recombinant Tnap1577. Lane M: Blue plus protein marker; lane 1: the crude extract; lane 2: the supernatant after heat treatment at 80 ◦ C for 10 min; lane 3: the recombinant ␤-galactosidase purified by Ni-NTA chromatography; lane 4: the recombinant ␤-galactosidase purified by Q-sepharose chromatography; lane 5: the whole cell products.

Fig. 3. Effects of temperature (A) and pH (B and C) on the activity of ␤-galactosidase from T. naphthophila. The temperature profile was measured in 50 mM phosphate buffer (pH 6.8) with o-NPG () or lactose () as substrates. pH effect was measured at 80 ◦ C for o-NPG (B) or at 70 ◦ C for lactose (C) in 50 mM different buffers: sodium acetate-acetic acid buffer (), sodium phosphate buffer (䊉) and Tris–HCl buffer ().

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a potential limitation, the ␤-galactosidase from Thermotoga naphthophila RUK-10 showed considerable thermal stability compared to other thermostable ␤-galactosidases. 3.4. Kinetic parameters and effect of various cations on ˇ-galactosidase activity

Fig. 4. Thermostability (A) and pH stability (B) of the recombinant enzyme. To determine thermostability, the residual activities of the enzyme with time change in 50 mM phosphate buffer (pH 6.8) at 75(), 80(䊉), 85 () and 90 ◦ C () were measured, respectively. The remained activities of the enzyme at 70 ◦ C and different pH values (pH 6.0 (), 6.5 (䊉), 6.8 (), 7.0 (), 7.5(), 8.0 ()), used as an index to monitor its pH stability, were measured, respectively.

Table 2 Effect of various cations on catalytic activity of ␤-galactosidase from Thermotoga naphthophila RUK-10a Reagentb

Relative activity (%)

Blank EDTA K+ Na+ Mg2+ Ca2+ Mn2+ Ni2+ Cu2+ Zn2+

100 126 105 103 94 94 76.5 66.2 61.1 15.6

Values are the average of duplicate experiments. a Enzymatic reactions were carried out for 1 min at 80 ◦ C in 50 mM Tris–HCl buffer(pH 8.0) using o-NPG as substrate. b Each cation and reagent was added to the reaction mixture at final concentrations of 1 mM respectively.

The influence of different metal ions on enzymatic activity was investigated (Table 2). Individual cations were added to enzyme reaction mixtures to achieve final concentrations of 1.0 mM. Addition of the monovalent ions K+ and Na+ increased enzyme activity by 5% and 3%, over the control, respectively. The addition of Mg2+ and Ca2+ had negligible effects on enzyme activity, with each showing a relative activity of 94%. Divalent ions such as Mn2+ , Ni2+ , Cu2+ and Zn2+ decreased enzyme activity to 76.5%, 66.2%, 61.1% and 15.6% of the control, respectively. By contrast, EDTA exerted an activation effect of 126%, suggesting that the ␤-galactosidase from Thermotoga naphthophila RUK-10 does not require metal ions to catalyze reactions. The presence of Mg2+ is beneficial to the activity of most ␤-galactosidases reported to date (e.g., Kluyveromyces lactis [8] and Lactobacillus reuteri [21]). The addition of Ca2+ inhibits ␤galactosidase activity in some cases (e.g., Lactobacillus reuteri [21] and Paracoccus sp. 32d [22]). However, metal ions have no effect on the activity of some thermostable ␤-galactosidases, such as those from Sulfolobus solfataricus [13] and Bacillus coagulans [23]. This result has also been observed for some ␤-galactosidases from GH family 42 (e.g., Thermotoga maritima (BgalB) [14] and Planococcus isolate [6]). However, Mg2+ is required for the catalytic activity ␤galactosidases of GH family 42 from Thermotoga maritima (BgalC) [16] and Bacillus sp. [18]. Enzyme kinetic parameters were calculated using a Lineweaver–Burk plot of the Michaelis–Menten equations at different concentrations of substrate. The Km and Vmax values for o-NPG hydrolysis at 80 ◦ C were 1.31 mM and 3385.67 ␮mol min−1 mg−1 , respectively. The Km and Vmax values for lactose hydrolysis at 70 ◦ C were 1.43 mM and 2.67 ␮mol min−1 mg−1 , respectively. The kcat values calculated on the basis of the theoretical Vmax for the hydrolysis of o-NPG and lactose were 4040.79 s−1 and 3.18 s−1 , respectively. Many other thermostable ␤-galactosidases have been cloned and expressed in recent years. The properties of some reported thermostable ␤-galactosidases are presented in Table 3 for comparison. The recombinant Thermotoga naphthophila RUK-10 ␤galactosidase studied in this work showed optimum catalysis at 90 ◦ C, revealing its hyperthermophilic properties. These enzymatic properties would be significantly advantageous in applications such as glyco-conjugation reactions. The Michaelis constants for ␤-galactosidase from Thermotoga naphthophila RUK-10 with both o-NPG and lactose were much lower than those reported for most known ␤-galactosidases. ␤-Galactosidase from Thermotoga naphthophila RUK-10 was the most effective enzyme when o-NPG was used as the substrate; this enzyme hydrolyzes lactose poorly and prefers to hydrolyze ␤-linked galactosidic substrates such as oNPG. Our results indicate that ␤-galactosidase from Thermotoga naphthophila RUK-10 possesses unique characteristics that will be valuable for various aspects of future research on ␤-galactosidases. 3.5. Transglycosylation activity Many ␤-galactosidases have been employed to synthesize GOS [4] in monophasic aqueous systems. Our experiments indicate that ␤-galactosidase from Thermotoga naphthophila RUK-10 did not synthesize GOS in such a system (data not shown). However, when a mixed aqueous/organic solvent system was employed instead [4,5,24], ␤-galactosidase from Thermotoga naphthophila

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Table 3 Comparison of some thermostable ␤-galactosidases properties from GH family 42. Strain

Thermus sp.A4 Thermotoga maritima(BgalB) Bacillus licheniformis Thermotoga maritima(BgalC) Alicyclobacillus acidocaldarius subsp. rittmannii Thermus sp.IB-21(BgaA) Alicyclobacillus acidocaldarius Thermotoga naphthophila *

Optimum temperature(◦ C)

>90

Optimum pH

Substrate

6.5

o-NPG lactose

80

5.5

*

p-NPG

50 50 80

6.5 6.5 5.5

o-NPG lactose p-NPG lactose

65

6.0

o-NPG

90

5.0–6.0

p-NPG lactose

70

5.8

o-NPG

90 70

6.8 5.8

o-NPG lactose

Kinetic parameters

Km (mM)

Vmax (␮mol min−1 mg−1 )

5.9(70 ◦ C) 19(70 ◦ C) 2.74 ± 0.22 (70 ◦ C) 13.7 ± 0.1(70 ◦ C) 169 ± 0.8 (70 ◦ C) 1.21 (70 ◦ C) 6.5 (70 ◦ C) 8.9 (65 ◦ C)

– – 351 ± 11.3 299 ± 2.3 13 ± 0.04 – – –

0.41 (70 ◦ C) 42 (70 ◦ C) 6.0 (60 ◦ C)

140 8.5 –

◦

1.31 (80 C) 1.43 (70 ◦ C)

3385.67 2.67

Molecular mass (kDa)

Refs.

75

[7]

78

[14]

75/160

[15]

78/286

[16]

76/165

[17]

73

[20]

83

[26]

70/120

This study

p-Nitriphenyl-ˇ-d-galactopyranoside.

RUK-10 catalyzed the synthesis of alkyl galactopyranosides from lactose and alkyl alcohols. TLC analysis of the reaction mixtures (Fig. 5) indicated the appearance of alkylglycoside derivatives, suggesting that the ␤-galactosidase from Thermotoga naphthophila RUK-10 catalyzed transglycosylation of lactose. The yields of butyl galactopyranoside, hexyl galactopyranoside and octyl galactopyranoside were 9.5%, 6.91% and 1.44% separately, which were lower than previous reports [4,24]. Concomitant hydrolysis of lactose to glucose and galactose was also observed. HPLC-MS product ion spectra indicated peaks at m/z 259.1, 265.2 and 293.2, which correspond to the molecular weight of butyl (M + Na+ ), hexyl (M + H+ ) and octyl (M + H+ ) galactopyranoside, respectively. 1 H NMR (500 MHz, DMSO) data were obtained as follows: butyl galactopyranoside: ı/ppm 0.82–0.91(m, 3H), 1.28–1.38 (m, 2H), 1.46–1.54 (m, 2H), 3.21–3.27 (m, 2H), 3.28–3.30 (m, 1H), 3.36–3.49 (m, 2H), 3.49–3.57 (m, 1H), 3.59–3.66 (s, 1H), 3.68–3.79 (dt, 1H), 4.00–4.08 (m, 1H), 4.27–4.33 (d, 1H), 4.48–4.56 (t, 1H), 4.62–4.68 (d, 1H), 4.75–4.81 (t, 1H); hexyl galactopyranoside: ı/ppm 0.81–0.89(t, 3H), 1.21–1.34 (tdd, 6H), 1.46–1.53 (m, 2H), 3.22–3.26 (d, 2H), 3.26–3.30 (m, 1H), 3.38–3.48 (m, 2H), 3.48–3.54 (dd, 1H), 3.59–3.65 (s, 1H), 3.68–3.74 (m, 1H), 4.02–4.07 (d, 1H), 4.28–4.34 (d, 1H), 4.49–4.54 (t, 1H), 4.61–4.67 (d, 1H), 4.75–4.80 (d, 1H); octyl galactopyranoside: ı/ppm 0.82–0.91(t, 3H), 1.20–1.34 (m, 10H), 1.46–1.53 (m, 2H), 3.23–3.26 (m, 2H), 3.27–3.30 (dd, 1H), 3.37–3.48 (m, 2H), 3.49–3.54 (m, 1H), 3.60–3.64 (s, 1H), 3.68–3.74

(dt, 1H), 4.01–4.06 (t, 1H), 4.28–4.33 (d, 1H), 4.50–4.55 (t, 1H), 4.62–4.68 (d, 1H), 4.74–4.80 (d, 1H). Our results demonstrate that the yield of galactosylated derivatives decreased as the carbon chain length of the aliphatic alcohols increased. As shown in Fig. 5, the synthesis of octyl galactopyranoside (96 h) was much slower than the corresponding formation of butyl galactopyranoside (24 h). The yield of product derived from octanol was also lower. Alkyl glycosides behave as biosurfactants and have potential applications as pharmaceuticals, detergents, and food ingredients. It has been reported that short-chain alkyl glycosides are not effective detergents and that good surface-active properties are observed only when the alkyl chain length is greater than eight carbons [25]. The reaction system described in this work requires further optimization to improve the yield of galactosylated derivatives from higher alkyl alcohols. The parameters to be investigated include increased reaction temperatures or additional organic cosolvents. Further studies on the synthesis of alkyl glycosides are in progress in our lab.

4. Conclusions Recombinant ␤-galactosidase from Thermotoga naphthophila RUK-10 has been successfully cloned and expressed in E. coli. Sequence analysis suggests that this enzyme belongs to GH family 42. The enzyme showed maximum o-NPG hydrolysis activity at 90 ◦ C and pH 6.8, and it showed optimum lactose hydrolysis activity at 70 ◦ C and pH 5.8. The recombinant enzyme showed transglycosylation activity with aliphatic alcohols when lactose was used as the sugar donor in an aqueous/organic biphasic solvent system. The excellent thermostability and promising transglycosylation activity of this novel enzyme suggest that it will be a useful tool in industrial applications, especially for the synthesis of alkyl galactopyranosides.

Acknowledgements Fig. 5. TLC analysis of transglycosylation reaction products. Lane 1: 1% glucose; lane 2: mixture of 1% galactose and lactose; lane 3: product of n-butanol reaction system (butyl galactopyranoside, 24 h); lane 4: product of n-hexanol reaction system (hexyl galactopyranoside, 72 h); lane 5: product of n-octanol reaction system (octyl galactopyranoside, 96 h).

The authors are grateful to the National Natural Science Foundation of China (No. 20772046 and No. 21072075) and to the National High Technology Research and Development Program (“863” Program) of China (No 2013AA102104) for financial support.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.procbio.2014. 02.008. References [1] Kishore D, Kayastha AMA. ␤-Galactosidase from chick pea (Cicer arietinum) seeds: its purification, biochemical properties and industrial applications. Food Chem 2012;134:1113–22. [2] Song C, Chi Z, Li J, Wang X. ␤-Galactosidase production by the psychrotolerant yeast Guehomyces pullulans 17-1 isolated from sea sediment in Antarctica and lactose hydrolysis. Bioprocess Biosyst Eng 2010;33:1025–31. [3] Lu L, Xiao M, Li Z, Li Y, Wang F. A novel transglycosylating ␤-galactosidase from Enterobacter cloacae B5. Process Biochem 2009;44:232–6. [4] Makowski K, Białkowska A, Olczak J, Kur J, Turkiewicz M. Antarctic, coldadapted ␤-galactosidase of Pseudoalteromonas sp. 22b as an effective tool for alkyl galactopyranosides synthesis. Enzyme Microb Technol 2009;44:59–64. [5] Ismail A, Soultani S, Ghoul M. Enzymatic-catalyzed synthesis of alkyl glycosides in monophasic and biphasic systems. I. The transglycosylation reaction. J Biotechnol 1999;69:135–43. [6] Sheridan PP, Brenchley JE. Characterization of a salt-tolerant family 42 ␤galactosidase from a psychrophilic antarctic Planococcus isolate. Appl Environ Microbiol 2000;66:2438–44. [7] Ohtsu N, Motoshima H, Goto K, Tsukasaki F, Matsuzawa H. Thermostable ␤-galactosidase from an extreme thermophile, thermus sp. A4: Enzyme purification and characterization, and gene cloning and sequencing. Biosci Biotechnol Biochem 1998;62:1539–45. [8] Kim CS, Ji ES, Oh DK. Expression and characterization of Kluyveromyces lactis ␤-galactosidase in Escherichia coli. Biotechnol Lett 2003;25:1769–74. [9] Holmes ML, Scopes RK, Moritz RL, Simpson RJ, Englert C, Pfeifer F, et al. Purification and analysis of an extremely halophilic ␤-galactosidase from Haloferax alicantei. Biochim Biophys Acta 1997;1337:276–86. [10] García-Garibay M, López-Munguía A, Barzana E. Alcoholysis and reverse hydrolysis reactions in organic one-phase system with a hyperthermophilic ␤-glycosidase. Biotechnol Bioeng 2000;69:627–32. [11] Hidaka M, Fushinobu S, Ohtsu N, Motoshima H, Matsuzawa H, Shoun H, et al. Trimeric crystal structure of the glycoside hydrolase family 42 ␤-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose. J Mol Biol 2002;322:79–91. [12] Shaikh SA, Khire JM, Khan MI. Characterization of a thermostable extracellular ␤-galactosidase from a thermophilic fungus Rhizomucor sp. Biochim Biophys Acta 1999;1472:314–22.

[13] Pisani FM, Rella R, Raia CA, Rozzo C, Nucci R, Gambacorta A, et al. Thermostable ␤-galactosidase from the archaebacterium Sulfolobus solfataricus. Purification and properties. Eur J Biochem 1990;187:321–8. [14] Li L, Zhang M, Jiang Z, Tang L, Cong Q. Characterisation of a thermostable family 42 ␤-galactosidase from Thermotoga maritima. Food Chem 2009;112:844–50. [15] Juajun O, Nguyen TH, Maischberger T, Iqbal S, Haltrich D, Yamabhai M. Cloning, purification, and characterization of ␤-galactosidase from Bacillus licheniformis DSM 13. Appl Microbiol Biotechnol 2011;89:645–54. [16] Katrolia P, Zhang M, Yan Q, Jiang Z, Song C, Li L. Characterisation of a thermostable family 42 ␤-galactosidase (BgalC) family from Thermotoga maritima a showing efficient lactose hydrolysis. Food Chem 2011;125: 614–21. [17] Gul-Guven R, Guven K, Poli A, Nicolaus B. Purification and some properties of a ␤-galactosidase from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica. Enzyme Microb Technol 2007;40:1570–7. [18] Chakraborti S, Sani RK, Banerjee UC, Sobti RC. Purification and characterization of a novel ␤-galactosidase from Bacillus sp MTCC 3088. J Ind Microbiol Biotechnol 2000;24:58–63. ˛ ´ [19] Dabrowski S, Maciunska J, Synowiecki J. Cloning and nucleotide sequence of the thermostable ␤-galactosidase gene from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme. Mol Biotechnol 1998;10: 217–22. [20] Kang SK, Cho KK, Ahn JK, Bok JD, Kang SH, Woo JH, et al. Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization. J Biotechnol 2005;116:337–46. [21] Nguyen TH, Splechtna B, Steinböck M, Kneifel W, Lettner HP, Kulbe KD, et al. Purification and characterization of two novel ␤-galactosidases from Lactobacillus reuteri. J Agric Food Chem 2006;54:4989–98. ´ [22] Wierzbicka-Wo´s A, Cie´slinski H, Wanarska M, Kozłowska-Tylingo M, Hildebrandt P, Kur J. A novel cold-active ␤-d-galactosidase from the Paracoccus sp. 32d-gene cloning, purification and characterization. Microb Cell Fact 2011;10:108. [23] Batra N, Singh J, Banerjee UC, Patnaik PR, Sobti RC. Production and characterization of a thermostable ␤-galactosidase from Bacillus coagulans RCS3. Biotechnol Appl Biochem 2002;36:1–6. [24] Ismail A, Linder M, Ghoul M. Optimization of butylgalactoside synthesis by ␤-galactosidase from Aspergillus oryzae. Enzyme Microb Technol 1999;25:208–13. [25] Rather MY, Mishra S, Verma V, Chand S. Biotransformation of methyl-␤-dglucopyranoside to higher chain alkyl glucosides by cell bound ␤-glucosidase of Pichia etchellsii. Bioresour Technol 2012;107:287–94. [26] Yuan T, Yang P, Wang Y, Meng K, Luo H, Zhang W, et al. Heterologous expression of a gene encoding a thermostable ␤-galactosidase from Alicyclobacillus acidocaldarius. Biotechnol Lett 2008;30:343–8.