INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

-Galactosidases (-Gals) are known to occur widely in microorganisms, plants, and animals, and some of them have been purified and characterized (5). -Gals catalyze the hydrolysis of 1,6-linked -galactose residues from oligosaccharides and polymeric galactomannans (19, 27, 28). In the sugar beet industry, -Gals have been used to increase the sucrose yield by eliminating raffinose, which prevents the crystallization of beet sugar (31). Raffinose and stachyose in beans are known to cause flatulence. -Gal has the potential to alleviate these symptoms, for instance, in the treatment of soybean milk (6).

We have studied the substrate specificity of -Gals from eukaryotes by using galactomanno-oligosaccharides, such as 6³-mono--D-galactopyranosyl--1,4-mannotriose (Gal³Man₃) and 6³-mono--D-galactopyranosyl--1,4-mannotetraose (Gal³Man₄). The structures of these galactomanno-oligosaccharides are shown in Fig. 1. Mortierella vinacea -Gal I (11) and yeast -Gals (32) are specific for Gal³Man_3, having an -galactosyl residue (designated the terminal -galactosyl residue) attached to the O-to-6 position of the nonreducing end mannose of -1,4-mannotriose. On the other hand, Aspergillus niger 5-16 -Gal (12) and Penicillium purpurogenum -Gal (27) show a preference for Gal³Man₄, having an -galactosyl residue (designated as the side chain -galactosyl residue) attached to the O-to-6 position of the third mannose from the reducing end of -1,4-mannotetraose. The M. vinacea -Gal II (28) acts on both substrates to almost equal extents. These facts indicate that eukaryotic -Gals were classified into three groups based on the substrate specificity of these galactomanno-oligosaccharides.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Structures of galactomanno-oligosaccharides.

Genes encoding -Gals have been cloned from various sources, including humans (3), plants (20, 33), yeasts (12), filamentous fungi (4, 19, 26, 28), and bacteria (1, 2, 13, 17, 18). -Gals from eukaryotes show a significant degree of similarity and are grouped into family 27. On the other hand, bacterial -Gals have been placed in family 36 (10), even though these enzymes display a low-level amino acid sequence similarity among them.

The genes encoding the thermostable - and -galactosidases from a thermophilic bacterium, Thermus sp. strain T2, have been cloned in Escherichia coli (14). The -Gal gene was located just downstream from the -galactosidase gene, and the -Gal gene was expressed in E. coli by using the expression vector pQE30. Here we describe the sequencing of the -Gal gene of the Thermus sp. strain T2, its expression in E. coli, and the purification and characterization of the recombinant enzyme.

	MATERIALS AND METHODS

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Materials. Melibiose, raffinose, stachyose, p-nitrophenyl--D-galactopyranoside (pNP--Gal), other p-nitrophenyl glycosides, and other chemicals were purchased from the Sigma Chemical Co. Restriction endonucleases and other enzymes were purchased from the Takara Shuzo Co. and used in accordance with the manufacturer's instructions.

Bacterial strains, plasmids, growth conditions, and sequencing procedures. E. coli JM109 and M15 (30), pQE30, and plasmid pOS105 carrying the Thermus sp. strain T2 -Gal gene were used for cloning and gene expression. E. coli cells were cultured in Luria-Bertani (LB) broth at 30°C with ampicillin (100 µg/ml). Recombinant DNA techniques were performed by conventional protocols (21). DNA sequencing was performed by the dideoxy chain termination method (22) with a dRhodamine Terminator Cycle Sequencing Reaction kit and ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, Calif.).

Construction of expression system. PCR amplification of the gene was performed with 2.5 U of Taq DNA polymerase (Takara Shuzo Co.), 10 ng of plasmid pOS105, a 0.2 µM concentration of each synthetic primer, a 200 µM concentration of each deoxynucleoside triphosphate, and 2 mM MgCl₂ in the buffer recommended by the manufacturer. Amplification was achieved with 30 cycles of 0.5 min of denaturation at 95°C, 0.5 min of annealing at 50°C, and 2.5 min of polymerase extension at 72°C, plus an additional extension at 72°C for 7 min using a Perkin-Elmer thermal cycler (GeneAmp PCR System 2400). The synthetic oligonucleotide primers used for the PCR amplification were P1 (5'-GGGGGATCCATGAGGCTTGTACTGG-3') and P2 (5'-GGGAAGCTTATGGAAAGGGGGCATA-3') (the BamHI and HindIII restriction sites are underlined). The obtained PCR product cloned in pCRII was digested with BamHI and HindIII and was ligated with pQE30 between the BamHI and HindIII sites. The plasmid was transferred into competent E. coli M15 cells.

Site-directed mutagenesis. Site-directed mutagenesis replacing Cys159 by Ala was performed by the improved megaprimer PCR mutagenesis strategy that was originally described by Seraphin and Kandels-Lewis (25).

Enzymatic assay and measurement of protein concentration. -Gal standard assays were performed with pNP--Gal at 70°C in 50 mM sodium phosphate, pH 6.0. After 10 min, an equal volume of 0.2 M Na₂CO₃ was added to stop the reaction and absorbance at 408 nm was measured. pNP--Gal was dissolved in 0.1 M sodium phosphate buffer (pH 6.0) and used at a final concentration of 10 mM. One unit of purified -Gal activity was defined as 1 µmol of p-nitrophenol released per min under the conditions described above.

Enzyme purification. E. coli M15 cells were grown in 500 ml of LB broth supplemented with ampicillin (100 µg/ml) at 37°C for 16 h with shaking. After the A₆₀₀ reached 0.6, 2.5 mM isopropyl--D-thiogalactopyranoside (IPTG) was added to the culture and the culture continued to grow at 37°C for 5 h. The cells were harvested; suspended in 5 ml of 50 mM sodium phosphate, pH 7.8, containing 300 mM NaCl; and sonicated on ice. The majority of the heat-labile proteins were precipitated by the heat treatment at 70°C for 10 min and removed by centrifugation. The supernatant was applied to the Chelating Sepharose FF column (0.6 by 4.5 cm; Amersham, Pharmacia Biotech, Little Chalfont, Buckinghamshire, England), which was equilibrated with 10 ml of 20 mM sodium phosphate, pH 7.4, containing 10 mM imidazole and 0.5 M NaCl. After a washing with the buffer containing 150 mM imidazole, the enzyme was eluted with 4 ml of the buffer containing 300 mM imidazole. The enzyme solution was desalted by dialysis against 20 mM sodium phosphate, pH 7.0, and stored at 4°C.

Electrophoretic analysis. Sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously by Laemmli (16). Native PAGE was carried out using an acrylamide gradient gel (4 to 15% [wt/vol]) that was electrophoresed in 192 mM glycine buffer, pH 8.4. After electrophoresis, the protein band was stained with CBB R-250.

Amino acid sequencing of recombinant -Gal. After the protein in the SDS-polyacrylamide gel was blotted on a polyvinylidene difluoride membrane, the membrane was stained with CBB R-250 to detect the protein. The protein band was cut out and put on a protein sequencer (G1005A; Hewlett-Packard Co.).

Preparation of galactomanno-oligosaccharides. The galactomanno-oligosaccharide having an -1,6-galactosyl side chain on -1,4-mannotetraose, Gal³Man₄, was prepared from a hydrolyzate of copra galactomannan using Streptomyces -mannanase (11). In addition, galactomanno-oligosaccharide with a terminal galactose at the nonreducing end of -1,4-mannotriose, Gal³Man₃, was prepared from Gal³Man₄ by cutting off the nonreducing end mannosyl residue of the saccharide with A. niger -mannosidase (15). The structures of Gal³Man₃ and Gal³Man₄ are shown in Fig. 1.

Substrate specificity. Hydrolysis of the galacto-oligosaccharides (such as melibiose, raffinose, and stachyose) and of the galactomanno-oligosaccharides (such as Gal³Man₃ and Gal³Man₄) by the purified -Gal was done at pH 6.0 (0.1 M sodium phosphate buffer) and 70°C. The sugar sample obtained after the enzyme reaction was analyzed by thin-layer chromatography (TLC) (Silica gel 60; Merck) for the characterization of the hydrolysis products. The reaction products were developed with 1-propanol-nitromethane-water (5:2:3, vol/vol). The sugars on the plate were detected by heating at 140°C for 5 min after spraying with sulfuric acid.

Determination of kinetic properties. The K_m and V_max values were graphically determined from the Lineweaver-Burk plots of the initial rate of the hydrolyzing reactions. The enzyme reactions were performed in 0.1 M sodium phosphate buffer, pH 6.0, at 70°C for pNP--Gal. pNP--Gal was used in the range of 0.1 to 10 mM.

Inhibition study. The purified enzyme was incubated with 1 mM chemicals, including pCMB and HgCl₂, at 30°C for 30 min. The remaining activity was then determined as described above.

Nucleotide sequence accession number. The -Gal DNA sequence and the 16S rRNA gene sequence are available in the DDBJ, EMBL, and GenBank databases under accession no. AB018548 and AB054646, respectively.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Sequencing analysis of 16S rRNA gene of Thermus sp. strain T2. The sequence of the 16S rRNA gene of Thermus sp. strain T2 was determined. Compared with other Thermus species, the sequence of Thermus sp. strain T2 exhibited the highest similarity (99.9%) to the sequence of Thermus oshimai (EMBL database accession no. Y18416). There are only two nucleotide differences between the 16S rRNA genes of Thermus sp. strain T2 and T. oshimai, suggesting that these strains are closely related to each other.

Sequencing analysis of the DNA encoding Thermus sp. strain T2 -Gal. The plasmid pOS105, containing one open reading frame (ORF) of 1,425 bp, was sequenced. The gene encodes a polypeptide of 474 amino acids with a calculated molecular mass of 53,514 Da. The deduced amino acid sequence of the -Gal gene was compared with -Gal sequences available from DDBJ. The sequence identities of Thermus T2 -Gal with the enzymes from Thermus brockianus (8), Thermotoga neapolitana (7), and Thermotoga maritima (17) were 74.7, 25.7, and 25.7%, respectively (Fig. 2). The G+C content of the ORF is 62.3%, and no similarity with the eukaryotic -Gals of family 27 was observed. There was only one cysteine residue in the molecule of Thermus sp. strain T2 -Gal. This cysteine residue (residue 189 in Fig. 2) is conserved among all the enzymes.

View larger version (59K): [in this window] [in a new window]   FIG. 2.   Sequence homology of -Gals from different sources. The amino acid sequences of Thermus sp. strain T2, T. brockianus (GenBank/EMBL accession no. AF135398), Thermotoga neapolitana (accession no. AF011400), and Thermotoga maritima (AJ001776) were aligned for optimal sequence similarity using the program CLUSTAL W, which is available in the DDBJ site (www.ddbj.nig.ac.jp). Hyphens indicate gaps. Identical amino acid residues, three of four or more at the same position, are shaded.

Purification of recombinant -Gal and its molecular mass. Purification was carried out using a Chelating Sepharose FF column because it has a histidine tag (His tag) at its N-terminal end. More than a 2,000-fold purification was obtained, with 53% recovery of the activity from the crude enzyme solution (Table 1). SDS-PAGE of the fraction corresponding to the peak of activity revealed a single protein band with a molecular mass of 55 kDa, which agrees with the sum of the molecular mass of -Gal (53.5 kDa) calculated from the nucleotide sequence and the additional His tag sequence (1.4 kDa).

View this table: [in this window] [in a new window]   TABLE 1.   Purification of Thermus sp. strain T2 -Gal

N-terminal amino acid sequencing of purified -Gal. The purified -Gal was subjected to SDS-PAGE and blotted on a polyvinylidene difluoride membrane. The N-terminal amino acid sequence was determined as M-R-G-S-H-H-H-H-H-H-G-S-M-R-L-V-L-G-G-L-E-V-P-L-K-A. It corresponds to the His tag sequence followed by the N-terminal deduced amino acid sequence of the -Gal ORF, which is underlined.

Enzymatic properties. The purified Thermus sp. strain T2 -Gal was most active at 75°C for pNP--Gal hydrolysis and was stable up to 60°C at pH 7.0 for 1 h. The maximum activity of the enzyme was observed at pH 6.0, and the enzyme was stable between pH 6.0 and 13.0 at 40°C for a 1-h incubation. This temperature dependence of the activity of the enzyme is the same as that of B. stearothermophilus (75°C) and is not as high as those of the T. brockianus and Thermotoga enzymes (90 to 95°C).

Substrate specificity. The -Gal was specific for -galactopyranosidic compounds. In contrast to pNP--Gal, it did not hydrolyze pNP--fucopyranoside, pNP--fucopyranoside, pNP--arabinofuranoside, pNP--arabinopyranoside, pNP--glucopyranoside, pNP--glucopyranoside, pNP--galactopyranoside, pNP--xylopyranoside, pNP--xylopyranoside, pNP--rhamnopyranoside, pNP--mannopyranoside, or pNP--mannopyranoside. Thermus sp. strain T2 -Gal exhibited a K_m for pNP--Gal of 4.7 mM, which is similar to the one obtained for -Gal from T. brockianus (2.5 mM).

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Action of -Gal on galacto-oligosaccharides. The reaction mixture was composed of 80 µl of 1% (wt/vol) substrate, 80 µl of 0.2 M sodium phosphate buffer (pH 6.0), and 40 µl (0.4 U) of enzyme solution. The reaction was done at 70°C, and 20 µl of the reaction mixture was withdrawn at each indicated time. Three microliters of the mixture was used for the TLC. Gal, authentic galactose; Mel, authentic melibiose; Raf, authentic raffinose; Sta, authentic stachyose.

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Action of -Gal on galactomanno-oligosaccharides. The enzymatic reaction was done as described for Fig. 3. Gal, authentic galactose. The structures of Gal3Man3 and Gal3Man4 are shown in Fig. 1.

Inhibition study. Some -Gals are reported to be inhibited by SH reagents, such as pCMB. Thermus sp. strain T2 -Gal was also completely inactivated (less than 1% of the control) after the treatment with 1 mM pCMB at 30°C for 30 min. One millimolar metal ions, including Co²⁺, Ca²⁺, Mg²⁺, Ni²⁺, Cu²⁺, Zn²⁺, and Mn²⁺, did not affect the enzymatic activity, but Hg²⁺ and Ag⁺ significantly inactivated the enzyme (less than 5 and 20% of the control, respectively). Fridjonsson et al. (8) reported that -Gal from T. brockianus contained three Cys residues and was almost completely inhibited by HgCl₂ and pCMB. They determined the presence of a thiol group at or near the catalytic site of the enzyme. Among the two conserved Cys residues (161 and 336), Cys residue 336, according to T. brockianus -Gal numbering, could be the conserved Cys residue found in their alignment with T. maritima and T. neapolitana. However, Cys residue 189, not residue 371 (according to our numbering in Fig. 2), corresponding to residue 161 of T. brockianus, could be considered the Cys residue which is modified by pCMB, because the Thermus sp. strain T2 enzyme has only 1 Cys residue in the molecule.

Purification and characterization of mutant enzyme Cys159Ala. The replacement of Cys by Ala of the enzyme was carried out to analyze the role of the enzyme's Cys residue. The expression and purification of the mutant enzyme were carried out as described in Materials and Methods. The purified mutant enzyme showed a single protein band in SDS-PAGE (data not shown). The mutant enzyme also showed the same specific activity against pNP--Gal with the native enzyme, and p-chloromercuribenzoic acid did not affect the enzymatic activity of the mutant enzyme, indicating that the Cys residue is not responsible for the catalytic function. This modification of the Cys residue of the native enzyme by pCMB probably introduced a conformational change in the enzyme by adding the large hydrophobicity and also the negative charge of the compound into the protein.