INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

-Galactosidase (Gal) (EC 3.2.1.22) is of particular interest in view of its biotechnological applications. Gal from coffee beans demonstrates a relatively broad substrate specificity, cleaving a variety of terminal -galactosyl residues, including blood group B antigens on the erythrocyte surface. Treatment of type B erythrocytes with coffee bean Gal results in specific removal of the terminal -galactosyl residues, thus generating serological type O erythrocytes (8). Cyamopsis tetragonoloba (guar) Gal effectively liberates the -galactosyl residue of galactomannan. Removal of a quantitative proportion of galactose moieties from guar gum by Gal improves the gelling properties of the polysaccharide and makes them comparable to those of locust bean gum (18). In the sugar beet industry, Gal has been used to increase the sucrose yield by eliminating raffinose, which prevents normal crystallization of beet sugar (28). 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 (16).

Gals are also known to occur widely in microorganisms, plants, and animals, and some of them have been purified and characterized (5). Dey et al. showed that Gals are classified into two groups based on their substrate specificity. One group is specific for low-M_r -galactosides such as pNPGal (p-nitrophenyl--D-galactopyranoside), melibiose, and the raffinose family of oligosaccharides. The other group of Gals acts on galactomannans and also hydrolyzes low-M_r substrates to various extents (6).

We have studied the substrate specificity of Gals by using galactomanno-oligosaccharides such as Gal³Man₃ (6³-mono--D-galactopyranosyl--1,4-mannotriose) and Gal³Man₄ (6³-mono--D-galactopyranosyl--1,4-mannotetraose). The structures of these galactomanno-oligosaccharides are shown in Fig. 1. Mortierella vinacea Gal I (11) and yeast Gals (29) are specific for the Gal³Man₃ having an -galactosyl residue (designated the terminal -galactosyl residue) attached to the O-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 (25) show a preference for the Gal³Man₄ having an -galactosyl residue (designated the stubbed -galactosyl residue) attached to the O-6 position of the third mannose from the reducing end of -1,4-mannotetraose. The M. vinacea Gal II (26) acts on both substrates to almost equal extents. The difference in specificity may be ascribed to the tertiary structures of these enzymes.

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, 32), yeasts (27), filamentous fungi (4, 17, 24, 26), and bacteria (1, 2, 15). Gals from eukaryotes show a considerable degree of similarity and are grouped into family 27 (10).

Here we describe the cloning of P. purpurogenum Gal cDNA, its expression in Saccharomyces cerevisiae, and the purification and characterization of the recombinant enzyme.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Strains, plasmids, media, and cultivation conditions. P. purpurogenum no. 618 was isolated from soil and maintained on a medium containing 2.0% (wt/vol) agar, 4.0% (wt/vol) malt extract, 0.2% (wt/vol) NH₄NO₃, 0.1% (wt/vol) KH₂PO₄, and 0.05% (wt/vol) MgSO₄ · 7H₂O. Escherichia coli INVaF' and plasmid pCRII (Invitrogen) were used for TA cloning of amplified DNA fragments and for preparation of single-stranded plasmid DNA. S. cerevisiae WS3-2A (MAT leu2 ura3 ade8 cys3) and plasmid YEp51 were kindly provided by Y. Jigami (National Institute of Bioscience and Human-Technology, Japan) and used for expression of the Gal cDNA.

Amino acid sequencing of Gal purified from P. purpurogenum. Gal was purified to homogeneity from the culture filtrate of P. purpurogenum as previously reported (25). The purified enzyme was treated with trypsin or V8 protease. The resulting peptides were isolated by reverse-phase high-performance liquid chromatography, and their N-terminal amino acid sequences were determined by a protein sequencer (G1005A; Hewlett-Packard Co.).

Cloning and sequencing analysis of Gal cDNA. Restriction endonucleases and other enzymes were purchased from Takara Shuzo Co. and used in accordance with the manufacturer's instructions. Total RNA was prepared from mycelia by the phenol-chloroform method (30), and poly(A)⁺ RNA was purified with an oligo(dT)-cellulose column. A DNA fragment encoding a portion of the P. purpurogenum Gal gene was amplified by the reverse transcription (RT)-PCR method with a set of P1 [5'-GCI(T/C)TIGGITGGAA(T/C)(A/T)(G/C)ITGGAA-3'] (I = inosine) and P2 [5'-(T/C)TTCAT(A/T/G)AT(A/T/G/C)GCCCA-3'] primers designed from the N-pep and V-pep sequences in Fig. 2, respectively.

View larger version (45K): [in this window] [in a new window]   FIG. 2.   Full-length cDNA encoding P. purpurogenum Gal. Four peptide sequences, N-pep, T1-pep, T2-pep, and V-pep, which were obtained from purified P. purpurogenum Gal are underlined. The designed oligonucleotide primers use for RT-PCR, P1 and P2, were based on N-pep and V-pep, respectively. The nucleotide sequences corresponding to the P1 and P2 primers are shown with arrows indicating the 5'-to-3' direction. Primers P3 to P6 were designed based on the nucleotide sequences of DNA fragment amplified by RT-PCR and used for 5' and 3' RACE. These primers are shown with arrows indicating the 5'-to-3' direction. The termination codon is indicated by an asterisk. Putative N-glycosylation sites are in shaded boxes.

Expression of P. purpurogenum Gal cDNA in yeast. To construct expression vector YEp-PGA the 5' and the 3' RACE products in pCRII were digested with ClaI and HindIII. They were then ligated at these sites. The full-length cDNA was amplified with SalP (5'-GCGGTCGACATGTTAAGTAGTGTAACTGTAGC-3') and M13 primer M4 (5'-GTTTTCCCAGTCACGAC-3'). SalP included a SalI cleavage site just before the initiation codon of the Gal gene. The cDNA was then digested with SalI and BamHI and ligated with YEp51 between the SalI and BamHI sites. The plasmid was transferred into S. cerevisiae WS3-2A by electroporation using 0.2-cm-diameter cuvettes at 7.5 kV/cm, 200 , and 25 µF with a Gene Pulser (Bio-Rad Laboratories). An SD minus Leu plate (0.67% [wt/vol] yeast nitrogen base; 2% [wt/vol] glucose; 20-µg/ml [each] His, adenosine, and uracil) was used for selection of the yeast transformants. S. cerevisiae WS3-2A carrying YEp-PGA was first grown in 20 ml of YPD medium at 30°C for 24 h. The cells were harvested by centrifugation and cultivated in 100 ml of YPGal medium (1% [wt/vol] yeast extract, 2% polypeptone, 2% [wt/vol] galactose) at 30°C with shaking to express the P. purpurogenum Gal gene.

Enzyme assay and measurement of protein concentration. Gal activity was assayed by measuring the amount of p-nitrophenol released from p-nitrophenyl--D-galactopyranoside (21). One unit of activity was defined as the amount of enzyme releasing 1 µmol of p-nitrophenol from pNPGal per min at pH 4.0 and 40°C.

Purification of recombinant Gal. The culture supernatant (100 ml) was harvested by centrifugation after 9 days of growth. The supernatant was concentrated with Centriprep 10 (Amicon) and dialyzed against 20 mM sodium acetate buffer, pH 5.4, and put on a DEAE-Sepharose Fast Flow column (1.3 by 21 cm; Pharmacia) equilibrated with the same buffer. Proteins were eluted with a linear gradient of 0 to 0.2 M NaCl. The active fractions were collected, concentrated, and dialyzed against 10 mM sodium acetate buffer, pH 3.5, and put on a Mono-S HR 5/5 column (Pharmacia) equilibrated with the same buffer. Proteins were eluted with a linear gradient of 0 to 0.2 M NaCl. Active fractions were collected, concentrated, and put on a HiPrep 16/60 Sephacryl S-200 HR column (Pharmacia) equilibrated with 10 mM sodium acetate buffer, pH 3.5, containing 0.15 M NaCl. The column was washed with the same buffer until protein could no longer be detected in the eluent.

Electrophoretic analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in 10% polyacrylamide gel as described by Laemmli (14). The proteins in the gel were visualized by staining with Coomassie brilliant blue R-250. The molecular mass was estimated with markers (10 kDa Protein Ladder; Gibco BRL).

Preparation of galactomanno-oligosaccharides. A galactomanno-oligosaccharide having an -1,6-galactosyl stub on -1,4-mannotetraose, Gal³Man₄, was prepared from a hydrolysate of copra galactomannan by using Streptomyces -mannanase (11). In addition, a galactomanno-oligosaccharide with a terminal galactose at the nonreducing end of -1,4-mannotriose, Gal³Man₃, was prepared from Gal³Man₄ by cutting off the nonreducing mannosyl residue end of the saccharide with Aspergillus niger -mannosidase (13).

Substrate specificity. The action of Gal on oligosaccharides and locust bean gum was monitored by determining the release of D-galactose by using D-galactose dehydrogenase (23). The reaction mixture in 0.5× McIlvaine buffer, pH 4.5, containing the 0.1% (wt/vol) substrate was incubated at 37°C for 24 h. The reaction was terminated by boiling for 5 min. The reaction mixture (40 µl) was added to 100 µl of 1 M Tris-HCl (pH 8.6), 10 µl of 10 mM NAD⁺, and water to make a final volume of 180 µl. The A₃₄₀ was measured as a blank, and 5 µl of D-galactose dehydrogenase was added to start the reaction. The solution was incubated at 37°C for 30 min, and the A₃₄₀ was measured.

Nucleotide sequence accession number. The Gal cDNA sequence is available in the DDBJ, EMBL, and GenBank databases under accession no. AB008367.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Cloning and characterization of the P. purpurogenum Gal cDNA. The gene encoding P. purpurogenum Gal was cloned by PCR with designed primers based on partial amino acid sequences of the purified protein. The nucleotide sequence and deduced amino acid sequence of the 5' and 3' RACE products are shown in Fig. 2. Examination of the sequence revealed the presence of one open reading frame of 1,371 bp. The nucleotide sequences of the overlap region of these fragments (between P6 and P4) were identical, and the amino acid sequences of the purified enzyme identified by Edman degradation (N-pep, T1-pep, T2-pep, and V-pep) were found in the sequence. The coding sequence consisted of 19 amino acids of signal sequence and 420 amino acids of mature Gal with a molecular mass of 46.3 kDa. Nine putative N-glycosylation sites were found in the sequence, and this is coincident with the reactivity with concanavalin A (25).

View larger version (87K): [in this window] [in a new window]   FIG. 3.   Sequence homology of Gals from different sources. The amino acid sequences of P. purpurogenum Gal (P.p.), T. reesei AGLI (T.r.), S. carlsbergensis Gal (Yeast), M. vinacea GalI (M.v.I), M. vinacea GalII (M.v.II), coffee bean Gal (Coffee), and human GalA (Human) were aligned for optimal sequence similarity by using the program GENETYX (Software Development, Tokyo, Japan). Hyphens indicated gaps, and the yeast and human Gal sequences were truncated at the C terminus as indicated by asterisks. Identical amino acid residues, five of seven or more at the same position, are shaded, and cysteine residues located at the insertion sequences are dotted.

Expression and purification of recombinant Gal in S. cerevisiae. P. purpurogenum Gal cDNA was expressed in S. cerevisiae under the control of the yeast GAL10 promoter. S. cerevisiae cells carrying YEp-PGA were cultured in YPGal medium, and Gal production was monitored. Gal was secreted into the medium, and the activity reached about 63 U/ml of medium at 216 h (equivalent to 0.21 g/liter of medium). Little Gal activity was detected in the periplasmic space or intracellular fractions throughout the culture period. No background activity was detected when the host cells carrying the expression vector YEp51 were cultured under the same conditions (data not shown). Recombinant Gal was purified to homogeneity by using three chromatographic steps (Table 1). Starting from the 100-ml culture medium, 6.75 mg of the purified Gal was obtained with 33% recovery.

View this table: [in this window] [in a new window]   TABLE 1.   Purification of recombinant Gal

Characterization of recombinant Gal. Purified recombinant Gal showed a single but broad protein band with the characteristics of a glycoprotein on SDS-PAGE, and its molecular mass was estimated to be in the range of 70 to 100 kDa (Fig. 4). The apparent molecular mass of the recombinant enzyme was 10 to 30 kDa larger than that of the native enzyme (25); however, no difference was found by SDS-PAGE between the recombinant and native enzymes after treatment with endoglycosidase H (data not shown). This suggests that the differences in the molecular masses existed in the carbohydrate moieties. The specific activity of the purified enzyme was 300 U/mg, which is slightly higher than that of the native enzyme (245 U/mg), and this may be due to the difference between the carbohydrate moieties or the purity of the enzymes.

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Homogeneity and molecular mass determination of recombinant Gal by SDS-PAGE. The enzyme (10 µg) was electrophoresed on 10.0% (wt/vol) polyacrylamide gel and stained with Coomassie brilliant blue R-250. Lanes: 1, molecular size markers; 2, recombinant Gal; 3, recombinant Gal digested with 2 mU of endo--N-acetylglucosaminidase H at pH 5.3 and incubated 37°C for 16 h.

View this table: [in this window] [in a new window]   TABLE 2.   Comparison of some properties of recombinant and native Gals

View larger version (58K): [in this window] [in a new window]   FIG. 5.   Action of native (a) and recombinant (b) Gals on galactomanno-oligosaccharides. The reaction mixture was composed of 80 µl of 1% (wt/vol) substrate, 80 µl of McIlvaine buffer (pH 4.5), and 40 µl (0.4 U) of enzyme solution. The reaction was done at 30°C, and 20 µl of the reaction mixture was withdrawn at each time indicated. Three microliters of the mixture was used for TLC. Gal, authentic galactose; M, authentic mannose to mannopentaose, from top to bottom.

View larger version (95K): [in this window] [in a new window]   FIG. 6.   Actions of native (a) and recombinant (b) Gals on galacto-oligosaccharides. The reaction mixture was composed of 40 µl of 1% (wt/vol) substrate, 40 µl of McIlvaine buffer (pH 4.5), and 20 µl (0.2 U) of enzyme solution. The reaction was performed at 30°C, and 20 µl of the reaction mixture was withdrawn at each time indicated. Three microliters of the mixture was used for TLC. Gal, authentic galactose; Mel, melibiose; Raf, raffinose; Sta, stachyose.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Gals are classified into two groups based on substrate specificity (5). Some enzymes are specific for low-M_r substrates, and others are able to efficiently hydrolyze polymer substrates. We have found that there are three kinds of Gals which act on lowM_r substrates (26). The first group acts only on the terminal -galactosyl residue of a substrate such as Gal³Man₃, the second one is specific only for the stubbed -galactosyl residue of a substrate such as Gal³Man₄, and the third shows a preference for both residues. Recombinant and native P. purpurogenum Gals could hardly act on the polymer substrate and showed a preference for the stubbed galactosyl residue among the low-M_r substrates. T. reesei Gal was reported to show a synergistic action on galactomannan with -mannanase and to effectively liberate galactose residues (31), suggesting that T. reesei Gal has a high ability to liberate the stubbed galactosyl residue from galactomanno-oligosaccharides having a stubbed -galactosyl residue with a high yield (13). Until now, only M. vinacea GalI and yeast Gals have shown specificity for the terminal -galactosyl residue and have rarely been seen to act on the stubbed -galactosyl residue of galactomanno-oligosaccharides. Other Gals show a preference for the stubbed -galactosyl residue of the galactomanno-oligosaccharides.

Three genes encoding Gals from T. reesei were isolated by expression cloning, and some properties of the enzymes produced by yeast were analyzed (17). Based on its substrate specificity, AGLI might correspond to the 50-kDa Gal which was purified from T. reesei RUT C-30 and previously characterized (31). The physicochemical properties and substrate specificity of T. reesei AGLI can resemble those of P. purpurogenum Gal. This might also be due to the similarity of the primary structures of the two enzymes. Although Gal from T. reesei RUT C-30 was nonglycosylated (31), P. purpurogenum Gal was highly glycosylated. P. purpurogenum secreted over 10 times more Gal into the culture medium than did T. reesei (25, 31). In this study, S. cerevisiae secreted P. purpurogenum Gal into the culture medium at about 200 mg/liter. This value is about 10 times higher than that of the native Gal produced by P. purpurogenum (25). In order to increase recombinant Gal production, optimization of the recombinant yeast culture conditions has also been studied (7, 22, 33). The level of P. purpurogenum Gal expressed in yeast is comparable to those of coffee Gal expressed in yeast and insect cells (32).

Many Gals have been purified and characterized, and genes encoding Gals have been isolated from several sources. Gals from T. reesei and humans have been crystallized, and X-ray diffraction studies are in progress (9, 19). However, only a few Gals were studied based on structure-function relationships. Thus, the structures of the active sites and catalytically important amino acid residues still remain largely unknown, and little is known about the structure-function relationship of Gal. The experimental data obtained and the expression system used in this study will be useful in studying the structure-function relationships of Gals.