Cloning, functional expression and purification of endo-β-galactosidase from Flavobacterium keratolyticus

Gene 222 (1998) 187–194

Cloning, functional expression and purification of endo-b-galactosidase from Flavobacterium keratolyticus Lin Leng, Alex Zhu, Zhenfan Zhang, Rosa Hurst, Jack Goldstein * Cell Biochemistry Laboratory, Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67 Street, New York, NY 10021, USA Received 16 June 1998; received in revised form 11 September 1998; accepted 11 September 1998; Received by J. Wild

Abstract Endo-b-galactosidase (EC 3.2.1.103) is an enzyme that hydrolyzes internal endo-b-galactosyl linkages in keratan sulfate, and glycoconjugates with N-acetyl-lactosamine repeating units. Here, we report the cloning of the endo-b-galactosidase-encoding gene from Flavobacterium keratolyticus, its expression in Escherichia coli and the purification of the enzyme. The enzyme was purified over 15 000-fold to apparent homogeneity. The purified endo-b-galactosidase consists of a single band of about 43 kDa on SDS–PAGE and has a specific activity of 148 u/mg. Based on peptide sequences derived from the purified enzyme, a full-length clone encoding endo-b-galactosidase was isolated from F. keratolyticus genomic DNA. The gene contains a single open reading frame coding for a protein of 422 amino acid residues with a putative N-terminal signal peptide. Its authenticity was confirmed by colinearity of deduced amino acid sequences with the peptide sequences, and synthesis of enzyme in E. coli. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Endo-b-galactosidase gene; Flavobacterium keratolyticus; Glycosidase; PCR

1. Introduction The endo-b-galactosidase found in Flavobacterium keratolyticus ( Kitamikado et al., 1981) is one of several enzymes that can hydrolyze internal galactosidic linkages of oligosaccharides containing repeating units of N-acetyl-lactosamine (Nakazawa and Suzuki, 1975; Nakazawa et al., 1975). Besides the enzyme from F. keratolyticus, the two other most commonly used enzymes have also been obtained from microbial sources, namely Escherichia freundii and Bacteroides fragilis (Fukuda and Matsumura, 1976; Scudder et al., 1983). All three have been shown to react with glycoconjugates from a variety of sources, including those

attached to the surface of human erythrocytes (Fukuda et al., 1979; Lenny and Goldstein, 1991). Endo-bgalactosidase from F. keratolyticus is believed to exhibit a wider substrate specificity (Amano et al., 1991), and to work more efficiently than its counterparts from E. freundii and B. fragilis ( Kitamikado et al., 1981). In order to obtain sufficient quantities for future studies of its molecular properties and its effect upon glyco-containing moieties of various cell surfaces, we purified the enzyme to homogeneity and then cloned the encoding gene and expressed it in E. coli.

2. Materials and methods

* Corresponding author. Tel: +1 212 570 3056; Fax: +1 212 879 0243; e-mail: [email protected]

2.1. Materials

Abbreviations: aa, amino acid(s); B, Bacillus; Ba, Bacteroides; C, Clostridium; CNBr, cyanogen bromide; E, Escherichia; F, Flavobacterium; IEF, isoelectric focusing; IPTG, isopropyl b--thiogalactopyranoside; oligo, oligodeoxyribonucleotide; ORF, open reading frame; PCR, polymerase chain reaction; Pfu, Pyrococcus furiosus; PMSF, phenylmethylsulfonyl fluoride; R, Rhodothermus; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TFA, triflouracetic acid; u, unit; [ ], plasmid carrier state.

All the chromatographic resins were purchased from Pharmacia LKB Biotechnology (Piscataway, NJ ). The Easy DNA Kit, Original TA cloning kit and pLEX expression system were from Invitrogen (Carlsbad, CA). The PCR in-vitro cloning kit was from Takara Shuzo (Madison, WI ). The Pfu DNA polymerase was purchased from Stratagene (La Jolla, CA). Tryptophan,

0378-1119/98/$ – see front matter © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 49 6 - X

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PMSF, leupeptin and pepstatin A were from Sigma (St. Louis, MO).

described by Huang et al., 1990, using the multiphor system (Pharmacia Biotech).

2.2. Culture medium and bacterial growth

2.5. N-terminal and internal amino acid sequences of endo-b-galactosidase

The medium for culturing F. keratolyticus contained 1% bacto-tryptone (Difco, Detroit, MI ), 0.1% selected yeast extract (Gibco BRL, Bethesda, MD) and 0.2% NaCl. The seed culture (150 ml ) was first incubated at 25°C, without shaking, for 2 days and then transferred to 4 l of liquid medium for continuing growth, without shaking, for 5 days ( Kitamikado et al., 1981). 2.3. Isolation and purification of endo-b-galactosidase from F. keratolyticus All purification procedures were performed at 4°C, following the method developed by Kitamikado et al. (1981) with some modifications. Briefly, the culture supernatant was first concentrated by Milipore pellicon-10 000 prior to precipitation with ammonium sulfate at 80% saturation. The dissolved precipitate was then subjected to various chromatographic steps including G-100 Sephadex, a combination of CM-Sephadex C-50, DEAE-Sephadex A-50 and then Blue Sepharose CL6B chromatography. Finally, endo-b-galactosidase was purified by chromatofocusing as follows. The Blue Sepharose pool was concentrated and equilibrated with 25 mM imidazole, pH 6.8 (starting buffer). The enzyme solution was applied to a chromatofocusing column (1 ml of polybuffer exchanger 94), which was equilibrated with starting buffer. The column was washed with starting buffer and eluted with polybuffer 74-HCl, pH 5.0. The unabsorbed fractions contained all of the endo-b-galactosidase activity; these were pooled, concentrated and stored at −20°C for further analysis. 2.4. Characterization of endo-b-galactosidase from F. keratolyticus Endo-b-galactosidase was assayed by using keratan sulfate isolated from bovine cornea according to the method set forth in Scudder et al. (1983). One unit of the enzyme was defined as the amount required to release 1 mmol of reducing galactose per minute at 37°C. The specific activity of the enzyme was expressed as units per mg of protein. Protein was determined by the BCA method using BSA as the standard ( Wiechelman et al., 1988). Enzyme samples from different purification steps were boiled with Laemmli buffer (Laemmli, 1970) and subjected to SDS–PAGE. Gels were visualized by Coomassie Blue stain. Isoelectric focusing (IEF ) analysis of endo-b-galactosidase purified from F. keratolyticus was performed as

For N-terminal aa sequencing, the purified endo-bgalactosidase (0.7 nmol ) was added to a pre-cycled, Biobrene-coated filter in the reaction cartridge of the ABI 477 protein sequencer. PTH-amino acids were automatically injected into an ABI 120 A liquid chromatography column for separation and characterization. For internal peptide sequencing, the purified endo-bgalactosidase (4 nmol ) was treated with 18 mmol of CNBr in 70% formic acid. After incubating at room temperature for 24 h, in the dark, with occasional agitation, the liquid in the tube was evaporated in a Speed Vac Concentrator, and the procedure was repeated once after the pellet was resuspended in water. The pellet was then dissolved in 0.1% TFA, and the digested fragments were separated on a C-18 reverse phase column. The aa sequence was determined using an ABI 477 A/120A aa sequencer. 2.6. Cloning of the endo-b-galactosidase-encoding gene Based on the N-terminal aa sequence of purified endob-galactosidase (pep-1 in Fig. 3), two oppositely oriented degenerate oligos were made; ED-1*, 5∞-AA(C/T )GCIACIACIGTIGCIACIACIGA-3∞, and ED-2*, 5∞-TT(G/A)AA( T/C ) TC(G/A)TCN(GA)/ (CT )CCA-3∞, where N is either A, G, T or C, and I is inosine. To reduce the primer sequence complexity, we used inosine in positions that otherwise would have required all four nucleotides for complete codon usage. Similarly, ED-3*, 5∞-ATGGA(C/T )AA(C/T )GCNGTNGT-3∞ and ED-4*, 5∞-GTI(GA)/(CT ) ( T/C )TTIACICCICCIGC(G/T )TG(G/A)TA-3∞ were synthesized, based on the sequence of pep-2 peptide, one of the six internal peptides obtained from digestion of F. keratolyticus endo-b-galactosidase with CNBr ( Fig. 3). The genomic DNA was isolated from F. keratolyticus by using the EASY DNA kit (Invitrogen) according to a procedure recommended by the manufacturer. Using the genomic DNA as a template and ED-1* and ED-4* as primers, a DNA fragment of 0.24 kb was amplified in a hot-start PCR according to the conditions reported before ( Zhu and Goldstein, 1994). The PCR reaction consisted of 35 cycles of 1 min, 94°C; 2 min, 50°C; 3 min, 72°C. After the authenticity of the PCR-amplified fragment was confirmed by DNA sequencing, the remaining portion of the endo-b-galactosidase gene was isolated by adding a ‘cassette’ to a restriction endonuclease-digested DNA fragment (PCR in-vitro Cloning Kit). The EcoRI-cassette and SalI-cassette were used to amplify the 5∞ end and 3∞ end, respectively, of the endo-

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b-galactosidase gene. Based on these amplified sequences, two primers, ED-11 and ED-12, residing upstream of the initiation codon and downstream of the stop codon, respectively, were synthesized (see Fig. 3). Finally, using F. keratolyticus cell genomic DNA as a template, ED-11 and ED-12 as primers, a 1.4-kb fragment containing the full-length endo-b-galactosidase gene was amplified. The PCR product was then ligated to the pCRll vector, generating the plasmid pCR-ED for DNA sequencing. 2.7. Functional expression of endo-b-galactosidase in E. coli The DNA containing the endo-b-galactosidase coding region was amplified from the plasmid pCR-ED using Pfu DNA polymerase in order to create a NdeI site at the 5∞ end of the coding sequence. The DNA fragment was then subcloned into the NdeI and EcoRI sites of the expression vector pLEX, generating the plasmid pLEX-ED. The plasmid pLEX-ED was then transformed into E. coli strain GI 724, and its sequence was confirmed by DNA sequencing. To express the endo-bgalactosidase-encoding gene, the bacterial transformant was first grown at 30°C. When the A reached 550 nm 0.60–0.65, the culture was induced by adding tryptophan to a final concentration of 100 mg/ml. After a 1-h induction at 37°C, the bacteria were harvested and resuspended in 50 mM sodium acetate, pH 6.0, containing 0.5 mg/ml of pepstatin A, 0.5 mg/ml of leupeptin, and 0.1 mM PMSF. After sonication, the lysate was centrifuged and both supernatant and pellet stored at −20°C for the endo-b-galactosidase activity assay. To study the distribution of endo-b-galactosidase in E. coli, cells from 50 ml of induced culture were harvested by centrifugation, resuspended in 2.4 ml of 50 mM sodium acetate, pH 6.0, 2 mM calcium acetate and 0.5 mg/ml of pepstatin A, 0.5 mg/ml of leupeptin, 0.1 mM PMSF, and sonicated three times for 1 min with a Brason sonifer 450. After centrifugation for 30 min at 48 000×g, the pellet was re-extracted with 0.5 ml of the same buffer containing 0.5% Triton X-100 (v/v) by stirring for 15 min at 4°C. The pellet obtained after further centrifugation at 48 000×g for 30 min was resuspended in 0.5 ml of the same buffer containing 0.5% Triton X-100.

3. Results and discussion 3.1. Purification and characterization of endo-bgalactosidase from F. keratolyticus Table 1 summarizes each step of the purification of endo-b-galactosidase from 100 l of F. keratolyticus culture supernatant. Chromatofocusing, rather than the

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second DEAE Sephadex chromatography step employed by Kitamikado et al. (1981), was used in order to obtain the purity necessary for aa sequence analysis. This modification produced endo-b-galactosidase having a specific activity of 148 u/mg, as compared to a specific activity of 44 u/mg previously reported for the enzyme ( Kitamikado et al., 1981). Also, recovery was four times higher in the method utilizing chromatofocusing. Besides the chromatofocusing step, our other modifications included concentrating the culture supernatant and increasing the amount of ammonium sulfate; both were needed in order to optimize recovery when dealing with large volumes. Samples from each step of enzyme purification were analyzed by SDS–PAGE stained with Coomassie Blue. As shown in Fig. 1, the final purification step yielded only one band indicating homogeneity of endo-b-galactosidase and having a molecular weight of approximately 43 kDa. Furthermore, the enzyme homogenity was confirmed by isoelectric focusing ( Fig. 2). 3.2. Molecular cloning of the endo-b-galactosidaseencoding gene In order to isolate the gene encoding endo-b-galactosidase, we first obtained an N-terminal peptide sequence (pep-1) and six CNBr digested peptide sequences (pep-2 to pep-7) from purified endo-b-galactosidase and then designed four highly degenerate primers based on the peptide sequences. These oligomers, ED-1* through ED-4*, were used in different combinations in the hotstart PCR with F. keratolyticus genomic DNA as a template. Primer pairs ED-1* and ED-2* or ED-3* and ED-4* amplified DNA fragments corresponding to the length of the two peptides, pep-1 and pep-2, respectively derived from purified endo-b-galactosidase ( Fig. 3). Furthermore, a PCR product of approximately 0.24 kb was visualized in agarose gel by using ED-1* and ED-4* as primers. The 0.24-kb fragment was then subcloned into a pCRII vector for sequencing. The deduced aa sequence from this DNA fragment matched perfectly with peptides, pep-1 and pep-2, which were derived from F. keratolyticus endo-b-galactosidase, indicating that the PCR-generated, 0.24-kb fragment was part of the gene encoding endo-b-galactosidase. The next step was to isolate the upstream and downstream sequences from the 0.24-kb fragment. We decided to apply the cassette PCR procedure by adding a ‘cassette’ to a restriction endonuclease-digested DNA fragment. In order to locate proper restriction enzyme sites flanking the endo-b-galactosidase gene, we first carried out a Southern blot of the genomic DNA by using the radioactivly labeled 0.24-kb fragment as a probe. A single band of 3.4 or 4.5 kb was visible from the genomic DNA digested with EcoRI or SaII, respectively (data not shown). Thus, these two restriction

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Table 1 Purification of endo-b-galactosidase from 100 l of culture medium of F. keratolyticus Step

Total units

Total protein (mg)

Specific activity (u/mg)

Recovery (%)

Purification fold

Culture supernatant Milipore concentrate (NH ) SO precipitation 42 4 Sephadex G-100 CM-C-50/DEAE-A-50 Sephadex Blue Sepharose Chromatofocusing

1132 1076 926 756 702 433 505

107 809 26 625 595 136 22.8 4.5 3.4

0.01 0.04 1.56 5.56 30.79 96.20 148.50

100 95 82 67 62 38.3 44.6

1 4 156 556 3079 9620 14 850

Fig. 1. Purification of endo-b-galactosidase from F. keratolyticus. Samples from each purification step listed in Table 1 were analyzed by 10% SDS–PAGE and the gel stained with Coomassie Blue. Lanes: 1, MW markers; 2 (NH ) SO precipitate; 3, Sephadex G-100; 4, DEAE42 4 Sephadex A-50/CM-Sephadex C-50; 5, Blue Sepharose; 6, chromatofocusing.

Fig. 2. Isoelectric focusing (IEF ) analysis of endo-b-galactosidase purified from F. keratolyticus. Endo-b-galactosidase purified from F. keratolyticus was loaded on to a IEF–pH gradient polyacrylamide gel. The gel was stained with Coomassie Blue. Lane 1, pI markers; lane 2, endob-galactosidase.

enzymes were chosen for the cassette PCR. By using ED-6 and ED-5 as 3∞-primers (consecutively) and oligos from the EcoRI cassette as 5∞-primers, we were able to amplify a 0.8-kb fragment, which extended from the 5∞ end of the 0.24-kb fragment. Similarly, by applying ED-7, ED-8 and SalI-cassette, we isolated a 1.0-kb fragment, which extended from the 3∞ end of the 0.24-kb fragment. The DNA sequence analysis indicated that these three fragments, 0.8, 0.24 and 1.0 kb, cover the entire coding region for endo-b-galactosidase. To generate an intact endo-b-galactosidase-encoding gene and to verify the sequences that we had obtained, we amplified a 1.4-kb fragment by using F. keratolyticus genomic DNA as a template, and ED-11 and ED-12 as primers. This fragment was directly subcloned into the pCRII vector, generating the plasmid pCR-ED, for further characterization. As shown in Fig. 3, this 1.4-kb DNA contains a single ORF coding for a protein of 422 aa residues. Its authenticity was established by colinearity of the deduced aa sequence with seven peptides (underlined sequences, from pep-1 to pep-7, in Fig. 3) isolated from purified endo-b-galactosidase from F. keratolyticus. In addition, based on the DNA sequence, the mature endo-b-galactosidase has a molecular mass of 41 kDa, which closely resembles that of the native enzyme (43 kDa) estimated by SDS–PAGE ( Fig. 1). As indicated by N-terminal sequencing, the mature enzyme starts at position 47, suggesting that the nascent protein contains a signal peptide of 46 residues for its secretion into the F. keratolyticus culture medium. Long signal sequences are typical for proteins secreted from Gram-positive organisms, like Streptomyces (Robbins et al., 1984; Palva et al., 1984) and unusual for Gramnegative bacteria such as Flavobacterium. Common to these signal sequences is a cluster of hydrophobic and uncharged residues following positively charged N-terminals ( Von Heizne, 1984). The putative signal peptide of endo-b-galactosidase matches well with this general architecture, although at its C-terminus, it contains an unusual region of several charged amino acids. Such a composition and arrangement of endo-b-galactosidase in F. keratolyticus resemble those of N-glycosidase F in F. meningosepticum (Lamp et al., 1990). Whether this is a typical feature of the Flavobacterium

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Fig. 3. DNA and its deduced aa sequence for F. keratolyticus endo-b-galactosidase. The oligos, designated ED series, which were used in the cloning and sequencing, are shown under the nucleotide sequence with arrows indicating the 5∞ to 3∞ direction. Asterisks (*) next to the oligos ED-1* through ED-4* indicate the degenerate nature of these sequences, and all others (ED-5 through ED-12) have unambiguous sequences, as underlined. The first 46 aa residues may constitute a putative signal peptide. The underlined peptide sequences (pep-1 through pep-7) were derived from the native enzyme purified from F. keratolyticus. The DNA sequence has been submitted to the GenBank with Accession No. AF083896.

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signal peptide has yet to be determined by cloning and sequencing of more secreted proteins from Flavobacterium. 3.3. Sequence homology between endo-b-galactosidase and bacterial b-glucanases The endo-b-galactosidase gene that we isolated from F. keratolyticus represents the first cloned gene, although the enzyme activity has been identified in different species. A sequence homology search (BLAST ) of the available protein database revealed that F. keratolyticus endo-b-galactosidase was partially homologous to three bacterial b-glucanases: Clostridium thermocellum endo-1,3 (4)-b-glucanase (Schwarz et al., 1998) ( EC 3.2.1.6), Rhodothermus marinus endo-b-1.3-1.4 glucanase (Spilliaert et al., 1994) (EC 3.2.1.73) and Bacillus circulans glucan endo-1.3-b-glucosidase ( Yahata et al., 1990) ( EC 3.2.1.39). Their sequence homology was analyzed using the computer program CLUSTRALW and is shown in Fig. 4. Bacterial b-glucanases are a family of enzymes that hydrolyze polymeric natural b-glucans such as lichenan and barley b-glucan, which contain mixed b-1,3 and b-1,4 linkages. The three-dimensional structure of B. macerans endo-1,3-1,4-b-glucanase has been determined by X-ray crystallography revealing a jelly-roll protein structure with a deep channel at the active site ( Keitel et al., 1993; Hahn et al., 1995). Based on the crystal structure and site-directed mutagenesis, Hahn et al. (1995) identified Glu103 and Glu107, located within the

deep channel, as being the catalytic aa residues responsible for cleavage of the b-1,4 glycosidic bond in the substrate molecule. In addition, Juncosa et al. (1994) identified active site carboxylic residues in B. licheniformis 1,3-1,4-b--glucan-4-glucanohydrolase ( EC 3.2.1.73) by site-directed mutagenesis. Their data indicate that Glu138 appears as the most likely candidate to function as the general acid catalyst, and Asp136 may affect the pK of catalytic residues. Although F. keratolyticus endo-b-galactosidase has a substrate specificity that is different from these bacterial b-glucanases, the partial homology in their primary sequences suggests a possible resemblance in their tertiary structures. Thus, knowledge of the catalytic mechanism and three dimensional structure of endo-1.3-1.4-b-glucanase will certainly facilitate similar studies of endo-b-galactosidase. 3.4. Production of enzymatically active endo-bgalactosidase in E. coli Initially, we chose the expression vector pKK233-2 (Clonetech, Palo Alto, CA) for the expression of the endo-b-galactosidase gene in E. coli. After subcloning the gene into NcoI and HindIII sites of pKK233-2, the resultant plasmid PKK233-2-endo was sequenced and then transformed into E. coli strain JM 109 (Promega, Madison, WI ) or INVaF ∞ (Invitrogen). However, upon IPTG induction, neither the expression of the endo-bgalactosidase activity nor its protein was detectable. Furthermore, sequencing of several plasmids isolated from different transformants indicated various deletion

Fig. 4. Aa sequence homology. Maximal sequence homology is revealed by CLUSTALW analysis (http://dot.imgen.bem.tmc.edu:9331/multi-align/ options/clustaly.htm1) among the following four enzymes. CTL: C. thermocellum endo-1,3(4)-b-glucanase ( X89732) GUB: R. marinus endob-1,3-1,4-glucanase ( U04836) E13B: B. circulans glucanendo-1,3-b-glucosidase (M37752) and Endo: F. keratolyticus endo-b-galactosidase (AF083896). Accession numbers are in parentheses. Numbers on the left of the sequences are residue positions in each corresponding protein. The symbols under the sequences indicate different degrees of similarity. Identical residues are shown as (*). Positions marked (:) are more conserved than positions marked (Ω).

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and insertion mutations around the 5∞ end of the endob-galactosidase gene. This suggested that mutations may have occurred during expression of the endo-b-galactosidase gene. We then decided to transform the plasmid PKK-233-2-endo to the E. coli strain ABLE (Stratagene), which was designed to lower the plasmid copy number and enhance the probability of propagating a toxic clone. We were able to detect endo-bgalactosidase activity after IPTG induction of transformed ABLE cells, which peaked at 30 min. Our best results have been obtained by using the pLEX expression system. The pLEX vector contains a tightly controlled and tryptophan-inducible promoter for the expression of potentially toxic proteins. The transformed cell density and endo-b-galactosidase activity were examined at different time points in order to establish optimum conditions for the production of the endo-galactosidase. As shown in Fig. 5, endo-b-galactosidase activity became detectable shortly after induction. The activity peaked at 1 h after induction and dropped to 50% at 1.5 h, suggesting that the recombinant endob-galactosidase may be unstable and susceptible to protease digestion. As a control, neither E. coli containing pLEX-ED without induction nor E. coli containing pLEX-vector with induction expressed any endo-bgalactosidase activity. In addition, after 2 h of induction, the A of E. coli [pLEX-ED] reached 1.0 at 550 nm A , whereas the control cells (transformed with the 550 nm pLEX-vector) had an A of 1.75. The slower growth 550 nm

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of the cells containing the endo-b-galactosidase gene may result from the effect of high-level expression or the potential toxicity of the protein product toward the host cells. Under the conditions applied, the total cellular enzyme activity reached approximately 12 u/l culture. While 70% of the activity can be extracted by ultrasonication (Fig. 5), the remaining 30% is solubilized by Triton X-100, which indicates an association with membranes. Such a cellular distribution of activity is very similar to that of N-glycosidase F expressed in E. coli (Lamp et al., 1990). Recombinant and native endo-bgalactosidase appear to have identical physical and catalytic properties. They reveal the same molecular weight when analyzed by SDS–PAGE. Furthermore, both demonstrate the same specific activity when assayed using keratan sulfate as substrate. Most significantly, both exhibit identical substrate specificity toward isolated oligosaccharides as well as oligoconjugates attached to the red cell surface. In conclusion, we have purified, cloned and expressed F. keratolyticus endo-b-galactosidase. The hydrolysis of the internal b-galactosyl linkages in keratin sulfate by the recombinant enzyme expressed in E. coli confirms that the gene that we isolated from F. keratolyticus codes for endo-b-galactosidase. Its partial sequence homology with bacterial b-glucanases will be helpful in determining both its catalytic mechanism and the critical residues involved in its active site.

Acknowledgement We thank Dr Jim Farmer and Fanny Huang for peptide and DNA sequence determinations and oligo syntheses. These studies were supported in part by The Metropolitan Life Foundation and in part by the Office of Naval Research Grand N00014-9-j-1180 with funds provided by the Naval Medical Research and Development Command. A. Zhu is supported in part by the NIH grand HL 55482-01A1.

References

Fig. 5. Production of endo-b-galactosidase in E. coli. Aliquots were taken from cultures at the indicated time intervals, sonicated and the extract measured for endo-b-galactosidase activity, as described in Section 2. E. coli transformed with pLEX-ED was grown with (%) or without (6) tryptophan; E. coli transformed with vector pLEX was grown with tryptophan (( ).

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