Cloning of the sucrose phosphorylase gene from Leuconostoc mesenteroides and its overexpression using a ‘sleeper’ bacteriophage vector

JOURNALOFFERMENTATIONANDBIOENGINEERING VOI. 73, NO. 3, 179-184. 1992

Cloning of the Sucrose Phosphorylase Gene from Leuconostoc mesenteroides and Its Overexpression Using a 'Sleeper' Bacteriophage Vector SATOSHI KITAO* AND EIICHI NAKANO

Research and Development Division, Kikkoman Corporation, 399 Nocla, Noda-shi, Chiba 278, Japan Received 5 August 1991/Accepted 10 December 1991 The sucrose phosphorylase gene was cloned from Leuconostoc mesenteroides ATCC 12291 and was overexpressed in Escherichia coli using a 'Sleeper' bacteriophage vector. A recombinant phage slp-spl-1, which had four copies of the sucrose phosphorylase gene, was lysogenized into E. coil 1100. After heat induction, this lysogen produced 55.7 units per ml culture of sucrose phosphorylase, Le. 80-times higher than that in L. mesenteroides. As the amount of this recombinant enzyme was over 30% of the soluble protein in the cell, E. coli 1100 (slp-spl-1) succeeded in overcoming problems such as, inhibited enzymes, in the case of L. mesenteroides. Thus it is possible to achieve an industrial-scale production of sucrose phosphorylase. The complete nucleotide sequence showed that it coded for a 490-amino acid protein of Mr 55,749. Homology between the deduced amino acid sequences for the L. mesenteroides sucrose phosphorylase gene and Streptococcus mutans gtfA, sucrose phosphorylase gene, was 68%.

Sucrose phosphorylase (sucrose: orthophosphate, a-Dglucosyltransferase, EC 2.4.1.7) catalyzes the following reaction: sucrose+inorganic phosphate ~ " a-D-glucose1-phosphate (G-1-P)+D-fructose. Industrial use of this enzyme has been studied mainly for the specific determination of inorganic phosphate (1). Other applications have been considered for the synthesis of novel disaccharides (2), the production of G-1-P and fructose from sucrose (3), the specific determination of sucrose (4), G-1P regeneration in amylose synthesis (5), and sucrose production from starch (6). Sucrose phosphorylase was discovered in Leuconostoc mesenteroides ATCC 12291 (7), Pseudomonas saccharophila ATCC 159,46 (8) and P. putrefaciens (9). A comparative study of these three strains revealed that L. mesenteroides had the highest sucrose phosphorylase activity (9). Furthermore, it was shown that the L. mesenteroides sucrose phosphorylase was a constitutive enzyme (10), unlike the enzyme produced by P. saccharophila (8). From these results, environmental optimization of the L. mesenteroides sucrose phosphorylase production has been studied in detail (10). Even at the optimal condition, however, the productivity of sucrose phosphorylase in L. mesenteroides was very low. Accumulation of mannitol in the medium and increase of medium viscosity by dextran secretion made the purification process complicated. Moreover activities of NADH oxidase and 6-phosphogluconate dehydrogenase were rather high, and these enzymes interfered with the specific determination of inorganic phosphate using the sucrose phosphorylase-phosphoglucomutase-6-phosphogluconate dehydrogenase system. To resolve these problems, we attempted to construct a sucrose phosphorylase-overproducing strain using a Sleeper bacteriophage vector (11). The Sleeper vectors are hybrid phages between AcIs57 and ~b80. As foreign DNA fragments to be cloned replace most of the DNA regions for the production of phage coat

proteins, the recombinant DNA molecule thus constructed loses the ability to produce phage particles in a host cell, and can be maintained stably in a lysogenic state on a host chromosome. Heat induction of the lysogens brings replication of the recombinant DNA molecule, resulting in amplification of the inserted genetic information. Therefore, the Sleeper vectors are suitable for industrial use. Actually, the glycerol kinase of E. coli was overproduced using this bacteriophage vector (12). In this report, we isolated a gene which codes for the sucrose phosphorylase of L. mesenteroides ATCC 12291, determined the nucleotide sequence and constructed a sucrose phosphorylase-overproducing strain using a Sleeper bacteriophage vector. MATERIALS AND METHODS

Bacterial strains, media, plasmids and phage E. coil strain DHI (F-recA 1 endA 1 gyrA 96 thi-1 hsdR17 supE44) was used as a carrier for recombinant plasmids. E. coli JM101 [A(lac-pro) thi laclqZAM15] was used for the DNA sequencing and the expression of the sucrose phosphorylase gene. E. coli strain 1100 (thi-, endA) was used for the expression using a Sleeper vector. E. coli cells were grown in Luria-Bertani (LB; 1% Bacto tryptone, 0.5% yeast extract, 1.0% NaCI) medium. L. mesenteroides ATCC 12291 was grown without aeration in ACI-B medium (13) at 30°C. Plasmid p U C l l 9 and pBR322 were purchased from Takara Shuzo (Kyoto). Bacteriophage slpS01S-Tc (Fig. 3) was constructed by Nakano (unpublished data). Enzymes and chemicals Restriction and modification enzymes were purchased from Takara Shuzo or Boehringer Mannheim Yamanouchi (Tokyo). Phosphoglucomutase and glucose-6-phosphate dehydrogenase were purchased from Boehringer Mannheim Yamanouchi and Oriental Yeast (Tokyo), respectively. IPTG (isopropyl-fl-D-thiogalactoside) was purchased from Wako Pure Chemical Industries (Osaka). Other chemicals used were

* Corresponding author. 179

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KITAOAND NAKANO

of reagent grade. Construction for genomic libraries from L. mesenteroides and library screening Chromosomal DNA of L. mesenteroides ATCC 12291 was prepared by the method of Saito and Miura (14). The chromosomal DNA was digested with EcoRI and ligated to EcoRI-digested pBR322. To construct the first library, the resulting plasmids were transformed into E. coli D H I and plated on a LB agar plate containing 100gg/ml of ampicillin and 10pg/ml of tetracycline. As the six NH2-terminal amino acids sequence of the sucrose phosphorylase was Met-GluIle-Gln-Asn-Lys (15), the predicted DNA sequences A T G G A A A T T C A A A A T A A were synthesized using a G C G C A System 1 Plus DNA synthesizer (Beckman Instruments, Inc., FuUerton, CA, USA). Using this oligonucleotide probe, the first library was screened by colony hybridization. The second library was constructed as follows. The chromosomal DNA of L. mesenteroides was digested with Sau3AI and ligated to the BamHI-digested pBR322. The resulting plasmids were introduced into E. coil DH1 and plated on an LB agar plate containing 100gg/ml of ampicillin. This library was screened by colony hybridization using labeled DNA fragments. D N A manipulations The procedures used for radioactive labeling of oligonucleotide DNA and DNA fragments, colony hybridization, Southern blot analysis, in vitro packaging, digestion with endonucleases and transformation of E. coil were carried out by the methods of Maniatis et aL (15). D N A sequencing The nucleotide sequence was determined by the dideoxy-chain termination method (17) using a 7-DEAZA sequencing kit (Takara Shuzo) and [a-32p]dCTP (Amersham Japan). Enzyme assay To prepare the crude extract for measurements of enzyme activities, E. coli pellets (10 ml cup ture) were resuspended in 2 ml of 50 mM potassium phosphate pH 6.8, disrupted by ultrasonication and centrifuged at 12,000rpm for 10rain. A sucrose phosphorylase was assayed by the method of Silverstein et al. (18) in which the production of G-1-P from sucrose and Pi were coupled to the reduction of NADP + in the presence of phosphoglucomutase and gincose-6-phosphate dehydrogenase. The standard assay medium contained 50 mM potassium phosphate buffer (pH6.8), 140raM sucrose, l mM EDTA-2Na, 150raM MgC12, 1 mg of HAl)P +, 1/4g of giucose-1,6-diphosphate, 100pg of phosphoglucomutase, 20 units of glucose-5phosphate dehydrogenase and the enzyme solution (20/zl) in a final volume of 3.3 ml. Increase in absorbance of NADPH at 340 um was measured at 25°C. One unit of sucrose phosphorylase activity was defined as the amount of enzyme which caused the reduction of 1/zmol of NADP + per minute under the above assay conditions. An extinction coefficient of 6.22 x 103 M-~cm-~ was used for calculation. The activity of N A D H oxidase was assayed by the reduction of NADH. The reaction mixture contained 100 mM potassium phosphate buffer (pH 7.0), 0.34 mg of N A D H and the enzyme solution (0.1 ml) in a final volume of 3.Oral. The rate o f reaction was followed by measuring the decrease in absorbance at 340 nm. One unit of N A D H oxidase activity was defined as the amount of enzyme which caused the reduction of 1/zmol of N A D H per minute.

J. FERMENT.BIOENG., 6-Phosphogluconate dehydrogenase was assayed by the method of De Moss (19). The reaction mixture contained 100 mM glycylglycine buffer (pH 7.5), 1.89 mg of 6phosphogluconate, 4.3 mg of NAD and the enzyme solution (0.5 ml) in a total volume of 2.85 ml. Increase in absorbance of NADH at 340 nm was measured at 25°C. One unit of 6-phosphogluconate dehydrogenase activity was defined as the amount of enzyme which caused the reduction of I pmol of NAD per minute. Sodium dodecyl snlfate-polyacrylamide gel electrophoresis (SDS-PAGE) Cell pellets (10 ml culture) were suspended in 2 ml of water, disrupted by ultrasonication and centrifuged at 12,000rpm for 10min. The supernatant mixed with an equal volume of sample buffer (20) was boiled for 5 rain and 10pl of the mixture was on a 4-20% gradient gel (Daiichi Pure Chemicals, Tokyo). RESULTS Cloning of the gene encoding sucrose phosphorylase Out of I0,000 transformants with ampicillin and tetracycline resistance, only one strain hybridized with the NH2-terminal oligonucleotide probe using the first genomic library. The recombinant plasmid containing 1.3-kb insert of DNA from L. rnesenteroides was designated pSPL02 and the E. coli DH 1 harboring pSPL02 had no sucrose phosphorylase activity. By sequencing and Southern blotting analysis, we found that plasmid pSPL02 did not cover the full-length sucrose phosphorylase structural gene, lacking the C-terminal region of the enzyme. To obtain the full-length sucrose phosphorylase gene, the second E 3 T 33H

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FIG. 1. Construction of p|asmJds for the expressionof sucrose phosphorylase. Blackened segmentsshow the coding re,on o£ the sucrose phosphorylasegene, and open segments show its non-coding regions. Abbreviations: lacP,/acZ promoter of E. col|; Ap, ampicfllin-resistance gene; Tc, tetracycline-resistance gene; E, EcoRI; H,

HindIII; P, Pstl; T, EcoTl4I; 3, Sau3AI.

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CLONING OF SUCROSE PHOSPHORYLASE GENE

the molecular weight, we concluded that the full-length sucrose phosphorylase structural gene was contained within the 1.8-kb HindlII fragment of pSPL10. A plasmid pSPL11 was constructed by inserting this 1.8-kb fragment into the HindlII site of pUC119 so that the protein encoded by the sucrose phosphorylase gene was expressed under control of the lac promoter. When pSPL11 was transformed into E. coli JM101, the sucrose phosphorylase activity was increased to 19.75 units per ml culture in the presence of I mM IPTG, i.e. 30-times higher than that in L. me-

library was screened using a labeled 0.34-kb Sau3AIEcoRl fragment of pSPL02 and, as the result, pSPL08 was selected. As plasmid pSPL08 had a 2.0-kb DNA fragment from L. mesenteroides containing the 0.34-kb Sau3AIEcoRI fragment on pSPL02 and the following DNA region for the C-terminal of sucrose phosphorylase, we estimated that the cloned DNA fragments from pSPL02 and pSPL08 might cover the structural gene completely. Figure 1 shows the procedure used for construction of a plasmid that allowed expression of the sucrose phosphorylase gene in E. coli. Plasmid pSPL09 was constructed by inserting the 2.0-kb Sau3AI fragment of pSPL08 into the B a m H I site of pUC119. The 0.75-kb EcoRI-PstI fragment of pSPL02 was replaced by the 1.66-kb EcoRI-PstI fragment of pSPL09. The resulting plasmid pSPLI0 was then transformed into E. coli DH. The sucrose phosphorylase activity of E. coli DH1 harboring pSPL10 was 0.64 units per ml culture, almost the same as that of L. rnesen-

senteroides. Nucleotide sequence of sucrose phosphorylase gene

Figure 2 shows an open reading frame (ORF) starting at position 191 and terminating at position 1660. This ORF codes for a 490-amino acid protein of molecular weight 55,749. The deduced amino acid sequence was consistent with the amino acid composition of sucrose phosphorylase and with the sequence of the 30 amino acids from NH2-terminal region analyzed by a protein sequencer (15). From these results, we concluded that this ORF corresponds to the sucrose phosphorylase gene. The coding region of the

teroides.

From the results of Southern blot analysis using the NH2terminal oligonucleotide probe and from the calculation of 1

181

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1777 GTTCA,~GCTT FIG. 2. Nucleotide sequence of the sucrose phosphorylase gene and its flanking regions and the deduced amino acid sequence of sucros~ phosphorylase. Thick underline indicates a probable Shine-Dalgarno sequence. Thin underline indicates the sequence o f amino acids from the 30

NH2-terrninal region analyzed by a protein sequencer.

182

KITAOAND NAKANO

J. FERMENT.BIOENG.,

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FIG. 3. Construction of slp-spl-I phage for the overexpression of sucrose phosphorylase. Italics are genetic markers of %80 or 2. 20/E is fused site of .080 and 2. Left region is ~80 and right one is 2. Abbreviation is same described in Fig. l, except for B, BamHI; Bg, BgllI; K, Kpnl; X, XbaI. sucrose phosphorylase gone was preceded by the sequence A G G A G , a probable ribosome-binding site (21) located 8 bp upstream from the initiation codon. However, no potential promoter sequences homologous to the Streptococcus (22) or E. coli consensus sequence were found. Sucrose phosphorylase production by the lysogen using a 'Sleeper' bacteriophage vector As shown in Fig. 3, the 0.2-kb lac promoter from pUC119 and 1.8-kb HindIII fragment of pSPL11 were inserted into the pBR322 to obtain pSPL14. Plasmid pSPL14-2 has a 4.0-kb KpnI insert containing two copies of the sucrose phosphorylase gone with lac promoter. This plasmid was digested with KpnI and the 4.0-kb fragment was isolated. This fragment was ligated to KpnI-digested Sleeper bacteriophage slp501STc DNA. The ligated DNA mixture was introduced into E. coil 1100 by the method of in vitro packaging. Resultant lysogens were plated on M9 minimal agar plate (16) containing 1.5 Hg/ml of tetracycline. Lysogenized clones were selected by temperature sensitivity and deficiency of plaque formation. Then sucrose phosphorylase-over-

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producing lysogen was selected and designated E. coli 1100 (slp-spl-1). Slp-spl-1 has two of 4.0-kb fragments, i.e. four copies of sucrose phosphorylase genes. The production of sucrose phosphorylase by E. coli 1100 (slp-spl-1) was performed in l0 ml of LB medium. E. coil cell was grown at 30°C until the growth had reached 100 Klett units. The temperature was then shifted to 43°C and the cultivation was maintained for 20 min at this temperature for heat induction. Finally, the temperature was lowered to 30°C and the shaking culture was continued. Six hours after induction, E. coli 1100 (slp-spl-1) produced 55.7 units per ml culture of sucrose phosphorylase, i.e. 80times higher than that in L. mesenteroides. The a m o u n t of this enzyme was about 30% of the soluble protein in the cell (Fig. 4). Purification of sucrose phosphorylase was done basically as previously described (15). Recombinant enzyme was purified to homogeneity by QAE-Sepharose and hydrophobic H P L C on TSK gel Ether-5PW. The overall purification was approximately 2.3-fold with a yield of 8 0 ~ . The specific activity of recombinant enzyme was 185.77 units per mg protein. This value was much almost the same as that produced in the original strain (173.8 units per mg protein) (15). As shown in Table 1, the productivity of interfered enzymes in E. coli 1100 (slp-spl-1) was fairly low compared with that of L. mesenteroides.

97.4-

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TABLE 1. Sucrosephosphorylase, NADH oxidase, and O-phosphogluconate debydrogenase activities in E. coli 1lO0 (slp-spl-l ) and L. mesenteroides Strains E. coil (slp-spl-1)

20.1 L. mesenteroides FIG. 4.

SDS-PAGE o f cell extracts from E. coil 1100 (slp-spl-I).

The numbers indicated hours after heat induction. The arrow indicates sucrose phosphorylase.

Enzyme activities (units/ml culture) Sucrose NADH 6-Phosphogluconate phosphorylase oxidase dehydrogenase 55.70 0.0042 0.011 (100%) (0.0076%) (0.020%) 0.69 0.070 O.138 (100%) (0.1%) (20.00%)

Parentheses indicate relative activities regard sucrose phosphorylase activities as 100%.

VOL. 73, 1992

CLONING OF SUCROSE PHOSPHORYLASE GENE

183

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DISCUSSION

Sucrose phosphorylase is used for a diagnostic reagent for the specific determination of inorganic phosphate, therefore a high quality of this enzyme is required. In this report, we have shown that the isolation of the sucrose phosphorylase gene from L. mesenteroides and the construction of the overproducing strain using a Sleeper bacteriophage vector. Using the recombinant strain, there are three advantages in the purification process. First, E. coli 1100 (slp-spl-1) produces sucrose phosphorylase 80-times higher than that in L. mesenteroid, es and this enzyme is the most major product in soluble proteins (Fig. 4), because the gene coded for sucrose phosphorylase was replaced instead of that of phage major coat protein. Second, E. coli scarcely secretes mannitol and dextran in the medium. In the case of L. mesenteroides, these sugars caused difficulty of purification, especially on cell washing and concentration by ultra filtration. Finally, as shown in Table 1, the activities of N A D H oxidase and 6-phosphogluconate dehydrogenase were rather high and therefore the purification process was complicated in L. mesenteroides. In the recombinant strain, however, the productivity of interfered enzymes was fairly low. Because of these advantages, high productivity of the sucrose phosphorylase by the recombinant strain led to simplification of the purification process and improvement of the quality of the enzyme. The specific activity of the recombinant enzyme was not appreciably different from that of the native one. Therefore, we guess that the sucrose phosphorylase was produced as an active form in E. coli 1100 (slp-spl-1). Comparison of the deduced amino acid sequence of the L. mesenteroides sucrose phosphorylase gene with that of S. mutans g t f A (23) showed that the overall homology of the two proteins was 68% (Fig. 5). The g t f A genes were representative of glucosyltransferases (GTFs; EC 2.4.1.5) participating in glucan synthesis. Subsequently, Russell et

al. (24) showed that sucrose phosphorylase activity was detectable in an E. coli recombinant harboring the plasmid containing g t f A . Our result also supports the idea that S. mutans gtfA codes for sucrose phosphorylase. ACKNOWLEDGMENTS

We are very grateful to Drs. T. Masuda and Y. Koyama for their valuable discussions and critical readings of this manuscript. We are indebted to Drs. S. Suigiyama and H. Sekine for their encouragement. We also thank Mrs. K. Saitoh for her technical assistance. REFERENCES 1. Vandamme, E.J., Loo, J.V., Machtelinckx, L., and Laporte, A.D.: Microbial sucrose phosphorylase: fermentation process, properties, and biotechnical applications. Adv. Appl. Microbiol., 32, 163-201 0987). 2. Doudoroff, M.W., Hassid, Z., and Baker, H. A.: Studies with bacterial sucrose phosphorylase. II. Enzymatic synthesis of a new reducing and of a new non-reducing disaccharide. J. Biol. Chem., 168, 733-746 (1947). 3. GuiberL A. and Monsan, M.: Production and purification of sucrose phosphorylase from Leuconostoc mesenteroides: application to the production of glucose-l-phosphate. Ann. N. Y. Acad. Sci., 542, 307-311 0988). 4. Birnberg, P. R. and Brenner, M. L.: A one-step enzymatic assay for sucrose with sucrose phosphorylase. Anal. Biochem., 142, 556-561 0984). 5. Waldomann, H., Gygax, D., Bednarski, M.D., Shangraw, W. R., and Withsides, G. M.: The enzymic utilization of sucrose in the synthesis of amylose and derivatives of amylose, using phosphorylases. Carbohydr. Res., 157, e4-c7 (1986). 6. Kelley, S. J. and Butler, L. G.: Enzymatic approaches to production of sucrose from starch. Bioteehnol. Bioeng., 22, 1501-1507 (1980). 7. Kagan, B.O., Latker, S.N., and Zfasman, E.M.: Phosphorolysis of saccharose by cultures of Leuconostoc mesenteroides. Biokhimiya, 7, 93-108 (1942). 8. Doudoroff, M.: Studies on the phosphorolysis of sucrose. J. Biol. Chem., 151, 351-361 (1943).

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9. Weimberg, R. and Doudoroff, M.: Studies with three bacterial sucrose phosphorylase. J. Bacteriol., 68, 381-388 (1954). 10. Vandamme, E. J., Loo, J. V., and Laport, A. D.: Dynamics and regulation of sucrose phosphorylase formation in Leuconostoc mesenteroides fermentations. Biotechnol. Bioeng., 29, 8-15 (1987). 11. Nakano, E. and Masuda, T.: Construction of 'Sleeper' cloning vehicles for enzyme overproduction. Agric. Biol. Chem., 46, 313-315 (1982). 12. Koyama, Y. and Nakano, E.: Cloning of the glpK gene of Escherichia ¢oli K-12 and its overexpression using a 'Sleeper' bacteriophage vector. Agric. Biol. Chem., 54, 1315-1316 (1990). 13. Doudoroif, M.: Disaccharide phosphorylases. Methods. Enzymol., 1, 225-229 (1955). 14. Saito, H. and Miura, K.: Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta, 72, 619-629 (1963). 15. Koga, T., Nakamura, K., Shirokane, Y., Mizusawa, K., Kitao, S., and Kikuchi, M.: Purification and some properties of sucrose phosphorylase from Leuconostoc mesenteroides. Agric. Biol. Chem., 55, 1805-1810 (1991). 16. Maniatis, T., Fritsch, E. F., and Sambrook, J.: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring, New York (1982).

J. FERMENT. BIOEN~ 17. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., a Roe, B. A.: Cloning in single-stranded bacteriophage as an aid rapid DNA sequencing. J. Mol. Biol., 143, 161-178 (1980). 18. Silverstein, R., Voet, J., Reed, D., and Abeles, R. H.: Purific tion and mechanism of action of sucrose phosphorylase. J. Bi~ Chem., 242, 1338-1346 (1963). 19. de Moss, R.D.: Glucose-6-phosphate and 6-phosphoglucor dehydrogenases from Leuconostoc mesenteroides. Methoc Enzymol., 1,328-334 0955). 20. Laemmli, U. K.: Cleavage of structural proteins during the assen bly of the head of bacteriophage T4. Nature, 227, 680-6 (1970). 21. Shine, J. and Dalgarno, L.: The 3'-terminal sequence of cherichia coli 16S ribosomal RNA: complementarity to nonsen triplets and ribosome binding sites. Proc. Natl. Acad. Sci. US, 71, 1342-1346 (1974). 22. de Vos, W.M.: Gene cloning and expression in lactic strept cocci. FEMS Microbiol. Rev., 46, 281-295 (1987). 23. Ferretti, J.J., Huang, T.T., and Russell, R. R. B.: Sequen analysis of the glucosyltransferase A (gtfA) gene from Streptoco cus mutans Ingbritt. Infect. Immun., 56, 1585-1588 (1988). 24. Russell, R. R. B., Mukasa, H., Shimamura, A., and Ferret J. J.: Streptococcus mutans gtfA gene specifies sucrose phosph rylase. Infect. Immun., 56, 1585-1588 (1988).