1 NOTES

Purification and characterization of a P-glucosidase from Streptomyces lividans 66

ANCA MIHOC Institut de biotechnologie, Conseil national de recherche du Canada, Montrc'al (Quc'bec), Canada H4P 2R2

I AND

DIETER KLUEPFEL' Centre de recherche en microbiologie appliquc'e, Institut Armand-Frappier, Universite' du Quc'bec, 531 boulevard des

Prairies, Laval-des-Rapides (Quc'bec), Canada H7N 423

Received August 15, 1989

Accepted October 19, 1989

MIHOC, A , , and KLUEPFEL, D. 1990. Purification and characterization of a P-glucosidase from Streptomyces lividans 66. Can. J. Microbiol. 36: 53-56.

An intracellular P-1 ,4-D-glucosidase (EC 3.2.1.21) was isolated from the mutant strain HP-3 of Streptomyces lividans 66 which produced about 12 times more enzyme than the wild-type strain. The purification was carried out by anion exchange column chromatography followed by high-performance liquid chromatography on DEAE and on molecular sieve columns. The enzyme is glycosylated and has an apparent M, of 51 000 and a pI of 4.3. Its activity was optimal at pH 6.5 and at a temperature of 40°C. The K, and the Vm, on cellobiose were 3.1 mM and 65.6 pmol min-' mg-I of enzyme.

Key words: P-glucosidase, Streptomyces lividans, purification, characterization.

MIHOC, A., et KLUEPFEL, D. 1990. Purification and characterization of a P-glucosidase from Streptomyces lividans 66. Can. J. Microbiol. 36: 53-56.

Une p-1 ,4-D-glucosidase intracellulaire (EC 3.2.1.21) a Ctk isolte de la souche mutante HP-3 de Streptomyces lividans 66 qui produisait environ 12 fois plus d'enzymes que la souche sauvage. La purification de cet enzyme s'est effectuCe par chromatographie sur colonne Cchangeuse d'ions puis par chromatographie liquide a haute performance (HPLC) sur colonnes DEAE et tamis moltculaire. Cet enzyme est glycosylt et a un M, de 51 000 et un pI de 4,3. Son activitt Ctait optimale &pH 6,5 et B 40°C. Le Km et le Vm, sont de 3,1 mM et 65,6 pmol min-' mg-' d'enzyme, respectivement.

Mots clis : P-glucosidase, Streptomyces lividans, purification, caractkrisation.

Cellobiase (P-glucosidase; EC 3.2.1.21) plays an important role in the biodegradation of cellulose. Together with the P- 1,4-glucan-cellobiohydrolase (exo-glucanase; EC 3.2.1.91) and the p- l,4-glucan-glucanohydrolase (endo-glucanase; EC 3.2.1.4), it forms the cellulase enzyme system and catalyzes the breakdown of cellobiose into two molecules of glucose. Through this action, the P-glucosidase is generally responsible for the regulation of the whole cellulolytic process and is often the rate-limiting factor (Kadam and Demain 1989).

Among the Actinomycetales, the biosynthesis of this enzyme has been reported as part of the cellulase system by several authors (Stutzenberger 1972; Hagerdal et al . 1979; Ishaque and Kluepfel 1980). Small quantities of cellobiase had also been

I found in Streptomyces lividans by Kluepfel et al. (1986). In preparation for the cloning and characterization of the P- glucosidase gene, we isolated and purified the enzyme from S . lividans and determined some of its chemical and physical properties.

The enzyme was produced by cultures of S . lividans HP-3, a mutant strain obtained from S . lividans 66 (wild-type strain 1326) by mutagenesis with N-methyl-N1-nitro-N-

1 nitmsoguanidine using the method of Dklic et al . (1970). It was selected for increased cellulase or P-glucosidase activities by the methods of Daigneault-Sylvestre and Kluepfel (1979) and Kluepfel (1 988). Streptomyces lividans HP-3 produced about 12 times more intracellular P-glucosidase than was reported earlier by Kluepfel et al . (1986) for the wild-type strain.

' ~ u t h o r to whom all correspondence should be addressed.

The strain was maintained and grown as described by Moldoveanu and Kluepfel (1983). Either oat spelts xylan (Sigma Chemical Co., St. Louis, MO, USA) or Avicel PH 105 (FMC Corp., Marcus Hook, PA, USA) at concentration of 1% (w/v) served as main carbon sources.

The mycelium was separated from the fermentation broth in a Beckman J2-21 centrifuge at 11 000 X g and washed twice with sterile distilled water. A homogenous cell suspension was prepared in a minute volume of 5 rnM sodium phosphate buffer, pH 6.5, and sonicated under constant cooling, applying at intervals, 5 to 6 sonic bursts of 20 s with a Biosonik IV apparatus (Bronwill-VWR Sci. Co., San Francisco, CA, USA). Imrned- iately after sonication, 50 pg/mL of phenylmethylsulfonyl- fluoride (Gibco Canada Ltd.) dissolved in ethanol was added to inhibit any proteolysis by serine proteases. The cell debris was removed by centrifugation at 11 000 X g for 1 h. Additional activity was recovered after resuspending the sediments and repeating the sonication. To the combined supernatants, repre- senting the crude enzyme preparation, 10% glycerol were added to increase enzyme stability. This mixture could be stored for several months at - 20°C.

The crude enzyme was adsorbed to a DEAE Biogel A ion-exchange resin (Bio-Rad Laboratories) that had been equilibrated with 5 mM sodium buffer, pH 6.5, containing 10% glycerol. The column was eluted stepwise with the same buffer containing 50,100,150, and 200 mM NaC1. The fractions were analyzed for P-glucosidase activity using methylumbelliferyl- P-D-glucoside (MUG) assay (described below). The active fractions were p l e d , concentrated, and desalted in an Arnicon

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54 CAN. J . MICROBIOL. VOL. 36, 1990

ultrafiltration cell using a PM 10 membrane, changing the buffer to 20mM Tris-HC1, pH 8.5, containing 10% glycerol. The enzyme was purified further on a HPLC Protein Pak DEAE 5 PW column (Waters-Millipore Inc., St. Laurent, Que.) using the same buffer with a linear NaCl gradient (0-500 mM) and a reverse linear pH gradient (pH 8.5-7.0) at a flow rate of 1 mL/min. The elution was monitored at 280nm. The active fractions were desalted and analyzed by the MUG assay. A second HPLC separation was carried out under the same conditions and the active fractions were pooled and concentrat- ed. Final purification was achieved on two Protein Pak SW 300 HPLC columns (Waters-Millipore) joined in series using a 5 mM sodium phosphate buffer, pH 6.5, containing 10% glycerol using a flow rate of 0.5 mL/min. The columns were calibrated with molecular protein standards (M,, 12 500, 25 000,45 000,68 000; BMC Canada Ltd., Dorval, Que.). The pure enzyme solution was concentrated in a CentriconTM microconcentrator (Amicon Division, Danvers, MA, USA). The pure enzyme was either kept at 4°C or for prolonged periods at -70°C.

P-Glucosidase activity was assayed on either of the three following substrates: ( i ) cellobiose using the method reported by Moldoveanu and Kluepfel(1983) (This assay was generally used throughout these investigations to determine enzyme activity); ( i i ) p-nitrophenyl-/3-D-glucoside (PNG; Sigma Chem- ical Go.) using the method described by Ishaque and Kluepfel ( 1 980); and ( i i i ) 4-methylurnbelliferyl-P-D-glucoside (MUG; Sigma Chemical Co.) which was used for rapid qualitative detection of enzyme activity. For this assay, 250 pL of 1 mM MUG dissolved in 50 mM sodium phosphate buffer, pH 6.5, was placed into wells of ELISA plates; 2 to ~ - F L aliquots of the enzyme solutions were added and the plates were incubated for 15 min at 50°C. Activity was revealed by blue fluorescence under shortwave ultraviolet light.

The activity of all enzyme assays was expressed in nanokatals (nkat) corresponding to nanomoles of glucose released per second. The results were averages of at least three independent determinations. The protein content of the enzyme preparations was determined by the method of Lowly et al. (1951).

Nondenaturing electrophoresis was carried out in gels con- taining 8% of polyacrylamide according to the method of Johnstone and Thorpe (1985). The M, of the purified P- glucosidase was estimated by SDS - polyacrylamide gel electroporesis as described by Laemmli (1970) using the high-range MI (14 300 - 200 000) protein standards (BRL). The gels were stained with either Coomassie blue for proteins or Schiff's reagent for the detection of glycoproteins (Glossmann and Neville 197 1). Analytical isoelectric-focusing gels were run as described earlier by Morosoli et al. (1986). For the detection of the P-glucosidase activity, 2 rnrn wide slices of these gels were incubated individually for 15 min at 40°C in 1 mL of 5 mM sodium phosphate buffer, pH 6.5, containing 1 mM of MUG.

The cellobiase levels of S. lividans HP-3 were determined at 4.50-5.0nkat.mL-' of cell extract which was about 12 times higher than those of the wild-type strain. It had shown rapid cellulolytic action on solid media containing filter paper. No extracellular P-glucosidase activity was detected in the culture filtrates. A comparison of the different cellulase activities with those of the wild-type strain S. lividans 1326 had shown that mutant HP-3 differed only in its intracellular P-glucosidase levels (F. Aumont, unpublished results). Thus it offered a considerable advantage for the purification of the enzyme. As reported for other actinomycetes (Hagerdal et al. 1979; Ishaque

and Kluepfel 1980), the enzyme located in the cytoplasm could be recovered from the supernatants after sonication.

P-Glucosidase production was studied on different carbon sources such as Avicel, xylan, cellobiose, or lactose. Best intracellular enzyme levels were obtained after 24 h, on either xylan or Avicel. Values of 7.5 and 5.8 nkat . m ~ - ' of cell extract were obtained corresponding to specific activities of 2.8 and 3.5 nkat .mg-' of protein, respectively. An increase to 5.21kat.m~-' was achieved on Avicel by adjusting the pH of the medium to pH 7.1. Similar results have been reported in CellulomonasJimi by Wakarchuk et al. (1984) and by Kluepfel and Ishaque (1982) for S. jlavogriseus. In contrast, the extracellular P-glucosidases secreted by a noncellulolytic Streptomyces sp. isolated by Kusama et al . (1986) demon- strated very weak activity against cellobiose.

Sonication of the mycelium and separation of the cell debris yielded between 4.5 to 4.7 nkat of crude enzyme per milligram of protein in the supernatants. An additional 17% of enzyme could be obtained by sonicating the pellet again. Phenylmethyl- sulfonylfluoride, added routinely to the extracts, did not prevent the rapid inactivation of the P-glucosidase. The effect of adding known stabilizing agents, such as glycerol, inositol, and cellobiose, was studied at different concentrations. Among these, best results were obtained with 10% glycerol, which reduced the enzyme inactivation to about 10% over a period of 120 h. It was therefore included in all solutions.

Initial purification of the enzyme on a DEAE anion-exchange column was achieved with buffer containing 150mM NaCl. This was followed by two separations on a DEAE HPLC column and a final passage on a gel HPLC column. The enzyme activity was followed by the MUG assay. The M, of the purified enzyme was determined at 47 kDa on the Protein Pak SW 300 columns calibrated with standard proteins. Table 1 summarizes the yields obtained with the different steps as well as the purification factors achieved. The enzyme was purified 11 3-fold with an overall yield of about 10%. The average specific activity of the pure P-glucosidase was 600 nkat-mg-' of protein.

Analysis of the purified enzyme by nondenaturing polyacryl- amide gel electrophoresis revealed a single protein band that corresponded to the maximum activity as determined with MUG.

The stepwise purification of the cellobiase is shown in Fig. 1. I i

The apparent MI of 5 1 000 was calculated from a standard linear 1 regression curve of reference proteins. The difference between ! the M, obtained by the HPLC gel chromatography and SDS- !

I PAGE was probably due to the fact that molecular sieves separate proteins on the basis of size and tertiary configuration, ,

whereas in SDS-PAGE their migration is determined by the size of the reduced and random-coil form. In nondenaturing , electrophoresis, isoelectric focusing as well as during the : purification steps on HPLC both on DEAE and molecular sieve I columns, the active protein migrated or was eluted as a ' homogenous compound. The M, of 5 1 000 is of the same order as those reported for the other bacterial cellobiases (Woodward and Wiseman 1982). The enzyme was glycosylated, as revealed by a positive stain with Schiff's reagent (results not shown). I

In analytical isoelectric focusing on a pH gradient of pH 3- 10, the enzyme showed a pI of 4.3 and the band corresponded to the activity as determined by the MUG assay (Fig. 2).

The stability of the purified cellobiase at different temperat- ures determined in the absence of cellobiose was considerably lower than that of the crude enzyme preparations in spite of the presence of glycerol. The optimal temperature and pH for

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TABLE 1. Summary of the purification of a P-glucosidase from Streptomyces lividans HP-3 based upon 7.5 L of fermentation broth

Total Specific Purification Volume activity activity Purification Yield

step (&at) (nkat . mg- ') factor (%) -

Cell extract 1550 6060 5.30 1.00 100.0 Bio-Gel A 420 2870 22.78 4.30 47.4 HPLC DEAE anion exchange 92 1534 166.70 31.45 25.3 HPLC DEAE anion exchange 25 834 583.45 110.08 13.7 HPLC gel filtration 5 633 603.30 113.83 10.4

FIG. 1. SDS-PAGE electrophoresis of P-glucosidase from Strep- romyces livi&ns W-3 at various stages of purification. Lane I , M, marker proteins; lane 2, crude ceH extract: lane 3, Biogel A column chmatagraphy; lane 4, fmt passage Protein Pak DEAE 5 PW HPLC column; lane 5, second passage on same column; lane 6, purified & glucosidase (10 pg) after passage through Protein Pak 300 SW HPLC molecular sieve columns. Gel was stained with Coomassie blue.

enzyme activity on cellobiose were 40°C and pH 7.0, respectiv- ely, while with PNPG as substrate they were 35-40°C and pH 6.5.

The Michaelis constant was determined with cellobiose concentrations ranging from 0.36 to 12.3 1 mg/mL. From a Lineweaver-Burk plot, the K , was found to be 3.1 rnM and the V,, was 65.6 pmol min-' mg-' of enzyme. On PNPG these values were 2.01 rnM and 45.4 pmol min-' mg-I, respectively.

The pwified 8-glucosidase showed neither P-xylosidase activity when tested with p-nitrophenyl-0-n-xylwside by the method of Kluepfel and Ishaque (1982) nor xylobiase activity. However, 4-methylumbelliferyl-p-~-celhbioside, generally used as substrate for the detection of cellobiohydrolase activity, was hydrolyzed which indicated a possible affinity of the enzyme towards cellotriose or celloletraose. A similar a- glucosidse has been purified by Uuang et al. (C. M. Huang, W. J . Kelly, M. M. Cuny, P. L. Yu, and R. V. Asmundson. 1987. FEMS Symposium on Biochemistry and Genetics of CeIlulose Degradation,53eptember 7-9, P i s , France. Abstract P3-08) from Ruminococcus jlavofaciens.

The other properties of the purified cellobiase were compara- ble with those reported for cellobiases from other prokaryotes. The pH optimum at 6.5 to 7.0 was similar to the values reported

LENGTH OF GEL (cm) FIG. 2. Is~eIectric focusing of the purified P-glucosidase fmm

Strepromyces lividans HP-3. Lane a, pI markers; lane b, purified P- gtucosidase (5 kg). One part of the sIab gel was stained with Coomas- sie blue (A), whlle the other was sliced into 2-rnm strips which were analyzed individually for enzyme activity with the methylumbelli- feryl-P-D-glucoside assay (B).

for the P-glucosidase of Alcaligenes faecalis (Han and Srinivas- an 1969) and the crude preparations from Cellulomonas jimi (Wakarchuck et al. 1984). The optimal temperature for enzyme activity of the different cellobiases ranged between 40 and 65°C.

Antibodies raised with this pure cellobiase served to campare the enzyme produced by S. lividnns HP-3 with that of the wild-type strain 1326. Both enzymes cross-reacted in the immunoblot made after SDS-PAGE, indicating a likely homology. The antibodies will be useful for further studies of the cellulase system of S. lividans.

Acknowledgements This research was supported by a graduate student fellowship

to one of us (A.M.) and by an operating grant from the Natural Sciences and Engineering Research Council of Canada. We also would like to thank Liette Biron for her help and technical assistance.

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56 CAN. J. MICROBIOL. VOL. 36, 1990

DAIGNEAULT-SYLVESTRE, N., and KLUEPFEL, D. 1979. Method for the rapid screening of cellulolytic streptomycetes and their mutants. Can. J. Microbiol. 25: 258-260.

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HAGERDAL, B., HARRIS, H., and PYE, E. K. 1979. Association of P-glucosidases with the intact cells of Thermoactinomyces. Bio- technol. Bioeng. 21: 345-355.

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KADAM, S. K., and DEMAIN, A. L. 1989. Addition of cloned P-glucosidase enhances the degradation of crystalline cellulose by Closteridium thermocellum cellulase complex. Biochem. Biophys. Res. Commun. 161: 706-71 1.

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KUSAMA, S., KUSAKABE, I., and MURAKAMI, K. 1986. Purification and some properties of P-glucosidase of Streptomyces sp. Agric. Biol. Chem. 50: 2891-2898.

LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 277: 680-685.

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Determination of peptidyl dipeptidase activity in 24 bacterial species

JOANNE STEVENS, BARRY L. FANBURG, AND JOSEPH J. LANZILLO' Department of Medicine, New England Medical Center Hospital, Box 19, 750 Washington Street, Boston, MA 02111, U.S.A.

Received March 15, 1989

Accepted October 16, 1989

STEVENS, J., FANBURG, B.L., and LANZILLO, J. J. 1990. Determination of peptidyl dipeptidase activity in 24 bacterial species. Can. J. Microbiol. 36: 56-59.

Of 24 bacterial species examined for lisinopril refractive peptidyl dipeptidase activity, only 8 contained activity. Activity in Pseudomoms maltophilia was more than fourfold higher than that of any other species. Pseudomoms maltophilia may be unique among bacteria in possessing high peptidyl dipeptidase activity that is both EDTA inhibitable and lisinopril resistant.

Key words: Pseudomonas maltophilia, peptidyl dipeptidase.

STEVENS, J., FANBURG, B.L., et LANZILLO, J. J. 1990. Determination of peptidyl dipeptidase activity in 24 bacterial species. Can. J. Microbiol. 36: 56-59.

Sur 24 esp6ces bactkriennes examinees quant 1 la rkfringence au lisinoprile de I'activitk peptidyl dipeptidase, seules 8 d'entre elles posskdaient cette activitk. L'activitk de Pseudomoms maltophilia, comparativement 1 celle de toute autre espkce, a kt6 quatre fois plus klevke. Panni les bactkries, ce P. maltophilia pourrait s'avkrer etre une bactkrie unique en raison de son activitk peptidyl dipeptidase 6levCe, laquelle peut 1 la fois &tre inhiMe par I'EDTA et rksistante au lisinoprile.

Mots clks : Pseudomonas maltophilia, pepiidyl dipeptidase. [Traduit par la revue]

Peptidyl dipeptidases (PDP) occur in both eukaryotic and Wilson 1984; Lee eta!. 1971 ; Nakahara 1978; Neidle and Kelly prokaryotic cells (Angus et al. 1972; Cushman et al. 1983; 1984; Yaron et a/. 1972), the archetypical PDP being angioten- Demrner and Brand 1983; Grandino and Paiva 1974; Harris and sin converting enzyme (ACE; EC 3.4.15.1) (Skeggs et al.

1954). Previously, we reported structural and catalytic proper- '~uthor to whom correspondence should be addressed. ties of a putative cryptic endotheIia1 cell PDP (designated

Rinted in Canada / Imprim6 au Canada

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