A β-lactamase produced by a thermophilic Bacillus

FEMS Microbiology Letters 140 (1996) 61-64

A P-lactamase produced by a thermophilic Fouzia Rhazi-Filali

a3b,Abdelhamid Zaid a, Philippe Ledent ‘, Marc Vanhove b, Jozef Van Beeumen ‘, Jean-Marie F&-e b**

a Faculte’ h Centre for Protein

’

Vakgroep

Biochemie.

BaciZZus

des Sciences. Universit&

Engineering, Fysiologie

UrGersite’

de Meknes. P.O. Box 4010, Meknes. Morocco

de L&e.

en Microbiologic.

lnstitut

de Chin&,

Unirersiteit

B6. Sart-Tihnan.

Gent, Ledeganckstmat

84000

Liege, Belgium

35, BY000 Ghent. Be/gium

Received 10 April 1996; accepted I2 April 1996

Abstract A P-lactamase was purified from a thermophilic Bacil1u.s strain, that had been isolated from a traditional hot bath in the Meknes area (Morocco). The properties of the enzyme were very similar to those of the p-lactamase produced by Bacillus lichenifiwmis 749C but it exhibited a somewhat increased thermostability and a higher activation energy with cefazolin as substrate. These properties were expected for an enzyme produced by a thermophilic strain. Kevwordsc

Thermophile;

Bacillus:

P-Lactamase;

Penicillin:

Cephalosporin

1. Introduction

2. Materials

We have recently compared the stabilities of plactamases produced by some mesophilic bacterial species [I ]. In order to increase the stability range of the studied enzymes and since p-lactamase production by thermophilic strains has not been described, we isolated thermophilic strains from traditional hot public baths in Meknes (Morocco) and analyzed them for p-lactamase production. Three Bacillus strains were found to produce the enzyme and the strain yielding the highest activity was further investigated. The properties of the enzyme, a close relative of that produced by Bacillus licheniformis 749C, are described in the following communication.

2. I. Strain isolation and characterization

and methods

Water samples were collected in traditional hot baths in the Meknes area. Aliquots (1 ml) were used to inoculate IO ml of liquid TK medium, pH 7.5 (1.6% bactotryptone, 0.5% yeast extract and 0.25% K2HPO;I). The cultures were grown for 48 h at 55°C and drops were subsequently streaked on agar containing the same TK medium and 5 mg/l of ampicillin. Three resistant strains were thus isolated and characterized as belonging to the Bacillus genus on the basis of morphological, physiological and biochemical properties, with the help of the Minitek [2] and API systems [3]. 2.2. Enzyme actilsity

* Corresponding author. Tel.: + 32 (41) 663 398; Fax: (41) 663 364; E-mail: U217902Bvml .ulg.ac.be 037%1097/96/$12.00 0 1996 Federation PII SO378-lO97(96)OOl61-9

of European

+32

Microbiological

The enzyme activity was routinely assayed with 100 PM nitrocefin [4]. One unit hydrolyses 1 pmol Societies.

All rights reserved

62

F. Rhazi-Filali

et al. / FEM.5 Microhiolog~

of substrate per minute at 30°C. The hydrolysis of the other plactam antibiotics was monitored spectrophotometrically as described by Matagne et al. [5]. Kinetic parameters were determined with the help of one of the three following methods: 1. Analysis of complete time-courses with the help of the integrated Henri-Michaelis equation [6]. 2. Analysis of initial rates according to the Hanes linearization. 3. Determining K, as a K, by using the compound as a competitive inhibitor (with nitrocefin as substrate) and V,,, as the initial rate at [S] > 10 X K Ai’kinetic experiments were performed at 30°C in 50 mM sodium phosphate, pH 7.0. 2.3. Enzyme production

and purijkation

Fifteen l-l Erlenmeyers, each containing 500 m1 of M4 medium (1% bactotryptone, 0.5% yeast extract, 1% NaCl and 0.375% glycine) were inoculated with 2 ml of a 18-h preculture and the culture was grown at 50°C under orbital agitation (250 rpm). After 12 h, cefazolin (0.1 mg/l, final concentration) was added and the culture grown for an additional 36 h. The culture supematant was concentrated by ultrafiltration, dialysed against 10 mM Tris HCl buffer, pH 9.0 and diluted 20-fold with the same buffer. The enzyme was adsorbed on 200 ml of DEAE-Sephacel equilibrated against the same buffer. After elution (0.2 M NaCl in the same buffer), concentration and dialysis, the solution was loaded onto a Q-Sepharose Hi-Load 26/ 10 column (Pharmacia) and the enzyme eluted with a linear NaCl gradient. After concentration of the active fractions, the enzyme was further purified by chromatofocusing with the help of a Mono-P H 5/20 column (Pharmacia) in the pH range 7.1-4.0. After concentration, the active fractions were filtered through a Superdex 75 H lo/30 column in 10 mM Tris . HCl pH 8.0 containing 200 mM NaCl. 2.4. Protein chemistry

Letten

140

f lW6161-M

filter paper previously dipped in a 300 /IM nitrocefin solution. Chemicals: the origin of the plactam compounds was the same as before [5] and the B. lichen@rmis 749C plactamase was produced and purified as described in the same contribution [5].

3. Resa& Three penicilhn-resistant (5 pg/ml) strains were isolated. On solid medium, their optimum growth temperature was around 50°C. The three strains were characterized by bacillary-shaped cells, forming endospores. On the basis of their biochemical properties, they could be classified as thermophilic bacilli according to Norris et al. IS]. Preliminary experiments indicated strain BS3 as the best p-lactamase producer and it was retained for further studies. The strain was resistant to chloramphenicol (30 pg/ml), oxacillin (5 pg/ml), ampicillin (25 pg/ml), benzylpenicillin (10 pg/ml), carbenicillin (100 pg/ml) and cefazolin (30 pg/ml) and sensitive to gentamycin (IO pg/ml), pristinamycin (10 pg/ml), ciprofloxin (5 pg/ml), tetracycline (30 pg/ml) and kanamycin (30 pg/ml). In the liquid M4 medium, a minimum generation time of 50 min was found at 49”C, vs. 156 and 100 min at 37 and 43”C, respectively. At 55”C, the generation time increased to 95 min. By comparison, a probably related strain, B. lichenijbmis 749C (see Section 4), exhibited generation times of 28, 22 and 22 min at 37, 43 and 49°C and no growth was observed at 55°C. The basal level of plactamase production by strain BS3 was quite low (0.006-0.020 units/ml after 12-48 h) but it could be increased 20-fold by Table 1 Purification

of the BS3 plactamase Total vohune

Total pfobzin

Total activity

(ml)

(rnp)

(units) (ulrits/ mg protein)

7500 8

26250 31 8 2.1 0.3

3750 3200 854 330 200

techniques

The M, value was estimated by SDS-PAGE according to Laemmli and Favre [7]. The isoelectric pH was determined on a Multiphor II apparatus at 4°C and the enzyme localized by application of a piece of

supematm DEAE-Sephacel Q-Sephamse Mono-P SUpeRieX

1 I 2

Specific activity

0.14 96 105 157 700

Yield (a)

100 80 23 9 5

F. Rhazi-Filali et al. / FEMS Microbiology Letters 140 (1996) 61-64 Table 2 Kinetic parameters

Nitrocefin Ampicillin Benzylpenicillin Carbenicillin Oxacillin Ticarcillin Cephaloridine Cefazolin Cephalosporin C Ceftazidimc Cefamandole Cefuroxime Cefotaxime Cephalothin 7-ACA ’

63

of the BS3 ,!?-lactamase Method

[Substrate] ( /zM)

b (nm)

k,,, (s-

1 3 3 3 3 3

100 20-150 25-200 25-150 25-100 25-100

I I

200

482 235 235 235 260 235 260 260 260 260 260 260 260 260 260

430 330 315 22 18 14.5 160 320 24 9 53 23 11 170 0.02

100 25-150

2 2

M-500

1 I I I

100 100 100 Ial

3

25-100

’) *lo *5 + 9 *2 f 0.1 + 0.2 +4 *IO i3 *I +5 k I +I +7 f 0.005

When method 3 was used, the indicated range of concentrations was that for the competitve at which V,, was measured. a 7-ACA = 7-amino-cephalosporanic acid; n.d = not determined.

K, (/.rM)

k,,,,‘K,

33+ I 25+ I 26 f 0.2 < IO < 10 <5 77 f 0.5 114+3 134i 14 510*50 19* I 42 f 4 50* I 38 * 3 16kO.l

13 kO.5 13 + 0.2 12 * 0.4 n.d. nd. nd. 2.1 f 0.1 2.8 + 0.2 0.2 + 0.02 (2 & 0.2) x 10-l 2.8 + 0.2 0.6 i 0.03 0.2 + 0.005 4.5 i 2 (1.3 * 0.3) x lo-’

inhibition experiments

(PM-’

.s-‘)

and the wavelength

that

and gel electrofocusing. These two techniques also indicated an M, value of 30 000 f 1000 and an isoelectric pH of 4.8. Active-site titration was performed with /3-iodopenicillanate [9] and complete enzyme inactivation occurred at an inactivator/enzyme ratio of 1.05 * 0.1.

induction with 0.1 pg/ml cefazolin. Ampicillin and benzylpenicillin also induced the production of enzyme but were less efficient. Table 1 summarizes the purification of the enzyme, performed with a total culture volume of 7.5 liters. The enzyme was pure as shown by SDS-PAGE

I-80

1

I

30

40

50

60

70

80

Temperature (‘c) Fig. I. Thermal denaturation of the BS3 plactamase. The enzyme solution (0.5 PM) in 50 mM sodium phosphate, rate of I .3”C/min. Fluorescence was recorded at 340 nm (slitwidth, 20 nm). Excitation was at 280 nm (slidwidth,

pH 7.0, was heated at a 4 nm).

64

F. Rki-Filali

et al. / FEM.5 Microbiology

Despite the poor yield, enough enzyme was obtained for preliminary characterization. Table 2 lists the kinetic parameters determined on a representative set of penicillins and cephalosporins. The temperature dependence of the rate of cefazolin hydrolysis was studied between 20 and 60°C. Within the limits of experimental error, the Arrhenius plots were linear and indicated activation energies of 11.8 + 1.5 and 9.1 + 0.5 kcal/mol for k,,, respectively. For the B. licheniformis and k,,,/K, 3 enzyme, the corresponding values determined under the same conditions and with the same substrate were 11.1 and 7.3 kcal/mol, respectively. The thermal denaturation of the enzyme was monitored by recording the fluorescence at 340 nm of a 0.5 PM solution in 50 mM sodium phosphate, pH 7. As shown in Fig. 1, a T, value of 64.7 ? 0.2”C was found. If the sample was immediately cooled after reaching 70°C the unfolding phenomenon was found to be fully reversible. Similarly, after a l-h incubation at 70°C the enzyme retained more than 80% of its initial activity as measured at 30°C. We attempted unsuccessfully to determine the N-terminal amino acid sequence on a 500-pmol sample with the help of an Applied Biosystem 470-A gas phase sequencer, suggesting a blocked N-terminus.

Letters

140 (IYYcil 61-64

stant. Indeed, between 30 and 60°C the k,,,/K, value of the BS3 enzyme increased 4-fold, vs. 3-fold for its B. lichenjformis counterpart. Since the K, value for this substrate is rather high, the k,,,/K, parameter is probably the most significant since it reflects the rate of the reaction at [S] < K,. Thus, these observations are in agreement with the behavior of a thermophilic protein. However, it is surprising to note that the thermostability of the B. licheniformis enzyme is rather high and it could be argued that, since the P-lactamase is probably a dispensable enzyme under ‘normal’ conditions, its stability should not necessarily be related to the optimum temperature for the growth of the bacterium.

Acknowledgements F.R.F. is indebted to the AUPELF-UREF Scientific Council (Paris, France) for a fellowship which allowed her to travel to and work in Liege. The work in Liege was supported by the Belgian Program of Interuniversity Poles of Attraction (PA1 No. 19) and an Action Concert&e with the Belgian Government (93-98/ 1701.

References 4. Discussion [I] Vanhove. M.. Houba, S.. Lamotte-Brasseur.

A P-lactamase-producing thermophilic Bacillus strain was isolated. Its generation time was 3-fold shorter at 49°C than at 37°C and non-negligible growth was observed at 55”C, in contrast with the behavior of B. licheniformis. To our knowledge, this is the first time a P-lactamase has been isolated from a thermophilic strain. Preliminary experiments indicate that the enzyme was rather thermostable. It was purified and its kinetic parameters determined with several substrates. These were rather similar to those of the B. licheniformis 749C enzyme studied previously by Matagne et al. 151. Consequently, the thermostable enzyme was compared to the B. licheniformis /3-lactamase. It exhibited a moderate, but significant increase of the T, value (64.7 vs. 63.2”C) and, for the k,,,/K, parameter, its activation energy was 2 kcal/mol higher, reflecting a more pronounced temperature dependence of this kinetic con-

J. and F&e. J.M. (1995) Biochem. J. 308, 859-864. [2] Sullivan, N.M., Mills. D.C.. Piemann, H.P. and Aron, S.S. (1987) Appl. Environ. Microbial. 53, 2980-2982. 131 Logan. N.A. and Berkeley, R.C.W. (1981) in: The Aerobic. Endospore-forming Bacteria: Classification and Identification (Berkeley, R.C.W. and Goodfellow, M.. Eds.), pp. 105-140. Academic Press, London. [4] O’Callaghan, C.H., Morris. A., Kirby, S. and Shingler, A.H. (1972) Antimicrob. Agents Chemother. I, 283-288. 151 Matagne, A., Misselyn-Bauduin, A.M., Joris. B.. Erpicum. T.. Granier. B. and F&e. J.M. (1990) Biochem. J. 265, 131- 146. [6] De Meester. F.. Joris, B.. Reckinger, G.. BellefroidBourguignon, C., F&e, J.M. and Waley, S.G. t I9871 Biochem. Pharmacol. 36. 2393-2403. [7] Laemmli. U.K. and Favre. M. (1973) J. Mol. Biol. 80, 575599. [8] Norris, J.R., Berkeley. R.C.W.. Logan. N.A. and Donnel. G (1977) in: The Prokaryotes. A Handbook on Habitats: Isolation and Identification. pp.I7 l3- 1734. Mortimer. New York. [9] De Meester, F.. F&e. J.M., Waley, S.G., Cartwright. S.J., Virden, R., Bush, K. and Sykes, R. (1986) Biochem. J. 239, 575-580.