N-terminal amino acid sequence of Brevibacterium sp. R312 wide-spectrum amidase

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Appl Microbiol Biotechnol (1991) 36:205-207

Applied Microbiology Biotechnology © Springer-Verlag 1991

N-Terminal amino acid sequence of Brevibacterium sp. R312 wide-spectrum amidase Chan K. N. Chan Kwo Chion, Robert Duran, Alain Arnaud, and Pierre Galzy Chaire de Microbiologie Industfielle et de G~n~tique des Microorganismes, Ecole Nationale Sup~rieure Agronomique de Montpellier, Place Viala, F-34060 Montpellier Cedex 1, France Received 3 June 1991/Accepted 11 July 1991

Summary. A w i d e - s p e c t r u m a m i d a s e f r o m Brevibacterium sp. R312 was partially purified. The e n z y m e subunit was purified by reversed p h a s e H P L C a n d the Nterminal a m i n o acid sequence was f o u n d to be identical to that o f Pseudomonas aeruoinosa aliphatic amidase.

lecular mass standards were supplied by BioRad (Richmond, Va., USA) and Serva (Heidelberg, FRG). Chemicals were obtained from Sigma (St. Louis, Mo., USA), except for ammonium sulphate (BRL, Gaithersburg, MD, USA) and n-butyric acid (Aldrich, Gellingham, Dorset, UK).

Microoroanism and culture conditions Introduction Brevibacterium sp. R312 is a nitrile-degrading strain involved in several industrial processes (Jallageas et al. 1980; W y a t t a n d L i n t o n 1988). Nitrile c o m p o u n d s are d e g r a d e d by two enzymes. The first step involves hyd r a t i o n o f nitrile to a m i d e by a nitrile hydratase; in the s e c o n d step a m i d e is h y d r a t e d to organic acid b y an amidase. A m i d a s e purification and characterization have b e e n r e p o r t e d for the following strains: Pseudornonas aeruoinosa ( B r o w n et al. 1973), Arthrobacter sp. J1 (Asano et al. 1982), Brevibacteriurn sp. R312 (Thi6ry et al. 1986), Corynebacterium sp. C5 (Tani et al. 1989), Rhodococcus sp. N-774 ( H a s h i m o t o et al. 1991) a n d Methylobacillus rnethylotrophus (Silman et al. 1991). T h e full a m i n o acid sequence o f P. aeruoinosa aliphatic a m i d a s e has b e e n d e t e r m i n e d previously (Ambler et al. 1987) a n d the N-terminal a m i n o acid sequence o f M. rnethylotrophus a m i d a s e was f o u n d to be h o m o l o g o u s with the P. aeruoinosa aliphatic amidase (Silman et al. I991). In the present study, we describe the purification a n d the N - t e r m i n a l a m i n o acid sequence o f Brevibacteriurn w i d e - s p e c t r u m amidase.

Materials and methods Materials Columns, Q-sepharose Fast Flow, Phenyl Sepharose CL4-B, Sephadex G-200 SF were from Pharmacia (Uppsala, Sweden). Mo-

Offprint requests to: A. Arnaud

Brevibacterium sp. R312 (Arnaud et al. 1976) was used for the purification of the wide-spectrum amidase. Cells were produced on a mineral medium of th~ following composition: KH2PO4, 1.2g1-1; Na2HPO4, 1.95 g1-1; CaCI2, 12mg1-1; ZnCl2, 1.2 mg 1- ~; FeSO4, 1.2 mg 1- ~; MnSO4, 1.2 mg 1- ~; MgSO4, 0.5 mg 1-1; thiamine ehlorhydrate, 2 mg 1-1; (NH~)2SO4, 5 g 1-1; glucose, 10 g 1-1. The cultures were incubated at 28°C in erlenmeyer fiasks filled to 10% of their capacity and shaken (80 oscillations rain -l, 8 cm amplitude). Purification of amidase Brevibacterium cells were produced on mineral medium supplemented with 30 mM N-methylacctamide to induce synthesis of the wide-spectrum amidase (Thi6ry et al. 1986). Fourteen litres of culture were harvested in the mid-exponential growth phase. All of the following steps were performed at + 4 ° C. The cells (116 g wet weight) were washed twice with 11 HEPES buffer (0.1 M HEPES/ KOH, pH 7.3, 40 mM n-butyric acid) and resuspended in the same buffer (0.3 mg 1-1) before sonication with a Sonifier 250 Branson (Danburg, Conn., USA) apparatus under the conditions described in Legras et al. (1989). The cell debris was removed by eentrifugation (10000O, 15 rain) and the cell-free extract (390 ml) obtained by ultracentrifugation (1800009, 90 min). The supernatant was fraetionated by ammonium sulphate precipitation at 40-70% saturation. The suspension was centrifuged (100009, 15 min), and the resulting pellet was dissolved in 100 ml HEPES buffer containing 0.1 M KC1 and dialysed overnight against 101 of the same buffer. Of total activity 91% was recovered at 40-70% ammonium sulphate fractionation. Step 1: Q-sepharose column chromatography. The dialysed solution was loaded on a Q-Sepharose Fast Flow column (26 x 350 mm) equilibrated with the HEPES-KC1 buffer. After one bed volume wash (360 ml h - ~ flow rate), a stepwise elution was performed successively with two bed volumes of 0.2 M KC1 (see Results and discussion) and 0.3 M KC1 in HEPES buffer (the fractions containing nitrile hydratase activity were eluted at 0.3 M KC1

206 and they were stored for other use). A 0.3-0.5 M KC1 gradient was used to elute the proteins containing amidase activity. The active 10-ml fractions were mixed and concentrated by Amicon cell ultrafiltration (PM 10 membrane). At this step, HEPES buffer was replaced by 0.1 M TRIS base/TRIS-HC1 (pH 7.5) because prolonged storage of the enzyme in the initial buffer decreased the activity. //

Step 2: phenyl Sepharose CL4B column ehromatooraphy. The protein solution from step 1 was brought to 20% saturation with ammonium sulphate and applied on a Phenyl Sepharose CL4B column (1.6 x 20 cm) equilibrated with 0.1 M TRIS buffer at 20% saturation with ammonium sulphate. The active 5 ml fractions were eluted by lowering the ionic strength of ammonium sulphate in the buffer (20-0% saturation) at 10 ml h -1 flow rate. The pooled fractions were concentrated by ultrafiltration using an Amicon cell (PM 10 membrane).

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Step 3: Sephadex G-2OOSFcolumn chromatooraphy. The protein solution from step 2 was loaded onto a Sephadex G-200SF column (1.6 x 100 era) equilibrated with 0.1 M TRIS buffer. The active 3 ml fractions were eluted at 3 ml h -1 flow rate, mixed and concentrated as described in step 2.

Protein sequenciny A gas-phase amino acid sequencer (Applied Biosystems, RoissyCharles de Gaulle, France, model 470A) was used for amino-terminal amino acid analysis.



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Protein assay Proteins were assayed according to the method of Lowry et al. (1951) using bovine serum albumin as standard.

Polyacrylamide 9el electrophoresis (PAGE) Sodium dodecyl sulphate (SDS) PAGE was performed according to the method of Laemmli (1970) in 12.5% polyacrylamide flat vertical gel. Gels were stained according to a method described by Andrews (1986), using Coomassie brillant blue R-250.

Results and discussion




F~g. 1. Q-Sepharose chromatography elution diagram of Brevibacterium sp. ~ 1 2 amidase purification (fraction size, 10 ml; ~ow rate£ 360 ml h-~): ~ - , absorbance at 280 rim; ~ , amidase activity; ., KC1 gradient

Table 1. Purification of Brevibacterium sp. R312 wide-spectrum amidase Fractions

Total protein (mg)

Total activity (U)

Specific Yield activity (%) (U mg- 1)

Cell-free extract

9450 3920 855 11 5

35 000 31850 28 370 3 340 2110

3.7 8.1 33.2 303.6 422.0

Enzyme assay and unit definition The standard amidase activity assays were carried out as described previously (Jallageas et al. 1978). One unit (U) of amidase activity was defined as the amount of enzyme required for hydrolysing 1 lxmol propionamide min -1.

] 50

Number of fractions

(NH4)2SO4 (40-70%)

Q-Sepharose Fast Flow Phenyl Sepharose CL-4B Sephadex G200 SF

100 91 81 9 6

U, units

dase described previously (Thi6ry et al. 1986). The enz y m e o f the first p e a k was further purified a n d f o u n d to differ f r o m the w i d e - s p e c t r u m a m i d a s e ( M o r e a u et al. in press). Therefore, the purification was further perf o r m e d with the e n z y m e o f the s e c o n d peak. After step 3, the a m i d a s e was purified l l 4 - f o l d c o m p a r e d to the cell-free extract, with 6% yield (Table 1) even t h o u g h the S D S - P A G E pattern o f the purified p r e p a r a t i o n s revealed the presence o f some c o n t a m i n a t i n g proteins (not shown). The purified enzyme, after the gel filtration step, catalysed the h y d r a t i o n o f p r o p i o n a m i d e at 422 U m g - 1 u n d e r s t a n d a r d reaction conditions.

Purification of amidase Two peaks c o n t a i n i n g amidase activity were eluted f r o m the Q - s e p h a r o s e fast flow c o l u m n (Fig. 1). T h e first p e a k was d e s o r b e d at 0.2 M KC1 a n d r e p r e s e n t e d 12% o f the total activity l o a d e d on the column, T h e seco n d p e a k was eluted with the 0.3-0.5 M KC1 gradient a n d represented 88% o f the total activity. O n l y the seco n d p e a k revealed a p r o t e i n o f relative m o l e c u l a r mass Mr = 43 000 identical to that o f the w i d e - s p e c t r u m ami-

Amidase subunit purification and N-terminal amino acid sequence The e n z y m e was further purified o n a C4 reversed p h a s e H P L C c o l u m n u n d e r the conditions described in the Materials a n d methods. The subunit was purified to h o m o g e n e i t y since the S D S - P A G E o f the subunit prep a r a t i o n s h o w e d a single b a n d o f Mr 43 000 (Fig. 2).

207 al. 1991) seemed identical to the enantiomer-selective a m i d a s e o f Brevibacterium sp. R312 ( M a y a u x et al. 1990). Acknowledgements. We thank J. F. Mayaux, D. Faucher and T. Cartwright (Rhrne Poulenc, Centre de Recherches de Vitry, Institut de Biotechnologie, France) for their help in microsequencing of the enzymes.


Fig. 2. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of Brevibacterium sp. R312 wide-spectrum amidase subunit: lane 1, molecular mass standards, a, phosphorylase b (97.4 kDa); b, bovine serum albumin (66.2 kDa); c, ovalbumin (42.7 kDa); d, carbonic anhydrase (31 kDa); e, trypsin inhibitor (21.5 kDa);f, lysozyme (14.4kDa); lane 2, HPLC-purified wide-spectrum amidase (100 lxg)

A u t o m a t e d E d m a n d e g r a d a t i o n o f 100 [xg o f the H P L C purified subunit e n a b l e d the identification o f 22 residues o f the 25 N-terminal residues present: 1 10 M(R)(H) G D I S S S N D T V G V A V V N Brevibacterium R312:




M I H (G) D I S S S N D T V G V A V V N ( N ) M. methylotrophus: X represents an u n k n o w n a m i n o acid residue a n d uncertain residues are given in parentheses. The identified sequence s h o w e d that Brevibacterium sp. R312 a m i d a s e h a d the same N - t e r m i n u s as P. aeruginosa amidase. Moreover, it seems that the bacterial amidases f r o m P. aeruginosa, Brevibacteriurn sp. R312 a n d M. methylotrophus h a d similar N-terminal a m i n o acid sequences. M o r e o v e r , Brevibacterium sp. R312 contains two different a m i d a s e s : a w i d e - s p e c t r u m amidase (Thirry et al. 1986) a n d an enantiomer-selective amidase ( M a y a u x et al. 1990). H o w e v e r , the N-terminal a m i n o acid seq u e n c e o f the w i d e - s p e c t r u m amidase is completely different f r o m that o f the enantiomer-selective amidase. The a m i d a s e o f Rhodococcus sp. N-774 ( H a s h i m o t o et

Ambler RP, Auffret AD, Clarke PH (1987) The amino acid sequence of the aliphatic amidase from Pseudomonas aeruginosa. FEBS Lett 215:285-290 Andrews AT (1986) Polyacrylamide gel electrophoresis (Page). Homogeneous gel and buffer systems. In: Peacocke AR, Harrington WF (eds) Electrophoresis. Theory, techniques and biochemical and clinical applications. Clarendon Press, Oxford, pp 26-44 Arnaud A, Galzy P, Jallageas JC (1976) Remarques sur l'activit~ nitrilasique de quelques bactrries. CR Acad Sci Paris 283:571573 Asano Y, Tachibana M, Tani Y, Yamada H (1982) Purification and characterization of amidase which participates in nitrile degradation. Agdc Biol Chem 46:1175-1181 Brown PR, Smith MJ, Clarke PH, Rosemeyer MA (1973) The subunit structure of the aliphatic amidase from Pseudomonas aeruginosa. Eur J Biochem 34:177-187 Hashimoto Y, Nishiyama M, Ikehata O, Horinouchi S, Beppu T (1991) Cloning and characterization of an amidase gene from Rhodoeoecus species N-774 and its expression in Escherichia eoli. Biochem Biophys Acta 1088:225-233 Jallageas JC, Arnaud A, Galzy P (1978) Application de la chromatographic en phase gazeuse ~ l'rtude des nitrilases et amidases. J Chromatogr 166:181-187 Jallageas JC, Amaud A, Galzy P (1980) Bioconversion of nitriles and their applications. Adv Biochem Eng 14:1-32 Laemli UK (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227:680-685 Legras JL, Kaakeh MR, Arnaud A, Galzy P (1989) Purification and properties of the fl-glucosidase from a nitrile hydrataseproducing Brevibaeterium sp. strain R312. J Basic Microbiol 29: 655-669 Lowry OH, Rosebrougb NH, Farr AL, Randall RJ (1951) Proteins measurement with the Folin phenol reagent. J Biol Chem 198:265-275 Mayaux JF, Cerbelaud E, Soubrier F, Faucher D, Petre D (1990) Purification, cloning, and primary structure of an enantiomerselective amidase from Brevibacterium sp. strain R312: structural evidence for a genetic coupling with nitrile hydratase. J Bacteriol 172:6764-6774 Silman NJ, Carver MA, Jones CW (1991) Directed evolution of amidase in Methylophilus methylotrophus; purification and properties of amidases from wild-type and mutant strain. J Gen Microbiol 137:169-178 Tani Y, Kurihara M, Nishise H (1989) Characterization of nitrile hydratase and amidase, which are responsable for the conversion of dinitriles to mononitriles, from Corynebacterium sp. Agric Biol Chem 53:3151-3158 Thiery A, Maestracci M, Arnaud A, Galzy P, Nicolas M (1986) Purification and properties of an acylamide amidohydrolase (E.C. with a wide activity spectrum from Brevibaeterium sp. R312. J Basic Mierobiol 26:299-311 Wyatt JM, Linton EA (1988) Microbial hydrolysis of organic nitriles and amides. In: Evered D, Harnett S (eds) Cyanide compounds in biology. Wiley, Chiehester, pp 32-48

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