Ahmed, Syed Ashrafuddin

Esaki, Nobuyoshi

Soda, Kenji

Tanaka, Hidehiko

Kyoto University Research Information Repository

Title Production and Stabilization of a-Amino-E-caprolactamRacemase from Achromobacter obaeAuthor(s)Ahmed, Syed Ashrafuddin; Esaki, Nobuyoshi; Tanaka,Hidehiko; Soda, KenjiCitationBulletin of the Institute for Chemical Research, KyotoUniversity (1982), 60(5-6): 342-346Issue Date1982-11-15URL http://hdl.handle.net/2433/77006RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityBull. Inst. Chem. Res., Kyoto Univ., Vol. 60, No. 1982     Production and Stabilization of a-Amino-E-caprolactam             Racemase from Achromobacter obae                Syed Ashrafuddin AHMED*, Nobuyoshi EsAKI*,                   Hidehiko TANAKA and Kenji SODA*                                Received Oct. 7, 1982    Achromobacter obae FERM-P 776 grown in both organic and inorganic nitrogen media produced a-amino-e-caprolactam racemase, and  ut,-a-amino-s-caprolactam stimulated the enzyme production. The enzyme was labile, but was stabilized considerably by addition of 40% ethylene glycol, 0.25 M sucrose and a mixture of glycerol,  dithiothreitol, superoxide dismutase and a pepsin inhibitor  S-PI. Approximately 90% of the enzyme activity was retained in the presence of sucrose even after storage at —20°C for four months.     KEY  WORDS  : Amino-caprolactam racemase/ Bacterial  enzyme/ Enzyme  production/ Enzyme stabilization/                            INTRODUCTION     a-Amino-s-caprolactam racemase (EC class 5.1.1) was demonstrated by  Fukumuran in some bacteria. Achromobacter obae that produces the enzyme most abundantly is utilized industrially to produce L-lysine from  DL-a-amino-s-capro-lactam with a high yield in a coupled system with  L-a-amino-z-caprolactamase (EC class 3.5.2) of Cryptococcus  laurentii.2) This enzyme has been purified to homo-geneity and  characterized.3) However,  Fukumura4) has made an attempt to purify the racemase with little success due to its lability.    In order to purify the enzyme and study its enzymological and physicochemical properties, it is essential to find out the optimum conditions for the enzyme production and stabilization. We here describe the studies of the growth medium for Achromobacter obae to produce the enzyme effectively, and its stabilization.                       MATERIALS AND METHODSChemicals     DL-a-Amino-s-caprolactam, L- and  n-a-amino-r-caprolactam hydrochlorides were obtained from Toray Industries Inc., pyridoxal 5'-phosphate from Kyowa Hakko and superoxide dismutase was from Sigma. A pepsin inhibitor  S-PI, produced by a Streptomyces  sp.5,6) was a kind gift from Drs. Murao and Arai of Osaka Prefecture University. L-a-Amino-s-caprolactamase was purified by a procedure previously  described.3) L-Lysine-a-oxidase purified as described  previously7) was kindly given by Dr. H. Kusakabe of Yamasa Shoyu, Choshi. The other * Syed Ashrafuddin Ahmed,  ilaq  [Eit  tIEEtt;g&: Laboratory of Microbial Bioche-  mistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu 611, Japan.  (  342  ) a-Amino-e-carolactam Racemase from Achromobacter obae chemicals were of analytical grade.  Microorganism and cultural conditions    Achromobacter obae FERM-P 7761) was obtained from Toray Industries Inc., and used throughout.    Unless otherwise stated, cells were grown aerobically at 28°C with shaking in a medium containing glucose 5.0 g,  KH2PO4 2.0 g,  MgSO4.7H20 0.5 g,  MnC12•4H20 0.2 g,  FeSO4.  7H20 0.03 g,  CaC12.  2H20 0.02 g, yeast extract 0.5 g, antifoam-AF-emulsion 0.03 g and indicated amounts of various nitrogen sources in 1 liter of tap water. The pH was adjusted to about 7.0 with NaOH or HC1. The harvested cells were washed with  0.85%  NaCl and then with 10 mM potassium phosphate buffer (pH 7.3) containing 20  iM pyridoxal 5'-phosphate and  0.01  % 2-mercapto-ethanol. Preparation of  cell  free extracts    We prepared cell-free extracts either by homogenizing the cells in a morter with sea sand and powdered alumina, and extracting the soluble protein with 10 mM potassium phosphate buffer (pH 7.3) containing  20  teM pyridoxal 5'-phosphate and 0.01 % 2-mercaptoethanol or by disruption of the cells by sonication (Kaijo Denki Oscillator, 19 KHz) in the same buffer for 5  min. The supernatant solution obtained was dialyzed against 500 volumes of the same buffer, and used as a crude a-amino-e-caprolactam racemase.  Enzyme assay    The reaction mixture (2.0 ml, pH 8.0) contained 320  timed of D- or  L-a-amino-e-caprolactam, 40 nmol of pyridoxal 5'-phosphate, 320  temol of potassium phosphate buffer and enzyme. The reaction was started by addition of enzyme. The race-mization was usually followed at 37°C by measurement of an initial optical rotation change at 546 nm with a Perkin-Elmer polarimeter (model  241). The reaction mixture containing  D-a-amino-s-caprolactam as a substrate was also incubated at 37°C for 5  —40  min, and then the L-isomer formed was determined by a coupled  reaction° with L-a-amino-s-caprolactamase3) and L-lysine-a-oxidase. Definition of enzyme unit     One unit of a-amino-e-caprolactam racemase was defined as the amount of enzyme that catalyzes the racemization of one  remole of D- or L-a-amino-s-capro-lactam per  min. The specific activity was units per mg of protein. Protein was determined by the method of Lowry et  al." with bovine serum albumin (Sigma) as a standard.                       RESULTS AND DISCUSSION    Cells were grown in various media containing glucose or glycerol a as carbon source and  DL-a-amino-s-caprolactam or peptone as a nitrogen source (Table I). The highest total and specific activities were obtained in the medium supplemented with  1.2%  DL-a-amino-e-caprolactam and  0.5% glucose (medium  H).    The cells were grown in the basal medium containing  2.18g of nitrogen of organic  (  343  )                      S. A. AHMED, N, ESAKI, H. TANAKA, and K. SODA      Table I. Effect of medium composition on  a-amino-s-caprolactam racemase production                                                Medium     Nutrients  (g/l)       A B C D E F  G H Glucose  - -  0.  5 -  5.0  5.0  Glycerol  2.0  2.0  -  -   -  -Peptone  2.0  2.0  -  -  -  -  DL-ACL  12.0  12.0  12.0  12.0  12.0  12.0  12.0  K2HPO4  1.0  1.0  1.0  1.0  -  -   - KH2PO4  1.0  1.0  1.0  1.0  2.0  2.0  2.0  2.0  MnCl2•  4H20 - -  0.  2  0.  2  0.2 0.2  FeSO4•7H20 - -  0.  03  0.  03  0.03  0.  03  CaC12•H20  0.02  0.02  0.02  0.02  Yeast extract  0.5  0.5  0.  5  0.  5  0.  5  0.  5  0.  5  0.5  Antifoam-AF-emulsion  0.03  0.03  0.03  0.03  0.03  0.03  0.03  0.03                                                   Results  Cell yield  (g/l)  2.8  1.3  2.8 0.5  2.  3  1.6  2.0  4.  7  Activity  (U/g cell)  5.0  3.0  2.7  4.0  8.3  1.4 n.d.  11.6  Specific activity 0. 17  0.06 0. 1 0. 1  0.  3 0. 1  0.4  0.  6  Cells were grown in 250 ml of each medium.  n.d.  =not determined.  ACL=  a-amino-s-capvolactam      Table II. Effect of nitrogen source on  a-amino-r-caprolactam racemase production.     Nitrogen (2.18 g/l) Cell yield (wet)  Racemase activity                                                                  Specific activity       source  (gfl)  (Units/g wet cell)  NaNO3  3.  1  2.  7 0.  18  (NH4)2SO4  7.  5  1.  1  0.  11 Urea  3,7  0.6  0.17  a-Amino-s-caprolactam  2, 9 2. 6  0.  44  a-Pyrrolidone  2.9  0.9  0.09  L-Aspartic acid  6.6  2.  2  0.  21  L-Glutamic acid  3.  8  1.  2  0.25  L-Lysine 3. 7  1.  2  0.20     Cells were grown in 250 ml of each medium as described in the MATERIALS AND      METHODS. Inoculum (6 ml each) was prepared  in a medium without nitrogen added. and inorganic nitrogen sources per liter. The cells grew well on all the media con-taining any nitrogen source tested, and all the extracts of the cells grown showed a-amino-s-caprolactam racemase activity more or less (Table II). Although ammonium sulfate supported the highest cell growth, the specific activity of the cells grown in the ammonium sulphate medium was very low. When L-aspartic acid was used as a nitrogen source, the highest total racemase activity was obtained. Though DL-a-amino-s-caprolactam added to the medium did not affect the total activity, the specific activity of the cells grown in this medium was about twice higher than that of the cells grown in the basal medium supplemented with L-aspartic acid. Thus, the cells can produce  a-amino-s-caprolactam racemase irrespective of addition of  (  344  )                    a-Amino-s-carolactam Racemase from Achromobacter obae  DL-a-amino-a-caprolactam to the medium, but the presence of  DL-a-amino-s-caprolactam stimulates the production of enzyme.     We have varied the ratio of glucose and  DL-a-amino-s-caprolactam (as a sole nitrogen source) added to the medium and examined the influence on the enzyme production (Table III). The highest total and specific activities of the enzyme were obtained when a medium containing 1.0 g per liter of glucose and 8.0 g of DL-a-amino-s-caprolactam was used. The addition of DL-a-amino-s-caprolactam to the basal medium is required for the maximum production of the racemase.      Table III. Effect of ratio of concentrations of glucose and  m..-a-amino-s-caprolactam                 in the growth medium on a-amino-s-caprolactam racemase production.      Concentration  (g/l) of                                Cell yield (wet)  Racernase activity .                                                                  Specific activity    Glucose DL-ACL  (g/1) (Units/g wet cell)  5.0  0.0  2.6  1.5  0.21  4.  0  2.  0  5.  6  9.  7  0.  74  3.0  4.0  4.7  11.3  0.88  2.0  6.0  4.1  11.0  0.78  1.0  8.0  4.5  12.5  0.93  0.0  10.0  1.9  9.0  0.20      Cells were grown in 250 ml of each medium.  ACL----  a-amino-s-caprolactam  a-Amino-t-caprolactam racemase is unstable as reported  previously') and this instability prevents one from purifying the enzyme effectively. Therefore, we have attempted to minimize a loss of the enzyme activity during its storage and purification.                       • •  100  •  - s\k  E  80  •  .0°  60  40  a)                      cc 20  00  o  4  8  12  130                                Storage period (days)      Fig. 1. Effect of various compounds on a-amino-s-caprolactam racemase stability.              Various compounds were added to crude  a-amino-e-caprolactam racemase pre-            parations (protein 15 mg/ml) and stored at -20°C. Activities of the enzyme             preparations (0.2 ml) were measured as described in MATERIALS AND             METHODS.  -0--, None;  -A-, ethylene glycol; -A-, a mixture of             superoxide  dismutase, S-PI, dithiothreitol and glycerol; and  -0-, sucrose.  (  345  )                      S. A. AHMED, N. ESAKI, H. TANAKA, and K. SODA Various cations, i.e., NH4+, Zn2+,  Fe2+., Mn2+,  Mg2+, Ca2+ (2 —10 mM), and pyridoxal 5'-phosphate (20  —200  pM) were added to the crude preparation in  10mM potassium phosphate buffer (pH 7.3), and the enzyme was stored at various temperatures (25°C, 4°C, —20°C and —195°C) for 3, 7, 15 and 32 days. None of them protected the enzyme from a decrease in the activity. Superoxide dismutase (0.2  mg/m1),  S-PI (0.2  mg/m1) or glycerol (0.4 —0.8  g/m1) was also ineffective, when added to the crude enzyme in 10 mM potassium phosphate  buffer (pH 7.3) containing 20 pM pyridoxal 5'-phosphate and  0.01  % 2-mercaptoethanol, and the mixture was stored at —20°C. However, when the enzyme solution was supplemented with a mixture of superoxide dismutase (0.2  mg/ml),  S-PI (0.2  mg/ml), dithiothreitol (2.0 mM) and glycerol (0.5  g/ml) and stored at —20°C, its activity was retained fully after 10 days (Fig. 1). Ethylene glycol  (40  %) also protected the enzyme from inactivation. However the enzyme lost its activity gradually on storage for a long period (i.e., more than 4 months) even in the presence of the effective stabilizers shown above. Sucrose (0.25 M) was found to stabilize the enzyme most effectively; about  90% of the enzyme activity was retained after storage at —20°C for four months. Sucrose and other polyhydroxy alcohols are known to stabilize labile enzymes owing to an increase in solvent cohesive  force,° hydration of the protein  molecule,") or by induction of a conformational change in a protein  molecule."  a-Amino-e-caprolactam racemase of Achromobacter obae probably is stabilized by sucrose through a similar mechanism, although further investigations are needed to clarify the detailed mechanism.     The purification of a-amino-e-caprolactam racemase is currently in progress to elucidate it  enzymologically.                       ACKNOWLEDGEMENT    We thank Dr. Kyosuke Yotsumoto of Toray  Ind. Inc. for generously supplying DL-, D- and  L-a-amino-e-caprolactams.                             REFERENCES  (  1  ) T. Fukumura, Agric. Biol. Chem., 41, 1321 (1977).  (  2  ) T. Fukumura, Agric. Biol. Chem., 41, 1327 (1977).  (  3  ) T. Fukumura, G. Talbot, H. Misono, Y. Teramura, K. Kato and K. Soda, FEBS Letters, 89,     299 (1978).  (  4  ) T. Fukumura,  Agric. Biol. Chem., 41, 1509 (1977).  (  5  ) S. Murao and S. Satoi, Agric. Biol. Chem., 34, 1265 (1970).  (  6  ) S. Satoi and S. Murao, Agric. Biol. Chem., 35, 1482 (1971).  (  7  ) H. Kusakabe, K. Kodama, A. Kuninaka, H. Yoshino, and K. Soda, Agric. Biol. Chem., 43, 1749     (1979).  (  8  ) 0. H. Lowry,  N.  J. Rosenbrough, A. L. Farr, and  R.  J. Randall, J. Biol. Chem., 193, 265  (1951).  (  9  ) J. C. Lee and S. N. Timasheff, J. Biol. Chem., 256, 7193 (1980). (10) R. P. Frigon and J. C. Lee, Arch. Biochem.  Biophys., 153, 587 (1972). (11) S. L. Bradbury and W. B. Jakoby, Proc.  Natl. Acad. Sci. U.S.A., 69, 2373 (1972).  (  346  )

Production and Stabilization of a-Amino-E-caprolactam Racemase from Achromobacter obae

http://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/77006/1/chd060_5-6_342.pdf

Production and Stabilization of a-Amino-E-caprolactam Racemase from Achromobacter obae

Abstract

Similar works

Full text

Available Versions

Kyoto University Research Information Repository