Journal of Biotechnology, 9 (1988) 29-38 Elsevier

JBT 00343

29

Isolation and characterization of enzymes hydrolysing amino acid carbamates

Erhard Lewke and Maria-Regina Kula Institute of Enzymetechnology, University of Diisseldorf, P.O. Box 20 50, D-5170 Jiilich, F.R.G.

(Received 11 July 1988; accepted 25 August 1988)

Summary

In a microbial screening, a bacterium has been isolated which produces two enzymes capable of hydrolysing stereospecifically either L- or D-amino acid carbamates. A mutant strain has been derived which produces the L-specific hydrolas e constitutively while the D-specific hydrolase is present only after induc- tion. The enzymes can be separated by gel filtration or ion-exchange chromato- graphy and have been purified to homogeneity. The substrate range has been in- vestigated in detail. The enzymes need Co 2÷ for stability and will cleave N-acetyl amino acids with about 5-fold higher apparent activity than the corresponding N-methoxycarbonyl amino acids.

Amino acid production; Stereospecific hydrolysis; Amino acid carbamate

Introduction

Besides extraction from waste proteins chiral amino acids are produced by two different biotechnological routes: by fermentation with selected strains mainly from the genera Corynebacterium and Brevibacterium utilizing molasses and ammonia as raw materials and yielding L-amino acids or by the enzymatic conversion of chemically prepared precursors such as N-acetyl amino acids, 5'-substituted hy- dantoins or 2-ketoacids, which may lead to L- or D-amino acids, respectively (Soda et al., 1983; Schmidt-Kastner and Egerer, 1984).

Correspondence to: M.-R. Kula, Institute of Enzymetechnology, University of Di~sseldorf, P.O. Box 20 50, D-5170 Jiilich, F.R.G.

0168-1656/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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A newly developed route starting with carbonic acids for chemical synthesis of amino acid carbamates has made this class of substances available as possible precursors (Effenberger and Drauz, 1981). Enzymatic cleavage of amino acid carbamates should yield only volatile byproducts which are easily removable, and besides, lead to complete conversion since backreactions are suppressed by fast chemical decomposition of the primary reaction products of the enzymic cleavage. Both aspects are advantageous for industrial production. In previous papers we described the enzymatic hydrolysis of various amino acid carbamates yielding L-amino acids (Sambale and Kula, 1987, 1988). Here we report an enzyme hydrolys- ing D-amino acid carbamates, as well as its purification and characterization together with a second enzyme from the same strain exhibiting opposite stereo- specificity in the cleavage of amino acid carbamates.

Materials and Methods

Screening Microorganisms capable of utilizing racemic N-(methoxycarbonyl)-valine or

-alanine as sole carbon and nitrogen source were enriched by liquid culture techniques in minimal medium (M1). Following two passages for 3 d, micro- organisms were plated after serial dilution and single colonies purified on agar. Medium M1 contained per liter: 0.8 g KH2PO 4, 1.76 g K2HPO4, 0.25 g NaCI, 0.2 g yeast extract, 2.5 g amino acid carbamate and 2 ml trace element solution. The trace element solution was composed of: 0.1 g ZnSO 4 • 7H20, 0.03 g MnC12 • 4H20 , 0.3 g H3BO3, 0.2 g CoC12 • 6H20 , 0.01 g CuC12 • 2HzO , 0.02 g NiC12 • 6H20 and 0.03 g N a z M o O 4 • 2 H 2 0 made up to 1 1. Media were sterifized for 30 rain at 121°C. For growth the basal medium M1 was supplemented with different amounts of yeast extract, carbamate or trace elements. Solid media were prepared by adding 2% agar.

Cultivation of cells During screening and media variations, cells were cultivated in 100 ml medium in

500 ml Erlenmeyer flasks on a rotary shaker at 100 rpm and 30 ° C. For enzyme isolation, cells were grown in a biostat E fermenter with a working volume of 10 1 (B. Braun-Melsungen, F.R.G.). Cells were separated at the end of the cultivation by centrifugation and washed once with 0.1 M potassium phosphate buffer, pH 7.5. The cells were suspended in the same buffer to give a 20% (v /v ) suspension and disintegrated discontinuously in a Dyno mill (Bachofen, Basel, Switzerland) equipped with a 300 ml grinding chamber filled with glassbeads ( ~ 0.3 mm) and agitated at 3 000 rpm. for 10 min. Cell debris was removed by high speed centrifugation. The supernatant was passed through a sterile filter and served as crude extract for further experiments.

A ssay Enzyme activity was measured at 37 °C in 0.1 M potassium phosphate buffer, pH

7.5, in a total volume of 1.00 ml for various times. The final carbamate concentra-

31

tion was 0.1 M. Specific activity is expressed in U mg -1 protein corresponding to the generation of 1 /xmol amino acid per min under the assay conditions. The reaction was started by addition of enzyme and stopped by acidification to p H 2. A suitable aliquot of the reaction mixture was then applied to the Biotronik LC 5001 (Maintal, F.R.G.) amino acid analyser equipped with an integrator and automatic sample changer. A standard amino acid mixture from Pierce (Rockford, U.S.A.) was used for calibration. For semi-quantitative measurements during screening, thin-layer chromatography of amino acids was carried out on silica plates 60 F 254 (E. Merck, Darmstadt , F.R.G.) using 70% ethanol to separate free amino acids from carba- mates and proteins. Amino acid enantiomers were separated on chiral plates (Macherey & Nagel, Dueren, F.R.G.) as described by Guenther et al. (1984). Amino acids were visualized by spraying with ninhydrin reagent and heating to 100 o C. The detectable amount was 0.5 nmol amino acid. A Shimadzu CS 930 (Duisburg, F.R.G.) TLC scanner was employed to evaluate enzymatic activity by comparison with an amino acid standard. Protein concentration was determined by the BCA assay (Smith et al., 1985) utilizing bovine serum albumin for calibration.

Chemicals The amino acid carbamates were in part donated by Degussa A G (Hanau,

F.R.G.) otherwise prepared in our laboratory by reacting amino acids with chloro- formic acid esters according to Taub and Hino (1964). The ester was obtained from Fluka (Buchs, Switzerland) while all other chemicals and solvents were purchased from E. Merck. Chromatographic materials were obtained from Pharmacia (Up- psala, Sweden) and LKB (Villeneuve, France).

Results and Discussion

A total of about 400 isolates have been obtained by screening soil samples and effluents from waste water treatment plants as described in the Screening section for the ability to grow on racemic N-(methoxyarbonyl)-valine or N-(methoxycarbonyl)- alanine as nitrogen and carbon source. The majority of the isolated strains produced only L-amino acids from the racemates. The specific activity is shown in Table 1 and is of the same order of magnitude as that described by Sambale and Kula (1988). One strain designated HB was noted for producing L- and D-amino acid from N-methoxycarbonyl amino acid racemates. By repeated early transfer of strain HB in liquid media M1 a mutant HB2 was derived with constitutive levels of a L-carbamatase. This result also demonstrated that the hydrolysis of racemic amino acid carbamates was due to two different enzymes in the parent strain and not to an unspecific hydrolysis of the substrate. Supplementation of trace elements and carbamate concentration in the basal medium were varied and enzyme production analysed. The results are summarized in Tables 2-4. The influence of various cations on enzyme production was investigated and a significant effect of cobalt ions was noted (Table 2). A rather high cobalt salt concentration of 50 mg 1-1 was found necessary to obtain the highest enzyme productivity (Table 3). Further

32

TABLE 1

ENZYMATIC ACTIVITIES OF SELECTED ISOLATES T O W A R D S N-(METHOXYCARBONYL)- D,L-VALINE

Strain Specific activity Enantiomer (U m g - 1 ) hydrolyzed

ZJ3 0,033 D HB 0.063 D, L P30 0.030 L VR2 0.024 L $3 0.031 L 41 0.015 L

TABLE 2

I N F L U E N C E OF T R A C E ELEMENTS IN THE M E D I U M ON ENZYME P R O D U C T I O N

Trace element solution Specific activity Volumetric activity without (U m g - 1 ) (U m l - 1 )

B 0.07 0.16 0.07 0.11

Co 0.03 0.04 0.02 0.04

Cu 0.08 0.20 0.08 0.19

Mn 0.08 0.13 0.08 0.18

Mo 0.08 0.18 0.09 0.17

Ni 0.08 0.15 0.08 0.18

Zn 0.08 0.16 0.08 0.17

Without trace elements 0.02 0.06 0.02 0.07

With all trace elements 0.08 0.21 0.08 0.19

TABLE 3

E N Z Y M E P R O D U C T I O N AS F U N C T I O N OF Co 2÷ C O N C E N T R A T I O N

CoC12 × 6H 20 Specific activity Volumetric activity (rag 1-1) (U mg -1 ) (U m1-1)

0 0.02 0.17 5 0.03 0.30

10 0.05 0.38 20 0.06 0.40 40 0.06 0.57 50 0.07 0.81 75 0.07 0.78

100 0.07 0.56 200 0.08 0.57

Substrate was N-(methoxycarbonyl)-D,L-valine.

TABLE 4

E N Z Y M E P R O D U C T I O N AS F U N C T I O N CARBONYL)-D,L-VALINE IN THE M E D I U M

OF C O N C E N T R A T I O N

33

OF N-(METHOXY-

Carbamate Specific activity Volumetric activity concentration (g 1 - ] ) (U m g - 1 ) (U ml - 1 )

0 0.048 0.40

1 0.055 0.59

2 0.059 0.53 3 0.068 0.67 4 0.075 0.78

5 0.073 0.81

10 0.064 0.59

increase did not alter the specific activity but led to progressive reduction of cell growth. Increasing carbamate concentrations lead to a rise in the specific activity and parallel in the volumetric activity, reaching an optimum of around 5 g carbamate per liter (Table 4). The constitutive L-specific carbamatase contributed about 50 m U mg -1 to the observed activity. The increase is due solely to the formation of D-carbamatase under these conditions. The time course of induction is shown in Fig. 1. The generation time in the log phase was 130 min. For enzyme isolation cells were grown under induction conditions and harvested at the onset of the stationary phase. Cell yields averaged 5 g wet cells per 1 medium.

Enzyme purification In preliminary experiments it was found that addition of 1 mM Co 2+ to all

buffers was essential to maintain enzymatic activity. Losses are particularly evident after ge l filtration or ultrafiltration steps. Purification was carried out at room temperature and is summarized in Table 5. For intermittent storage samples were kept at 4 o C.

~O~ 50

E

F: 4o

3o I .>

20 o .m

o (9 10 o.

0 1 2 3 4 5 6 7

t ime

. ° 1 O

o) t--

"O

.2

0

0

8 9 lO

t ( h )

Fig. 1. Time course of induction of D-carbamatase. (A) Specific activity; (e ) OD660.

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TABLE 5

PURIFICATION OF THE ENZYMES

L-Carbamatase D-Carbamatase (U mg -1) (U mg l)

Crude extract 0.06 0.06 DEAE-Cellulose 0.79 0.08 ACA 54 2.27 1.09 Mono Q 8.75 2.32 Superose TM12 12.10 4.50

Substrate: 100 mM of the corresponding enantiomer of N-(methoxycarbonyl)-valine.

Chromatographic steps 325 ml crude extract were applied to a DEAE-cellulose colunm (2.6 × 30)

equilibrated against 0.1 M Tris-HC1, pH 7.5, and 1 mM CoCI 2 (standard buffer) and washed with one column volume. The column was developed with a steep gradient of sodium chloride in starting buffer. Fractions were collected and assayed for enzymatic activity. Active fractions were combined and concentrated by ultra- filtration using an Amicon CEC 1 (Witten, F.R.G.) equipped with a cellulose acetate membrane, cut off of 10 kDa. The concentrate was applied to a gel filtration column (2.6 × 55) packed with ACA 54 resin and equilibrated against standard buffer. Gel filtration clearly separated L- and D-carbamatase. Further purification was achieved by FPLC using a Mono Q column, equilibrated against standard buffer. L-Carbamatase was eluted in a NaCI gradient at 0.19 M concentration. D-Carbamatase was purified in a similar way and eluted at 0.22 M NaC1 concentra- tion. In the steep gradient of the first ion-exchange chromatography both enzymes eluted together. Aliquots of 0.1 ml from the most active fractions of both enzymes obtained by FPLC were subjected to gel filtration over a column of Superose TM12 to determine the molecular weight. Peak fractions of the Superose column were analysed by SDS electrophoresis followed by silver staining.

Enzyme characterization The molecular weight of the enzymes was estimated by gel filtration and found to

be 160.000 Da for L-carbamatase and 90.000 Da for D-carbamatase. By gel electrophoresis in the presence of SDS two subunits of identical size (45.000 Da) were observed for D-carbamatase. Under the same conditions the L-carbamatase exhibited a strong band corresponding to a molecular weight of 28.000 Da and a weak band at 50.000 Da. Presently it is unclear whether the minor band arises from a contaminating protein or represents a second subunit.

To test the influence of various bivalent cations on enzymatic activity, enzyme assays were performed in the presence of 10 mM concentration of Mg 2+, Mn 2÷, Cu 2÷, Co 2+, Ca 2÷ and Zn 2÷ in 0.1 M Tris-HC1. These additions did not alter the enzymatic hydrolysis of N-methoxycarbonyl-DL-valine. Addition of benzamidine, EDTA, or dimethylmalonic acid in the same concentration or 1 mM PMSF had also no effect. The result with EDTA is somewhat surprising considering the influence of

35

110

% 1oo

9 0

~) 8 0

70: g 6 0

~ 5 0

~ 40 ~ 3 0

~ 2 0

~- 10

0

2O 3 0 4 0 5 0 6 0 70 8 0

temperature T (°C) Fig. 2. Ac t iv i ty o f c a r b a m a t a s e s as a f u n c t i o n o f t e m p e r a t u r e . (m) Ac t iv i ty o f L - c a r b a m a t a s e ; ( e ) activity

of D-carbamatase.

cobalt ions on the stability of the enzyme. Enzymatic hydrolysis by both enzymes was completely inhibited by HgC1 z, p-hydroxymercury-benzoate or sodium cyanide indicating an essential thiol group. To investigate the temperature optimum, enzyme assays were performed at various temperatures for 15 rain and the amount of amino acid liberated in this time was quantified. Results are presented in Fig. 2 and show that both enzymes have maximal activities around 60 ° C. The influence of pH on the reaction rate was investigated by incubating the enzymes in the presence of 0.5 M potassium phosphate in the range pH 6.5 to 9. Results are summarized in Fig. 3. L-Carbamatase has a wide optimum at pH 8, D-carbamatase at slightly more

% 1oo

• 8O

60 c 0

0 40

t,,.

-~ 20"

~ o i 6.0

110 %

100 • ~P t~

t- 90 0

t,..

80

7(1

6.5 7.0 7.5 8.0 8.5 9.0 9.5

pH

Fig. 3. Ac t iv i ty o f carbamatases as a function of pH. (m) Activity of L-carbamatase; (e) activity of D-carbamatase.

36

' - - 9 E

8 E , " 7 "

6

5

c ,£ 3

0 100 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0

._= E

50-- 0

40

30 ~

20 .~

~ 1 0 ~

L , 0

8O0

subs t ra te concen t ra t i on mM

Fig. 4. Activity of carbamatases as a function of substrate concentration. (11) Activity of L-carbamatase; (e) activity of D-carbamatase.

alkaline conditions around pH 8.8. The reaction rate was measured as a function of substrate concentration in the range 1 to 700 mM. Results are shown in Fig. 4. D-Carbamatase exhibits substrate surplus inhibition at concentrations above 200 mM D-N-(methoxycarbonyl)-valine. The K m value for D-carbamatase was de- termined to be 1.2 mM and falls within the expected range of similar enzymes. In contrast, the reaction rate of L-carbamatase was found to increase with substrate concentration over the entire range investigated. Saturation was not yet evident at 700 mM concentration, therefore the Michaelis-Menten parameters cannot be estimated with confidence. This kinetic behaviour also explains that 100% conver- sion of HB2 L-carbamatase requires a rather high enzyme concentration or long reaction times.

Stereospecificity Purified enzyme fractions were employed to analyse the stereospecificity of the

reaction. Both enzymes were incubated with pure enantiomeric substrates if availa- ble or with racemic substrates followed by a separation of the amino acids generated on chiral plates. According to Guenther et al. (1984) and our own experience a contamination of 0.5% of the opposite enantiomer would have been detected. Both enzymes showed a high stereoselectivity hydrolysing only one enantiomer.

Substrate range The substrate range of L- and D-carbamatase was tested with regard to the

amino acid sidechain and the nature of the alcohol component in the carbamate. Results are summarized in Table 6 and show distinct differences with regard to the amino acid sidechain. The observed influence of cobalt ions on enzymatic stability during the purification steps prompted us to investigate also N-acetyl amino acids as substrates. As shown in Table 7 N-acetyl derivatives are hydrolysed even faster than the corresponding carbamates by the newly isolated enzymes. From these

TABLE 6

SPECIFIC ACTIVITIES OF THE PURIFIED ENZYMES

37

Carbamate L-Carbamatase D-Carbamatase (U mg -1) (U mg -1)

N-(Methoxycarbonyl)-DL-vahne 12.1 N-(Ethoxycarbonyl)-DL-valine 1.1 N-(Methoxycarbonyl)-DL-alanine 25.6 N-( Ethoxycarbonyl)-DL-alanine 8.9 N-(Methoxycarbonyl)-DL-phenylalanine 42.6 N-(Methoxycarbonyl)-DL-leucine 3.3 N-(Methoxycarb0nyl)-DL-lysine 1.0

4.5 2.1

51.3 26.7

< 0.1 1.0 0.5

Substrate concentration: 200 mM.

resul t s it has to be c o n c l u d e d tha t the b a c t e r i u m i so la t ed in the sc reen ing possesses

t w o a m i n o ac id acy lases wh ich wil l a lso accep t c a r b a m a t e s as subs t r a t e ana logues .

C o m p a r e d wi th a m i n o ac id acy lase f r o m hog k i d n e y the speci f ic ac t iv i ty o f H B 2 is

r e l a t ive ly low, h o w e v e r the r a t io b e t w e e n acy lase and c a r b a m a t a s e ac t iv i ty is a b o u t

TABLE 7

SUBSTRATE RANGE INVESTIGATED WITH CRUDE EXTRACT

S u b s t r a t e U r a g - 1 U m l - 1

N-Ac~tyl- D-alanine D-vahne L-isoleucine L-leucine L-tyrosine DL-tryptophane DL-methionine DL-valine DL-phenylalanine

N-Methoxycarbonyl- D-valine L-vahne L-leucine DL-valine DL-alanine DL-phenylalanine

N-Ethoxycarbonyl- DL-valine DL-alanine

N-n-Butoxycarbonyl- DL-valine DL-glutamic acid

0.48 1.68 0.16 0.58 0.26 0.92 0.26 0.92 0.31 1.08 0.55 1.93 0.53 1.85 0.19 0.67 0.26 0.91

0.03 0.11 0.10 0.34 0.01 0.04 0.1i 0.40 0.17 0.60 0.11 0.40

0.02 0.08 0.10 0.36

0.01 0.04 0.01 0.04

In case of racemic substrates the sum of both enzyme activities was measured, with pure enantiomers 0nly the corresponding hydrolase was determined.

38

tenfold higher in HB2 than that observed for hog kidney acylase (Sambale and Kula, 1987). In contrast to other enzymes described in the literature (Suhara et al., 1984; Murao et al., 1984; Matsumura et al., 1985) hydrolysing urethane structures N-benzyloxycarbonyl amino acids are not hydrolysed by the enzymes from HB2. N-Benzyloxycarbonyl is the so called Z protecting group widely used in peptide synthesis. Z-L-glutamine, Z-L-lysine, Z-L-glutamate, Z-L-proline, Z-L-phenyl- alanine, Z-L-arginine and Z-L-tyrosine were tested as substrates and found inactive. The results are consistent with the observation, that enzyme activities decreased by increasing the number of carbon atoms in the alcohol component of the carbamates going from methanol to butanol. No activity was observed when incubating the HB2 enzymes with N-carbamoylalanine and N-carbamoylvaline or various hy- dantoins at 100 mM concentration as substrates.

Acknowledgement

We are obliged to S. LiSslein for carrying out the amino acid analysis.

References

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Guenther, K., Martens, J. and Schickedanz, M. (1984) Duennschichtchromatographische Enanti- omerentrennung rnittels Ligandenaustausch. Angew. Chem. 96, 514-515.

Matsumura, E., Shin, T., Murao, S. and Kawano, T. (1985) Substrate specificity and stoichiometry of N-a-benzyloxycarbonyl amino acid urethane hydrolase from Streptococcus faecalis R ATCC 8043. Agric. Biol. Chem. 49, 973-979.

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E.K., Goeke, N.M., Oison, B.J. and Klenk, D.C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85.

Soda, K., Tanaka, H. and Eskai, N. (1983) Amino Acids. Biotechnology 3, 481-530. Suhara, A., Itoh, S., Yokose, K., Ninomiya, R., Watanabe, K. and Maruyama, H.B. (1984) Microbial

resolution of DL-N-benzyloxycarbonyl-p-hydroxyphenylglycine by Streptomyces zaomyceticus. Can. J. Microbiol. 30, 1301-1304.

Taub, B. and Hino, J.B. (1964) Synthesis of N-carboalkoxy-L-aminocapronic acid esters. J. Chem. Eng. Data 9, 106-107.