FEMS Microbiology Letters 37 (1986) 335-339 335 Published by Elsevier

FEM 02617

Conservation of antigenic determinants in leucine dehydrogenase from thermophilic and mesophilic Bacillus strains

(Bacillus sp.; leucine dehydrogenase; thermophilic bacterium; antigenic determinant conservation)

Uwe K~irst, Hs in Tsai and H o r s t Schii t te

GBF, Gesellschaft ffir Biotechnologische Forschung mbH, D-3300 Braunschweig-Sti~ckheim, F.R.G.

Received 25 August 1986 Accepted 1 September 1986

1. SUMMARY

Antibodies against the purified octameric L- leucine dehydrogenase (LeuDH) from the meso- philic Bacillus cereus have been used to screen 16 thermophilic Bacillus strains for LeuDH. 4 of these strains, Bacillus sphaericus 461 and Bacillus

sp. 405, 406, and 411, showed a particularly strong cross reaction of the partial identity type when examined by Ouchterlony double diffusion assay, thus indicating that they were immunologically related to the B. cereus enzyme. The LeuDH from the thermophilic strains were very stable and highly active at elevated temperatures, and gave a down- ward bend at about 55°C in the Arrhenius plot. The pH optimum for L-leucine deamination was around pH 11 for all strains examined.

2. I N T R O D U C T I O N

LeuDH has been detected in a number of dif- ferent Bacillus strains [1-5]. It is a NAD(H)-de- pendent enzyme catalysing the oxidative deamina- tion of branched-chain amino acids and the reduc- tive amination of their corresponding a-keto acids. The enzyme from the mesophilic B. sphaericus [2] and B. cereus [3] has been purified and crystal-

lised. Electron microscopic analysis of the purified enzyme from B. cereus has revealed that the en- zyme is octameric, consisting of 8 identical sub- units [3,6]. With the recent advent of the enzyme- membrane reactor [7] and NAD(H) regenerating system [8], LeuDH has been tested for its use as a technical catalyst in pilot-scale manufacture of L-leucine and L-methionine [9,10], and L-tert- leucine (L-2-amino-3,3-dimethyl butylic acid) [11]. However, in order to strengthen the economical viability of the process, it is of importance to improve the catalyst further by modern genetic and protein engineering techniques [2,13]. For example, to prevent contamination of the reactor or to accelerate the enzyme-catalysed reaction it is often desirable to operate the enzyme-reactor at elevated temperatures. Furthermore, it is also sensible to clone and over-express the gene of thermophilic origin in a mesophilic host (see [5] and citations therein), because in this way the costs of production and downstream processing of the enzyme can be further reduced. With all these considerations in mind, we undertook screening of some extremely thermophilic Bacillus strains for heat-resistant LeuDHs. Moreover, for the purpose of future comparative biochemical studies of the structure, activity and stability relationships of the enzyme, the desired LeuDH producers were

0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies

336

selected on the basis not only of the enzyme's higher specific activity and thermostability, but also of its cross-reactivity with the polyclonal anti- body raised against the purified enzyme from B. cereus. In this paper we report the results of these studies.

3. MATERIALS AND METHODS

3.1. Organisms and cultivation All Bacillus strains examined were obtained

from the Deutsche Sammlung von Mikroorganis- men (DSM) in Gi3ttingen, F.R.G., and are listed in Table 1 Stock cultures were kept on agar slants and plates on the Bacillus medium recommended by the DSM, containing per 1 :5 g peptone, 3 g meat extract, 10 mg MnSO 4 • H 2 0 and 25 g agar, adjusted to pH 7.0. Bacillus aeidocaldarius was kept on the medium described by Darland and Brock [14] with 2.5% agar added, at pH 3.5. For the preparation of crude extracts the cells were grown at 60°C in 1-1 Erlenmeyer flasks with 100 ml induction medium containing per 1 :2 .25 g

KH2PO4, 7.2 g K2HPO4, 0.2 g MgSO 4 • 7 H20, 10 mg CaC12. 2 H20, 15 mg FeC13 .6 H20, 5 g glucose, 10 g yeast extract, 1 g L-leucine, and was adjusted to pH 7.5. B. acidocaldarius was grown in medium [14] supplemented with 5 g/1 of glu- cose and 1 g/1 of L-leucine. Extracts for analysis of enzyme activity and immunological cross-reac- tion were prepared by ultrasonic treatment of a concentrated cell suspension (40%, w/v ) for 10 min per ml with amplitude setting at 6 (Branson Sonifier Cell Disruptor B15). Cell debris was re- moved by centrifugation, and the supernatant was referred to as crude extract.

3.2. Enzyme assays Leucine dehydrogenase was assayed for both

oxidative deamination and reductive amination reactions as described [3]. The assay was routinely performed at 30°C in a Beckman DU-5 spectro- photometer. The pH optimum for oxidative deamination was determined in the respective buffer [3] adjusted to different pHs in the range pH 8-12. Stability against thermal denaturation was estimated by heating an aliquot of crude

Table 1

Screening for L-leucine dehydrogenase in thermophilic Bacillus strains

Strain Specific activity of LeuDH ( U/mg) b

deamination amination

pH optimum CRM c

B. acidocaldarius DSM446 0.077 B. coagulans DSM459 0.009 B. coagulans DSM2312 0.024 B. coagulans DSM2319 0.024 Bacillus sp. (caldolyticus) a DSM405 0.26 Bacillus sp. (caldotenax) DSM406 0.16 Bacillus sp. (caldovelox) DSM411 0.26 Bacillus sp. DSM465 0.021 Bacillus sp. (thermocatenlatus) DSM730 0.094 Bacillus sp. (flavothermus) DSM2641 0.095 B. sphaericus DSM461 0.24 B. sphaericus DSM463 0.014 B. stearothermophilus DSM22 0.03 B. stearothermophilus DSM2027 0.04 B. stearothermophilus DSM2349 0.006 B. thermoglucosidasius DSM2542 0.064

0.094 11 + , p 0.036 11 ncd 0.045 11 ncd 0.13 11 ncd 1.71 11 + , p 1.28 11 + , p 1.47 11 + , p 0.37 11 ncd 0.61 11 + , p 0.29 11 + , p 1.62 11 + , p 0.023 11 + 0.11 11 + , p 0.21 11 + , p 0.021 11 ncd 0.21 11 + , p

a Currently not approved bacterial names are given in parentheses. b Activity was measured at 30°C. c CRM, cross-reactive material; + , positive reaction; p, partial identity; ncd, no cross-reaction detected.

337

extract at different temperatures for 30 min, and equilibrating to 30°C before assaying the residual activity. Activity at elevated temperatures was de- termined using an LKB Ultrospec 4050 spectro- photometer (LKB, Bromma, Sweden) connected to an Apple IIc microcomputer.

Protein was determined using the Pierce BCA protein assay reagent (Pierce Eurochemie, Oud- Beijerland, The Netherlands), with bovine serum albumin as a standard.

3.3. Immunochemical analysis Polyclonal antibodies raised against the puri-

fied LeuDH from B. cereus [3] were used for detecting the cross-reacting material in crude ex- tracts prepared from thermophilic strains by Ouchterlony double diffusion assay. The assay was performed on microscope slides as described by Oakley [15].

4. RESULTS AND DISCUSSION

16 thermophilic Bacillus strains with an opti- mal growth temperature of at least 55°C were selected from DSM. With the exception of B. acidocaldarius, which needs an acidic medium, they could all be grown at 60°C in the neutral induc-

tion medium using L-leucine (7.6 raM) as the inducer for LeuDH.

All 16 strains examined were found to have LeuDH activity. The specific activities for the oxidative deamination reaction (at 30°C) were found in the range of 0.006-0.26 U/mg protein. Activities for the reductive amination reaction were 1.5-10-fold higher (Table 1). The pH optimum for the deamination reaction was about pH 11 for all strains, including B. acidocaldarius, and was found to be in the same range as the values reported for the enzymes from B. cereus [3] and the mesophilic B. sphaericus [2]. The highest specific activities for LeuDH were found in B. sphaericus 461, as well as in the 3 extremely thermophilic strains Bacillus sp. 405, 406, and 411, which were also the fastest growing strains we examined. The doubting times were about 0.5 h.

To detect a LeuDH immunologically related to the B. cereus enzyme, crude extracts from induced cells were analysed by Ouchterlony double diffu- sion assay for material cross-reacting with poly- clonal antibodies raised against the purified LeuDH from B. cereus. Although no reaction of identity was observed, 10 strains produced fused bands with a spur towards the B. cereus LeuDH when placed in juxtaposition to this enzyme, thus displaying a reaction of partial identity (Fig. 1).

Fig. 1. Immunochemical analysis of crude extracts from thermophilic Bacillus strains by Ouchterlony double diffusion assay. In each frame the central well contained 10 fsl (585 ~g) of antibody solution, and wells 1 and 4 were filled with 10 t~1 (2.1 izg) of purified B. cereus LeuDH. The other wells (2, 3, 5, 6) contained crude extracts (0.3-1.4 mg protein) from cells induced for LeuDH synthesis. Frame A: (2) B. acidocaldarius; (3) Bacillus sp. 405; (5) Bacillus sp. 406; (6) Bacillus sp. 411. Frame B: (2) Bacillus sp. 465; (3) Bacillus sp. 730; (5) Bacillus sp. 2641; (6) B. sphaericus 461. Frame C: (2) B. sphaericus 463; (3) B. stearothermophilus 22; (5) B. stearothermophilus 2027; (6) B. glucosidasius 2542.

338

120 7 ~00 J

~ r - - 8 o 4

>,

> ~ 6 o d o

¢0

2 0" 2 0

0 i

2 0 30

0

o

i I (? 40 50 60 7O 80 ~ O O

T e m p e r o t u r e [ °C]

Fig. 2. Temperature dependence of enzyme stability. The resis- tance to heat denaturation of the LeuDH from Bacillus sp. 405 (©) and B. cereus (I) was determined after incubation for 30 min at elevated temperatures, as described in MATERIALS AND METHODS.

One other strain gave a weak positive reaction whose type could not be determined without being unbiased. N o cross-reaction was found with 5 other strains (Table 1). Since the latter strains had the lowest specific activities, the amount of cross- reacting material, if any, might have been beyond the sensitivity limit of the Ouchterlony assay.

Based on their higher enzymatic activities, as well as on favourable growth parameters, we selected B. sphaer icus 461 and Baci l lus sp. 405, 406, and 411, for closer investigation of the LeuDH they produced. When examining the stability of LeuDH against thermal denaturation we found that all retained 50% activity after 30 rain incuba- tion at 80°C. A typical result is shown in Fig. 2. This is about 15°C higher than that determined for the B. cereus enzyme (Fig. 2). The temperature optima of the enzymes from Baci l lus sp. 406 and 411 were both at about 80°C, but those of Baci l -

lus sp. 405 and B. sphaer icus 461 were above 80 ° C (Fig. 3A). Thus, the temperature optima of the thermophilic LeuDHs were at least 20°C higher than that of the B. cereus enzyme (Fig. 3A). In an Arrhenius plot (Fig. 3B) the curve for the thermophilic enzyme showed a downward bend at about 55°C, a property often observed in thermo- stable enzymes, which has been attributed to con-

T e m p e r o t u r e [°C]

0.1

>

"~ - 0 . 3

0

U - 0 . 7

®

I/I -1 .1

0 ..J

- 1 . 5

2 0 3 0 & 0 5 0 6 0 7 0 8 0 9 0

1.2 A

>~ 1.0

>

4-, 0 . 8 13 D

13 0 . 6

£) 0 , L

O. I/} 0 . 2

- - i i [

i × 10 ~ [ K - q T

Fig. 3. (A) Temperature dependence of enzyme activity. The enzymes from Bacillus sp. 405 (O) and B. cereus (O) were assayed for oxidative deamination at different temperatures as described in MATERIALS AND METHODS. (B) Arrhenius plot of enzyme specific activities at different temperatures for LeuDH of Bacillus sp. 405 (©) and B. cereus (I). Specific activity was expressed as U per mg protein.

formational changes [16,17]. The calculated activa- tion energies (Ea) for the thermophilic enzymes above 55°C, 27-31 kJ/mol , were very similar to that of B. cereus LeuDH (E a = 32 kJ/mol). Below 55°C the E a values were about 15 kJ /mol higher.

From these studies we concluded that the LeuDH from the thermophilic strains have prop- erties quite similar to those of the enzyme from B. cereus except for their thermostability. However, since the antigenic determinants (or epitopes) were conserved in both thermally stable and unstable LeuDHs, this suggests that the genotype for ther- mostability, an intrinsic property of the enzyme,

could have evolved divergently under environmen- tal selective pressure.

ACKNOWLEDGEMENT

This work was supported in part by a grant to the GBF 'protein-design' project from the Bundesministerium fiar Forschung und Technolo- gie, F.R.G.

REFERENCES

[1] Zink, M.W. and Sanwal, B.D. (1962) Arch. Biochem. Biophys. 99, 72-77.

[2] Ohshima, T., Misono, H. and Soda, K. (1978) J. Biol. Chem. 253, 5719-5725.

[3] Schi~tte, H., Hummel, W., Tsai, H. and Kula, M.-R. (1985) Appl. Microbiol. Biotechnol. 22, 306-317.

[4] Ohshima, T., Nagata, S. and Soda, K. (1985) Arch. Micro- biol. 141,407-411.

339

[5] Ohshima, T., Wandrey, C., Sugiura, M. and Soda, K. (1985) Biotechnol. Lett. 7, 871-876.

[6] Li)nsdorf, H. and Tsai, H. (1985) FEBS Lett. 193,261-266. [7] Wichmann, R., Wandrey, C., Biickmann, A.F. and Kula,

M.-R. (1981) Biotechnol. Bioeng. 23, 2789-2802. [8] Bl)ckmann, A.F., Kula, M.-R., Wichmann, R. and

Wandrey, C. (1981) J. Appl. Biochem. 3, 301-315. [9] Wandrey, C., Wichmann, R., Berke, W., Morr, M. and

Kula, M.-R. (1984) Proc. 3rd Eur. Congr. Biotechnol., Vol. I, pp. 239-244. Vedag Chemie, Weinheim.

[10] Ohshima, T., Wandrey, C., Kula, M.-R. and Soda, K. (1985) Biotechnol. Bioeng. 27, 1616-1618.

[11] Wandrey, C. and Bossow, B. (1985) Proc. 3rd Int. Con- ference on Chemistry and Biotechnology of Biologically Active Natural Products, Vol. I, pp. 195-214. Sofia, Bulgaria.

[12] Ulmer, K.M. (1983) Science 219, 616-671. [13] Winter, G. and Fersht, A.R. (1984) Trends Biotechnol.

Sci. 2, 15-119. [14] Dadand, G. and Brock, T.D. (1971) J. Gen. Microbiol. 67,

9-15. [15] Oakley, C.L. (1971) In: Methods in Microbiology 5A

(Morris, J.R. and Ribbons, D.W., Eds.), pp. 173-218. [16] Ljungdahl, L.G. (1979) Adv. Microb. Physiol. 19, 150-243. [17] Mozhaev, V.V. and Martinek, K. (1984) Enzyme Microb.

Technol. 6, 50-59.