Journal of Molecular Catalysis, 58 U990) 393-403 393

EFFECT OF IMMOBILIZATION ON THE CATALYTIC PROPERTIES OF FERREDO2U.N~NITRITE REDUCTASE FROM CIHJMYDOMONAS REINHAR.DTII

JO& LE6N, FRANCISCO GALVh end JOSfi M. VEGA*

lkpartamento de Bioqutmica Vegetal y Biologtb Molecular, Facultad de Quimica, Universidad de Sevilla, Seville (Spain)

(Received March 21,1989; accepted July 11,1989)

The ferredoxin-nitrite reductase (EC 1.7.7.1) from Chlamya!omonas reinhardtii has been immobilized either by ionic binding to DEAE-cellulose or by covalent binding to alkylamine silica, giving immobilization yields higher than 60%. The use of DEAE-cellulose pretreated with glutaraldehyde as a matrix to immobilize nitrite reductase increases the stability of the system with respect to temperature and ionic strength, but decreases the ability to retain enzyme activity.

Immobilized nitrite reductase activity is more stable, and shows a lower apparent K, value for reduced ferredoxin than for free suspended enzyme, while the apparent K,,, of the enzyme for nitrite or reduced methyl viologen did not change substantially by the immobilization treatment.

Introduction

Immobilization techniques may significantly affect the properties of an enzyme because (a) its conformational changes are thus limited, (b) the microenvironment surrounding the enzyme is different, especially in terms of substrate and product concentrations, which involve the enzyme kinetic properties, and (c) the protein-protein interactions are conditioned, which is very important for aggregation processes and proteolytic degradations [ 1 I.

Immobilization of enzymes by ionic binding to an ion-exchanger matrix is the simplest and mildest procedure available, because it basically retains the original conformation of the protein, and thus its catalytic activity, the process being easily reversible. On the other hand, the immobilization of enzymes by covalent binding to inorganic and organic matrices gives very stable systems, in which enzyme activity and catalytic parameters may be significantly altered [21.

Recently, the use of immobilized oxidoreductases to perform synthetic transformations has become a major area of interest in biotechnology 131.

*Author to whom correspondence should be addressed.

0304-5102/90/$3.50 @ Elsevier Sequoia/Printed in The Netherlands

However, information on the immobilization of enzymes involved in the photosynthetic assimilation of nitrate is very scanty. Only the use of immobilized nitrate reductase has been reported for analytical applications r4, 51.

Ferredoxin-nitrite reductase (EC 1.7.7.1) catalyzes the reduction of nitrite to ammonium, involving the transfer of six electrons from reduced ferredoxin (Fd) (or methyl viologen) to nitrite. The enzyme from Chlamydonwnas reinhardtii has been purified to electrophoretic homo- geneity, resulting in a molecule, il& = 86 000, composed of two subunits of M, 63 000 and 25 000 respectively, and containing 1 siroheme and 1 iron-sulfur cluster of the [4Fe-4Sl type [Sl.

In this paper we have selected a suitable technique and optimized the conditions to immobilize Fd-nitrite reductase from C. reinhurdtii. We have studied the stability and catalytic properties of the immobilized enzyme and compare them here with those shown by free suspended nitrite reductase.

Materials and methods

Reagents Sodium alginate, DEAE-cellulose, glutaraldehyde, 1-ethyl-3-(3-dimethyl-

aminopropyl)carbodiimide, protamine sulfate and Tris buffer were obtained from Sigma (St. Louis, U.S.A.). Acrylamide and bis(acrylamide) were products of Bio-Rad (Richmond, U.S.A.); silica gel 60 (70-230 mesh ASTM), 3-(triethoxysilyl)propylamine, 2-mercaptoethanol, EDTA and reagents of analytical grade were obtained from Merck (Darmstadt, R.F.A. ).

Organism and growth conditions The unicellular green alga Chlamydomonas reinhardtii, mutant strain

104, lacking in NAD(P)H-nitrate reductase activity, was grown at 25 “C with continuous light of 50 W m-‘, at the surface of the culture, and using the medium previously described by Sueoka et al. 171, containing 6 mM KNOz as unique nitrogen source, which was flushed with air supplemented with 5% (v/v) COZ as carbon source.

Purification of Fd-nitrite reductase from C. reinhardtii About 50g of cells (fresh weight) were harvested, at the exponential

phase of growth, by low speed centrifugation and broken by freezing in liquid nitrogen and thawing slowly in 250 ml of 20 n&I Tris-HCl buffer, pH 8.0, containing 14 mM 2-mercaptoethanol and 0.5 mM EDTA (standard buffer). After centrifugation at 16 000 x g for 30 min, the supernatant was made up 8% (v/v) with a solution of 2% (w/v) protamine sulfate. After 15 min with gentle stirring, the suspension was centrifuged at high speed, and the resulting supernatant was filtered through a DEAE-cellulose column (1.6 x 36 cm) equilibrated with the standard buffer. The bed-column was washed with the same buffer and then with a solution of 40 mM NaCl in the buffer. Finally, nitrite reductase was eluted by adding 140 mM NaCl to the eluent,

The active fractions were pooled, concentrated by ultrafiltration through a PM-10 membrane, and dialyzed overnight against the standard buffer. All the steps were performed at 4 “C.

This purification procedure yielded a preparation of Fd-nitrite reduct- ase from C. reinhardtii with a specific activity of about 3.5 U mg-’ protein.

Puri.fication of ferredoxin Ferredozin was purified from C. reinhardtii by the method described

previously [81. The absorbance ratio, A&A 276 of the final protein prepara- tion was 0.54 or higher.

Analytical determinations Nitrite was determined calorimetrically using solutions of sulfanilamide

and N-( l-naphthyl)ethylenediammonium dichloride as previously described [9]. Soluble protein concentration was determined by the method of Bradford [ 101, while protein retained in the matrix was estimated indirectly by difference.

Nitrite reductase activity assay Nitrite reductase activity (NiR) was measured using methyl viologen,

chemically reduced with sodium dithionite, as electron donor and determin- ing the nitrite concentration in the medium after incubation at 40 “C for 10 min [ 111. The reaction mixture contained, in a final volume of 1 ml: 150 pm01 Tris-HCl buffer, pH 8; 2 pmol KNO,; 2 pmol methyl viologen; 50-150 mU of nitrite reductase and 10 pmol of sodium dithionite (freshly prepared in 0.3 M NaHC03). In the case of immobilized samples, the reaction mixture was gently shaken during incubation. One unit of nitrite reductase activity is the amount of enzyme which reduces 1 pmol of nitrite to ammonium per min.

Immobilization of nitrite reductase from C. reinhardtii A partially purified preparation of Fd-nitrite reductase, about 1 mg

protein ml-l, was used to obtain immobilized systems by different tech- niques. In all cases the immobilization yield is defined as the relation of the activity retained in the matrix to the total used to make the immobilized system.

Alginate systems Entrapment of nitrite reductase was obtained by ionotropic gelation of

sodium alginate solutions containing the enzyme (20 U (g support)-l) which is added dropwise to the bulk phase of a 0.1 M CaC12 (NiR-alginate-Ca) or 0.1 M BaC12 (NiR-alginate-Ba) solution and maintained with magnetic stirring at 4 “C during 2 h to harden the resulting gel. The immobilized system is composed of beads of 2 mm diameter suspended in standard buffer and containing a final concentration of 3% (w/v) of alginate.

Polyacrylamide system Nitrite reductase was also entrapped by polymerization of a solution

containing the enzyme, 1.25 U ml-‘, 5% (w/v) acrylamide and 0.2% (w/v) bis(acrylamide). Small amounts of the redox system, composed of tetra- methylenediamine and ammonium persulfate, were added to start the polymerization reaction. The resulting gel was cut in small segments 5 mm in diameter and 2 mm thick, which were washed and resuspended in standard buffer at 4 “C, until use.

NiR-BSA-G systems Co-crosslinking of nitrite reductase and bovine serum albumin (BSA)

was obtained by making up a 5% (w/v) in glutaraldehyde solution containing 46 mg ml-’ of BSA plus 3.7 U ml-’ of nitrite reductase. The resulting mixture was freeze-dried onto glass plate. Similarly, solutions of nitrite reductase (3.7 U ml-‘) and ferredoxin (3.5 mgml-‘) were co-crosslinked (NiR-Fd-G) by this method.

NiR-DEAE-cellulose systems Suspensions containing 0.15 g dry weight of DEAE-cellulose per ml

were used to obtain immobilized systems by ionic binding (NiR-DEAE) or combined with crosslinking (NiR-DEAE-G) by treating previously the support with different concentrations, between 140 and 830 mgg-’ dry weight DEAE-cellulose, of glutaraldehyde. A solution of nitrite reductase (5.2 Uml-‘1 was added to the resin suspension and the mixture was continuously shaken at 4 “C during 2 h. After washing with standard buffer, the pellet was resuspended (0.15 g dry weight per ml) in the same buffer and used as an immobilized nitrite reductase source.

NiR-Si02+zlkylamine systems Fd-nitrite reductase was immobilized by covalent binding to silica gels.

A commerical support was previously washed with a 5% (v/v) nitric acid solution at 80-90 “C during 1 h, then the acid was removed by rinsing with excess distilled water, and the support resuspended in standard buffer (O.OSg dry weight per ml). The matrix was silanized with 1% (v/v) of 3-(triethoxysilyl)propylamine and dried at 115”C, resulting in the alkyl- amine silica which was again resuspended in standard buffer (SiOZ- alkylamine). Furthermore, the carbonyl derivative of the alkylamine silica was obtained by adding 620 mg of glutaraldehyde per g of dry weight support. After 1 h at room temperature with gentle stirring, the gel was rinsed with excess distilled water and finally resuspended in standard buffer (SiO,-alkylamine-G) .

The NiR-SiOz-alkylamine system was prepared by mixing 2 volumes of a suspension (0.08 g of dry weight support per ml) of the corresponding matrix with one volume of nitrite reductase solution (5.1 U ml-‘). The mixture was made up 1% (w/v) with the condensing agent l-ethyl-3-(3- dimethylaminopropyl)carbodiimide, and after 6 h at 4 “C with gentle shak- ing, it was centrifuged at 15 000 x g for 15 min. The corresponding pellet was resuspended in standard buffer until a final concentration of 0.08g of dry

397

weight support per ml was reached. The enzyme is bound through its carboxyl groups to the matrix, and the immobilized system was ready to use.

We proceeded analogously to prepare the NiR-Si02-alkylamine-G system, except the condensing agent was not added. In this case nitrite reductase was bound to the support through its amino groups, by forming a Schiff base.

ResuIts

Involvement of amino and carboxyl groups of Fd-nitrite reductase from C. reinhardtii in the catalysis

Table 1 shows that treatment of nitrite reductase with 8 mM of TNRS, which blocks the free amino groups causes inactivation of the enzyme, which seems to be specifically protected by ferredoxin. On the other hand, if we add to the purified enzyme solution 8 mM of EDC (l-ethyl-&(&dimethylamino- propyl)carbodiimide), which, in the presence of an excess of glycine ethyl ester, reacts with the carboxyl groups of the protein, a partial inactivation is observed, which cannot be protected by substrates. These data indicate that, under equal concentration of reagents, the inactivation produced by mod- ification of the enzyme carboxyl groups is lower than that observed by modification of amino groups, and suggests that immobilization of nitrite reductase from C. reinhardtii should be obtained preferably through the carboxyl groups of the protein or by methods involving non-covalent enzyme- matrix interactions.

Immobilization of Fd-nitrite reductase from C. reinhardtii Several immobilization methods have been used, and in Table 2 they

are indicated with the corresponding values of the obtained immobilization

TABLE 1

Effects of specific reagents for amino and carboxyl groups on the nitrite reductaee activity from C. winhardtii”

Addition Nitrite reductaee activity (96)

3omin 60 min

None 95 94 TNBS 5 0 TNBS + nitrite 0 0 TNBS + ferredoxin 88 94 EDC + GEE 72 72 EDC + GEE + nitrite 41 35 EDC + GEE + ferredoxin 52 60

a Nitrite reductase (2.4 U per mg protein) wae incubated, in standard buffer at 4 “c, with 8 mM 2,4,6_trinitrobenzene sulfonic acid (TNBS) or with 8mM l-ethyl&(3dimethyl aminopropyl)- carbodiimide (EDC) in the presence of 0.5 M glycine ethyl ester (GEE). When indicated, 4 mM nitrite or 170 pM ferredoxin were included in the incubation mixture. At the indicated times, the nitrite reductase activity was estimated by adding aliquota of the corresponding incubation mixture to the reagents of the standard aeeay. 100% of activity was 2.7 U ml-‘.

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TABLE 2

Effect of different immobilization techniques end supports on the nitrite reductase activity from C. reinhurdtii”

System Immobilization procedure Immobilization yield ( % ) Activity (Uperg support)

NiB-alginate-Ca entrapment 13 2.9 NiB-alginate-Ba entrapment 15 18.4 NiB-polyacrylamide entrapment 23 -

NiB-DEAE ionic binding 94 13.1 NiB-DEAE-G ionic binding-crosslinking 48 3.2 NiB-BSA-G co-cros&nking 3.5 9.7 NiB-Fd-G co-crosshnking 16 5.9 NiB-SiOz-akylamine covalent binding 62 13.1 NiB-SiOz-akylamine-G covalent binding 20 4.2

“Details of nitrite reductase immobilization and measurements of immobilization yield and nitrite reductase activity are given in Materials and Methods.

yield. Ionic binding of nitrite reductase to DEAE-cellulose gives the best results, with an enzyme loading for the NiR-DEAE of 14 U g-l of support and a maximum immobilization yield of 94% (Fig. 1).

Table 2 also shows the entrapment of nitrite reductase from C. reinhardtii in alginate and polyacrylamide gels, with immobilization yields

10 30

ENZYME L-DING W/g support)

Fig. 1. Immobilization yield of NiB-DEAE oersup enzymatic loading on the support. Different amounts of partially purified nitrite reductase, with an activity of 5.2 U ml-‘, were added to 2 ml of suspension of DEAE-ceIIulose (0.15 gmI-‘1. The immobilization procedure, a~ weII as the nitrite reductase activity and immobilization determinations, are described in Materials and Methods.

lower than 25% in all cases. Covalent binding to silica produces immobi- lization systems whose corresponding yields improve if the enzyme is bound to the support through its carboxyl groups rather than if the amino groups are involved. In general, pretreatment of the immobilization matrix with glutaraldehyde is not recommended in these systems, because it causes significant reduction in the nitrite reductase activity, although it seems to confer high stability (see later).

Stability of free suspended and immobilized nitrite reductase Table 3 shows that immobilized nitrite reductase is more stable than

free suspended enzyme, when incubated either at 4°C or 60°C. The NiR-DEAE and NiR-DEAE-G-140 systems show, at 4 “C, half-life values of 250 and 120 days, respectively, as compared with the 40 days shown by free suspended enzyme. The addition of increasing concentrations of glutaral- dehyde to the system produces a progressive loss of the retained nitrite reductase activity in the immobilized system (results not shown) which, on the other hand, exhibits increasing resistance to thermal inactivation (Table 3). Thus, NiR-DEAE-G-830 has a half-life value at 60 “C of 20 min, which is at least 5-fold higher than that shown by free suspended nitrite reductase.

Effect of ionic strength on the stability of the NiR-DEAE and NiR-DEAE-G systems

The nitrite reductase bound to a DEAE-cellulose matrix can be easily recovered by increasing the ionic strength of the medium. Figure 2 shows that while NiR-DEAE alone does not retain any activity, with 0.1 M NaCl in the medium, NiR-DEAE-G-140 and NiR-DEAE-G-830 retained 50 and

TABLE 3

Stability of nitrite reductase activity from C. reinhrdtii in soluble and immobilized systema~

System Glutaraldehyde Nitrite reductase (mg per g support) activity

half-life (days) at 4°C

half-life (min) at 60 “C

NiR soluble NiR-DEAE NiR-DEAE-G-140 NiR-DEAE-G-280 NiR-DEAE-G-560 NiR-DEAE-G-830 NiR-SiOz-alkylamine NiR-SiOz-ah&mine-G

- 40 - 250 140 120 280 100 560 60 830 60 - 12 620 30

<5 cc5

5 10 15 20

<5 <5

e Solutions of partially purified nitrite reductase (0.11 mg protein/ml) or the corresponding immobilized systems were incubated at 4 “C and 60°C in standard buffer. In all cases, nitrite reductase activity was determined at different times by adding aliquots of the corresponding mizture to the reagenta of standard assay.

0 0.1 0.3 0.5

N&l CM)

Fig. 2. Effect of ionic strength on leakage of enzyme from NiR-DEAE CO), NiR-DEAE-G-140 (0) and NiR-DEAE-G-830 (Cl) systems. Samples of 0.15 ml of the immobilized systems were resuspended in standard buffer containing NaCl as indicated and maintained at 4°C with discontinuous and mild shaking during 3Omin. The suspensions were then centrifuged at 16 000 x g during 15 mm and the pellets resuspended in 0.5 ml of standard buffer. Nitrite reductase activity in the immobilized systems were determined by adding 0.1 ml aliquots to reagents of standard assay. 100% of immobilized activity were 6.27, 2.80 and 1.33 U per g of support for Nil-DEAR, NiR-DEAE-G-140 and NiR-DEAR-G-830, respectively.

100% of the immobilized nitrite reductase activity, respectively. NiR-DEAE- G-830 retains between 20 and 30% of the immobilized activity with 0.3M NaCl in the medium, indicating that simultaneous crosslinking with glutar- aldehyde improves the stability of the nitrite reductase when immobilized on DEAE-cellulose.

Kinetic parameters of immobilized nitrite reductase from C. reinhardtii Table 4 summarizes the kinetic parameters shown by several immobi-

lized nitrite reductase systems in comparison to those of the native enzyme previously determined by Romero et al. [61. It can be observed that nitrite reductase immobilized on alkylamine silica and its carbonylic derivative show optimum pH at 7.5 and 8.5, respectively, as compared with 8.0 for the free suspended enzyme. On the other hand, immobilization of nitrite reductase by ionic binding to DEAE-cellulose reduces the activation energy of the reaction, especially when glutaraldehyde is present in the immobilization system. A study of the immobilized nitrite reductase covalently bound to a matrix shows that the corresponding activation energy decreases in the temperature range between 20-35 “C, while it increases in the range 35-45 “C.

The atllnity of nitrite reductase for nitrite or reduced methyl viologen is

401

TABLE 4

Kinetic parameters of free suspended or immobilized nitrite reductaae from C. reinhurdtii’

Parameter NiR Nil-DEAE NiI-DEXEL NiR-SiOz- NiFt-SiOz- dissolved G-560 alhylamine alkylamine-G

optimum pH 8.0 8.0 8.0 7.5 8.5 optimum temp. (“C) 40 45 45 40 45 activ. energy 58.6 45.5 27.8 26.9 20.8 (20-35 “C, kJ mol-‘) activ. energy 23.9 25.5 18.8 102.3 44.1 (35-45 “C, ltJ mol-‘1 K,(app) nitrite 0.31 0.40 1.46 0.75 0.20

knM) K,,,(app) reduced 24 0.040 0.370 0.040 0.009

ferredoxin (PM) K,(app) MVH 0.91 0.98 2.39 0.41 0.20

(mV)

“All parameters were measured under similar conditions, either for soluble and immobilized enzyme systems, previously established according to [61.

not substantially changed by the immobilization procedure. In contrast, the apparent K, value for reduced ferredoxin shown by nitrite reductase from C. reinhardtii, immobilized either by ionic or covalent binding, is very low, indicating a high affinity of the enzyme for the substrate (Table 4).

Discussion

Among the different matrices studied to immobilize Fd-nitrite reduc- tase from Chlamydomonas reinhurdtii, the use of DEAF-cellulose, which binds the enzyme through ionic interactions, gives the optimal system in terms of immobilization yield and enzyme stability (Table 2). The addition of glutaraldehyde to such system causes crosslinking of the protein, which enhances the stability of the system against ionic strength and thermal inactivation. However, a decrease in the nitrite reductase activity is also observed.

In general, the immobilization provides additional stability of the enzyme activity 1121, probably because (a) the native protein conformation is protected against thermal or protease-dependent denaturation 113, 141, and/or (b) the enzyme is concentrated on the surface of the matrix. Our data are consistent with this observation because immobilized nitrite reductase from C. reinhardtii is more stable than free suspended enzyme (Table 3).

The catalytic properties of an enzyme are substantially altered by immobilization, because the microenvironment of the enzyme is changed in terms of local concentrations of substrates and products 1151. Particularly interesting is the low K, for reduced ferredoxin shown by immobilized C. reinhardtii nitrite reductase. In the NiR-DEAE system, the matrix is

402

positively charged at neutral pH, which in this case is particularly interest- ing because ferredoxin is an anionic protein at the indicated pH, and is bound to the matrix producing a high concentration of substrate in the nitrite reductase environment. Of course, this circumstance may affect the estima- tion of the corresponding apparent K,,,. On the other hand, when nitrite reductase is immobilized by covalent binding to SiO,-alkylamine or SiOz- alkylamine-G matrix, the native enzyme conformation is preserved, and thus the active site for reduced ferredoxin is in optimal condition to bind the substrate. This hypothesis is sustained because native dissolved Fd-nitrite reductase from C. reinhardtii may easily split into two subunits, one of M, 63 000 which retains the activity with reduced methyl viologen but has lost most of the ferredoxin-dependent activity; and a small subunit of M, 24 000 (called the coupling protein) which forms the essential part of the ferredoxin- binding active site 1161. Perhaps the immobilization of nitrite reductase may stabilize this domain.

A negative factor to consider in the case of immobilized enzymes is the diffusional restriction of substrates and products to or from the enzyme molecule [ 171. In our case, this may occur, because the NiR-DEAE-G-560 system shows high apparent K,,., values for the three susbstrates.

The activation energy of the reaction catalyzed by nitrite reductase is significatively decreased when immobilized enzyme is used, which improves its catalytic efficiency. This pattern is not followed by the NiR-SiOz- alkylamine systems, probably because of its temperature-dependent lability (Table 3).

Immobilized enzymes are good biological catalytic systems for industrial and analytical applications [18, 191. For instance, aminoacylase immobilized on DEAE-Sephadex has been used to produce L-amino acids [201. In addition, immobilization techniques provide a good approach to the study of enzyme-bound membranes or water-insoluble biological structures [ 21 I. Our data indicate that the immobilized nitrite reductase system may be a good model to study the native ferredoxin-binding domain.

Acknowledgements

This work was supported by the Comision Interministerial de Ciencia y Tecnologfa (Research Grant no BT87-002%C02). One of us (J.L.) is recipient of a fellowship from the Junta de Andalucia.

References

1 E. Katchalski, I. Silman and R. Goldman, Adu. Enzymol., 34 (1971) 445. 2 W. E. Hornby, M. D. Lilly and E. M. Crook, Biochem. J., 107 (1968) 669. 3 S. W. May and S. R. Padgette, &o/Technology, 1 (1983) 677. 4 R. R. Mohan and N. N. Li, Biotechml. Bineng., 16 (1974) 513. 5 D. R. Senn, P. W. Carr and L. N. Klatt, Anal. Chem, 48 (1976) 954.

403

6 L. C. Romero, F. Galv&n and J. M. Vega, Biochim. Biophys. Acta, 914 (1987) 55. 7 N. Sueoka, K. S. Wang and J. R. Kates, J. Mol. Biol., 25 (1967) 47. 8 F. GaIvti, A. J. M&rquez and E. FernBndez, Z. Naturforsch., 40 (1985) 373, 9 F. D. Snell and C. T. SnelI, in Calorimetric Methods of Analysis, Vol. 2, Van Nostrand

Reinhold, Princeton, New York, 1949, p. 804. 10 M. M. Bradford, Anal. B&hem., 72 (1976) 248. 11 J. M. Vega and H. Kamin, J. Biol. &em., 252 (1977) 896. 12 A. M. KIibanov, An&. B&hem., 93 (1979) 1. 13 I. V. Berezin, A. M. KIibanov and K. Martinek, Russ. Chem. Rev., 44 (1975) 9. 14 A. M. KIibanov, Adv. Appl. Microbial., 29 (1983) 1. 15 K. J. LaidIer and P. S. Bunting, Methods Emzymol., 64 (1980) 227. 16 L. C. Romero, C. G&or, A. J. M8rquez, B. G. Forde and J. M. Vega, Biachim. Biophys. Acta,

957 (1988) 152. 17 H. Ooshima and Y. Harano, Biotechrwl. Bioeng., 23 (1981) 1991. 18 A. Wiseman, J. Chem. Tech. Biotechnol., 30 (1980) 521. 19 L. J. Kricka and G. H. G. Thorpe, Trends Biotechnol.. 4 (1986) 253. 20 T. Tosa, T. Mori and I. Chibata, Agric Biol. Chem., 33 (1969) 1053. 21 K. Martinek and V. V. Mozhaev, Adv. Enzymol., 57 (1985) 179.