[methods in enzymology] immobilized enzymes and cells, part c volume 136 || [2] continuous enzymatic...

[2] CONTINUOUS ENZYMATIC TRANSFORMATION 9

cations of immobilized active NAD in analysis and as a component in enzyme reactors. In addition a polyethylene glycol-bound ATP ~9 and NADP 2° have recently been used in enzyme reactor studies. It should be kept in mind that native coenzymes for a number of applications remain an alternative (see, e.g., the contribution on ATP recycling). 2~ In this context a recent publication on "an affinity chromatographic reactor for highly efficient turnover of dissociable cofactors" applying free NAD in an ultrafiltration hollow fiber tube should be mentioned. 22

19 W. Berke, M. Morr, C. Wandrey, and M.-R. Kula, Ann. N. Y. Acad. Sci. 437, 257 (1984). 20 K. Okuda, I. Urabe, and H. Okada, Eur. J. Biochem. 151, 33 (1985). 2~ D. C. Crans, R. J. Kazlauskas, B. L. Hirschbein, C.-H. Wong, O. Abril, and G. M.

Whitesides, this volume [25]. 22 O. Miyawaki, N. Osato, and T. Yano, Agric. Biol. Chem. 49, 2063 (1985).

I y . Yamazaki and H. Maeda, this volume [3]. m j. Grunwald and T. M. S. Chang, J. Mol. Catal. 11, 83 (1981). n M. O. MAnsson, P. O. Larsson, and K. Mosbach, this series, Vol. 89, p. 457, o S. Gestrelius, M. O. MAnsson, and K. Mosbach, Eur. J. Biochem. 57, 529 (1975). p E. Schmidt, E. Fiolitakis, and C. Wandrey, Enzyme Eng. 8 (in press). q H. Suzuki and Y. Yamazaki, this volume [5]. • K. J. Laidler and M. A. Mazid, this volume [6]. s T. Yao and S. Musha, Anal. Chim. Acta 110, 203 (1979). t A. Malinauskas and J. Kulys, Anal. Chim. Acta 98, 31 (1978). " Y. Sakaguchi, M. Sugahara, J. Endo, and T. Murachi, J. Appl. Biochem. 3, 32 (1981).

[2] C o n t i n u o u s E n z y m a t i c T r a n s f o r m a t i o n in an E n z y m e - M e m b r a n e R e a c t o r wi th

S i m u l t a n e o u s N A D H R e g e n e r a t i o n

B y MARIA-REGINA KULA a n d CHRISTIAN WANDREY

Enzymes as catalysts are especially noted for their high stereo- and regiospecificity. To accomplish the synthesis of chiral compounds often not only an enzyme is needed as catalyst but in addition a low molecular weight coenzyme which participates in the reaction. For a large number of redox processes NAD(H) or NADP(H) serves as coenzymes for dehydrogenases and is utilized in stoichiometric amounts in the course of the reaction. These coenzymes are readily dissociable from the enzyme and require a separate second reaction for regeneration.

I S. S. Wang and C. K. King, Adv. Biochem. Eng. 12, 119 (1979).

Copyright © 1987 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 136 All rights of reproduction in any form reserved.

l 0 MULTISTEP ENZYME SYSTEMS AND COENZYMES [2]

Since NADH and NADPH are complex and rather labile organic chemicals they are quite expensive. For the application of dehydrogenases in the enzyme-catalyzed synthesis of chiral compounds an efficient coenzyme regeneration is therefore needed in order to make such a process economically viable. A simple comparison of cost of I mol of NADH (665 g/mol, value -$1000) with 1 mol of chiral a-hydroxy or a-amino acid ( -100 g/mol, value -$2) shows that separation, external regeneration, and recycling of the coenzyme would require an overall yield of 99.98% per pass to keep the coenzyme cost at 10% of the product value.

Simultaneous regeneration offers a chance to overcome the stoichiometric relation between product and coenzyme by an efficient internal recycling. In principle several experimental approaches are possible. ~-6 Here we concentrate on an enzymatic regeneration of NADH by a second enzyme-catalyzed reaction. Our approach follows the strategy developed in cellular metabolism in which NADH is produced and consumed by a series of balanced reactions. 6-~° Considering ultimately a technical application it should be noted that besides the anticipated conversion, a second substrate is now needed in a stoichiometric amount and two products are formed in equimolar concentration. Therefore the cosubstrate should be cheap enough and neither cosubstrate or coproduct should be obnoxious to the enzymes involved. In the authors' laboratories formate dehydrogenase is preferred as a general NADH-regenerating enzyme for several reasons: the enzyme is readily available in large amounts from Candida boidinii grown on methanolH,~2; the equilibrium of the reaction catalyzed lies far on the side of CO2 and therefore NADH formationS1; and the

2 H. Simon, J. Bader, H. Giinther, S. Neumann, and J. Thanos, Ann. N. Y. Acad. Sci. 434, 171 (1984).

3 Z. Shaked, J. J. Barber, and G. M. Whitesides, J. Org. Chem. 46, 4100 (1981). 4 M. O. M~nsson, P. O. Larsson, and K. Mosbach, this series, Vol. 89, p. 457.

M. Aizawa, R. W. Coughlin, and M. Charles, Biotechnol. Bioeng. 18, 209 (1976). 6 R. Wichmann, C. Wandrey, A. F. Biickmann, and M.-R. Kula, Biotechnol. Bioeng. 23,

2789 (1981). 7 C. Wandrey, R. Wichmann, W. Leuchtenberger, A. B/ickmann, and M.-R. Kula, U.S.

Patent 4,326,031, European Patent 80,104,346.4; U.S. Patent 4,304,858, European Patent 80,104,345.6.

s W. Leuchtenberger, C. Wandrey, and M.-R. Kula, DOS 3,307,094.6, Spanish Patent 530,155.

9 A. F. Bfickmann, U.S. Patent 4,443,594, European Patent 79,102,954.9. ~ C- Wandrey and R. Wichmann, in "Application of Isolated Enzymes and Immobilized

Cells to Biotechnology" (A. Laskin, ed.), p. 177. 1985. H H. Schtitte, J. Flossdorf, H. Sahm, and M.-R. Kula, Eur. J. Bioehem. 62, 151 (1976). ~2 K. H. Kroner, H. Schiitte, W. Stach, and M,-R. Kula, J. Chem. Technol. Bioteehnol.,

Biotechnol. 32B, 130 (1982).

[2] CONTINUOUS ENZYMATIC TRANSFORMATION 1 l

HCO0- reduced subsfrate

u . ~ PEG 2oooo-NAD" /z~

PE5 2oooo-NAOH • k0H ~A C0 z oxidized subsfrafe

FIG. 1. Reaction scheme. FDH, Formate dehydrogenase; E, enzyme; PEG 20000-NAD + NADH, coenzyme derivatives.

coproduct CO2 is easily separable. The overall reaction scheme is outlined in Fig. 1.

It is apparent that for fast NADH recycling, ready access to both enzymes becomes crucial and reactor systems with inherent mass transfer resistances should be avoided. Therefore an enzyme-membrane reactor was chosen. Here the reaction will proceed in homogenous solution. 6-1° The enzymes are retained in the reactor by virtue of their high molecular weight, utilizing an ultrafiltration membrane as a selective barrier. How- ever, the difference in molecular weight between product and coenzyme is not sufficient to achieve satisfactory results by a membrane separation. Therefore we attempted to increase the molecular weight of the coenzyme by attaching it covalently to a water-soluble polymer such as polyethylene glycol (PEG). 9 This way the retention of the coenzyme by an ultrafiltration membrane could be accomplished together with separation of the coenzyme from the product stream. Provided the coenzyme activity is not lost upon modification, the basic design of the reactor should allow continuous enzymatic conversion and regeneration of NADH to proceed.

Coenzyme Derivat ives

The synthesis of the PEG-NADH is outlined in Fig. 2 and has been described in detail by Bfickmann et al. 9,13 The modified coenzyme is readily soluble in water, and its concentration can be determined from absorb- ance at 340 or 259 nm, respectively. Assuming that the molar absorption coefficients are identical with the native coenzyme the following values have been used for calculation: 6220 M -~ cm -I for NADH at 340 nm, and 18,000 M -~ cm -~ for NAD at 259 nm. A number of dehydrogenases has been tested with regard to their ability to accept the modified coenzyme.

u A. F. Biickmann, M.-R. Kula, R. Wichmann, and C, Wandrey, J. Appl. Biochem. 3, 301 (1981).

12 MULTISTEP ENZYME SYSTEMS AND COENZYMES [2]

NH2

~NH~ \ N / "k 2H*

"~N/~"~ N LRib-- p--p --.,bJ

L. ib- - p-- p--Rin /

NH2

h R i b - - p - - P--Rib ~

PEG--COO-

C Q r b o d i i m i d e

r e d u c t i o n in

D i m r o t h r e o r r Q n g e m e n t

H P E G - - C - - N --CH~--CH~ ~'- NH H H O

o N N NH 2

L R i b - - p - - p - - R i b -]

FIG. 2. General synthesis route to modified coenzymes. Rib, Ribose; P, phosphate; PEG, polyethylene glycol.

TABLE I KINETIC PARAMETERS OF DEYHYDROGENASES FOR NATIVE NADH AND PEG-NADH a

Conditions Native NADH PEG-NADH

Temperature Vmax Km Vmax Krn Enzyme b Substrate pH (°C) (U/rag) (mM) (U/mg) (raM)

AIaDH Pyruvate 9 25 20.9 0.022 10.3 1.290 LeuDH Ketoleucine 8 25 13.2 0.033 11.5 0.029 PheDH Phenylpyruvate 8.5 25 37.2 0.047 17.7 0.099 L-LDH Pyruvate 9 25 308.0 0.001 150.0 0.080 D-LDH Pyruvate 9 25 37.0 0.289 4.3 0.060 L-HicDH Ketoleucine 8.5 25 511.0 0.044 214.0 0.144 b-HicHD Ketomethionine 8 25 259.0 0.170 240.0 0.240 FDH Formate 8 40 1.2 0.300 2.6 0.330

a Activity per milligram protein in the enzyme preparation. b AlaDH, Alanine dehydrogenase; LeuDH, leucine dehydrogenase; PheDH, phenylalanine dehy-

drogenase; L-LDH, L-lactate dehydrogenase; D-LDH, D-lactate dehydrogenase; L-HicDH, L-hydroxyisocaproate dehydrogenase; D-HicDH, D-hydroxyisocaproate dehydrogenase; FDH, formate dehydrogenase.


The results are listed in Table I. Often the Vmax value obtained with the derivative is in the range 50-100% in comparison with the native coenzyme, while the Km value may change by an order of magnitude. It should be noted that formate dehydrogenase exhibits an even higher initial reaction velocity with the modified coenzyme than with the native. 6 Besides the examples listed in Table I the following enzymes are known to accept PEG-NAD(H) as coenzyme: alcohol dehydrogenase, isopropanol dehydrogenase, malate dehydrogenase, glutamate dehydrogenase. So far, glu- cose dehydrogenase from Bacillus megaterium is the only dehydrogenase detected that does not accept PEG-NADH as a substrate.

Retent ion

The retention of the coenzyme in the reactor becomes very important for continuous operation. In Fig. 3 the loss of coenzyme by incomplete retention in the reactor is plotted as a function of the operating time. The data show that retentions better than 99.9% are necessary to avoid exces- sive loss of coenzyme at a typical mean residence time of 1 hr. The high

. . . . . . . . . . . . . . . . . . . . PEG_20000_NAD(H) \ " " " . . . . . . . . . R = 99 93

8 . 8 \ ~ . . . . . . . . . kELU = 1 7 X/d

O \ PEG-1000g-NAD C H)~""~ \ R = g g 8 2 X

\ \ kELU = 4 3 X/d e . 4

\, \ ,

N

0 . 2 ""

R = 9 9 . 0 0 ~'". kEL U 24 .0 ~/cl "'"--.....,...

I I I I I I I 1 I I I 8 2 4 I~ 8 18 1 2 1 4 1 0 1 8 2a 22 2 4

TIME l D

Flo. 3. Elution of P E G - N A D ( H ) across a YM5 (Amicon) membrane in a continuously operated membrane reactor; residence time Z = l hr. R, Retention (R = 99.00% for comparison); ke~u, elution loss (due to incomplete retention); PEG 10000 and PEG 20000, molecular weight 10,000 and 20,000.


retention is a very stringent requirement when choosing the right membrane and depends on the average molecular weight of the coenzyme derivative and also on the pore size distribution determining the actual cutoff behavior of a given membrane. The performance of a membrane selected should be checked by experiments lasting for several days in order to allow assessment of the data with sufficient confidence. Retention R is defined by Eq. (1):

R = ( C r - C f ) / C r (1)

where Cr and Cf are the concentration of the compound of interest in the retentate and filtrate, respectively. The washout can be described as a function of time by Eq. (2):

Cr/Cro = e -[~1 - R)/~-]t (2)

Cro is the retentate concentration at time zero, t is the elapsed time, and r the mean residence time. The apparent retention R can be calculated according to Eq. (2) from a plot of l n (Cr /Cro ) versus operating time. The elution loss Kelu is then defined by Eq. (3):

Kelu = (1 - R ) / r (3)

Even for rather high retention, washout becomes critical as residence times go down, since it depends on residence times as well as retention. In Fig. 4 the retention of PEG, carboxylated PEG, and the final preparation of the P E G - N A D H are represented. Experiments were performed within the membrane reactor as described below. Concentration of PEG was measured by refractive index; PEG-NADH was determined by UV ab- sorbance at 260 nm employing a cuvette with a 1-mm light path for the retentate and a 10-mm light path for the filtrate to compensate partly for the large difference in concentration of the different streams. It should be noted, however, that the apparent retention depends on the hydrodynam- ics of the system employed. Considering its importance for coenzyme- dependent processes the apparent retention should be reexamined if other reactor configurations are evaluated or hydrodynamic conditions are drastically changed. The absolute loss of coenzyme is also a function of the coenzyme concentration, which should therefore be as small as possible in the reactor. The lower limits of coenzyme concentration are defined by the necessity to saturate the enzymes involved in order to operate with their maximal catalytic efficiency. Table I shows that PEG-NADH derivatives exhibit comparatively low Km values, so that in many cases stationary coenzyme concentrations below 0.5 mM are sufficient for operation. Since the enzymes employed in the membrane reactor are much larger


t

8.6

8.4

8.2 A PE8-28888 D PEG-28088-COOH

o ¢ PEG-28888-NADH

8 -

0 I I I I I I

I 2 3 4 5 6 7

TIME / D

FIG. 4. Elution of PEG and PEG derivatives across a YM5 (Amicon) membrane ; residence time ~" = 1 hr.

than the coenzyme derivative, elution losses of the catalyst itself are negligible.

Experimental Setup of an Enzyme-Membrane Reactor

The ultimate goal of an experiment determines the degree of sophisti- cation the experimental setup of an enzyme-membrane reactor has to meet. A simple homemade version utilizing commercially available equip- ment is illustrated in Fig. 5. It has been successfully used for the determi- nation of the stereospecificity of enzymes in crude extracts during a screening procedure.~4 A similar version could be employed if the preparation of small amounts (1-100 mmol) of a certain chiral compound is of prime interest. However, if the enzyme-membrane reactor is operated to evaluate coenzyme utilization and to use the data for feasibility studies or the design of a large-scale process several, safety and control features as well as precise measuring devices have to be installed.

14 W. Hummel , H. Schiitte, and M.-R. Kula, Ann. N . Y. Acad. Sci. 434, 194 (1984).


p-

i

i . . . . . . . . . . . . . . .

FIG. 5. Simple version of a laboratory enzyme-membrane reactor. The glass vessel (1) contains the substrate solution in 0.3 M ammonium formate or sodium formate, pH 7.0-7.5. In the thermostated (25 ° water bath) reactor (2), 0.5 ~mol/ml PEG-NADH, 3 U/ml formate dehydrogenase, and 3-5 U/ml of the second dehydrogenase are placed in 10 ml substrate solution containing 0.3 M formate. The reaction mixture is circulated by means of a peri- staltic pump (4) passing through an ultrafiltration device (3). A CEC unit from Amicon or a similar device fitted with a YM5 membrane (Amicon) can be conveniently employed. With a CEC unit the circulating rate is set to approximately 30 ml/hr. This will create a small pressure differential and lead to a filtrate flux of approximately 3 ml/hr and a residence time of 3 hr. Filtrate flux is controlled and readjusted manually about twice a day if necessary. Filtrate is collected in bulk or with a fraction collector. The reactor (2) is closed airtight and the liquid level kept constant by the pneumatic overhead arrangement (1). The reactor is stirred using a magnetic stirrer to ensure proper mixing.

Figure 6 gives a flow diagram of a continuous process including all measuring points. Figure 7 shows a laboratory reactor which has been assembled in the mechanics workshop of the authors. The guiding princi- ples in the design were to achieve a high ratio of membrane surface to internal retentate volume, to incorporate temperature control, and to allow easy access to the membrane for setup and changes between experiments. Polarization control is effected by operating the membrane reactor

enzyme ~ ~ o ~ c ° e n z y m e ~ I ~ ~J I

g o

substrate

I METERINfi PUMP 4 PHOTOMETER 2 STERILE FILTER 5 POLARIMETER S ENZYME MEMBRANE REACTOR 6 RECORDER

F product

FIG. 6. Flow diagram of a laboratory-scale membrane reactor with magnetic stirrer and thermostating jacket.


se

mc

se

l id

st i

bc

sa ) s t ra te

FIG. 7. Details of a laboratory membrane reactor (the magnetic bar is inserted in a plastic disk to minimize the retentate volume).

on a magnetic stirrer and inserting a magnetic bar underneath the membrane. In general the speed of the stirrer is set to 200 rpm. For long-time operations special care has to be taken to achieve and maintain sterile conditions in the reactor. After complete assembly a solution containing a suitable disinfectant such as 0.1% peroxyacetic acid is pumped through the reactor, and at the same time all tubing and filters are flushed. The disinfectant is replaced after several hours, at least 5, by sterile water followed by substrate solution. At this point the integrity of all seals is also tested. Finally the selected enzymes and PEG-NADH are added aseptic- ally or pumped into the reactor and concentrated on the retentate side. Reaction starts at zero conversion and the time course until steady state is obtained can be followed and utilized to test the kinetic model based on measurement of initial rates, which has previously been developed to describe the reactor performance. 6,1°

Because chiral compounds are produced, polarimetry is the method of choice to follow conversion in the reactor effluent. A port to the retentate side, closed by a septum, serves to draw periodically samples from the reactor using sterile syringes and to measure coenzyme content and enzyme activities. If desired, on-line control of enzyme activity and coenzyme levels is possible, but this requires for each single case a detailed


1 0 0

g,g

z cl H

OC bJ

Z 0 (J

80

80

40

20 SPACE-TIME-YIELD = 214 G/(LND) 100 MMOL/L KETDISOCAPROATE 400 MMOL/L NH4-FORMATE pH = 8 ,0 , T = 2S * • = 7S MIN

0 | I I I } 1 I I

0 10 20 3B 48 SO 50 70 80

TIME / D

FIG. 8. L-Leucine product ion with FDH, L e u O H , and PEG 20000-NADH.

90

-I

(3 b.

298 U/KG PRODUCT

D

i~ rl

i~r~ O

FDH - ADDITION

I I I I 1 I I

0 2 0 3 0 40 5 0 8 0 7 0

T I M E / D

FIG. 9. L-Leucine product ion and deactivation of FDH.

i

80 90


._1

\

I

w 1

261 U/KG PRODUCT 1

t I LEUDH - ADDITION

o I I 1 I 1 I I I

0 10 20 30 40 SO 60 70 80 90

TIME / D

FIG. 10. L-Leucine production and deactivation of LeuDH.

kinetic model in order to differentiate the activity of the enzymes involved. 15 The two enzymes and the coenzymes utilized in the coupled reaction will have different rates of decomposition. The component limit- ing the performance of the reactor can be identified and added with the substrate stream in order to restore conversion and performance. After sufficiently long operating times the consumption of enzyme activity and coenzyme loss per unit weight of product can be determined with confidence and put into an economic model of the reaction in order to optimize the process studied. For calculation and design it is important to maintain a constant flow. A pulse-free piston pump (Reichelt Chemietechnik, Hei- delberg) was found satisfactory for prolonged operation with the low flow rates required for the laboratory reactor.

Performance of Reactor

Figures 8-11 demonstrate 3 months' performance of a laboratory enzyme-membrane reactor for the production of L-leucine. A cycle number of 80,000 was obtained for the coenzyme. The cycle number is defined

15 R. Wichmann and C. Wandrey, Enzyme Eng. 6, 311 (1982).


I 100000

O.B 00000

8 6 6 0 8 0 8

d 0.4 48000 ~

\

0.2 20000

0 0

0 10 20 38 40 50 60 70 80 90

TZME / D

FIG. 11. L-Leucine production, deactivation of PEG 20000-NADH, and development of cycle number.

here as moles of product obtained per moles of coenzyme lost in the process (for whatever reasons). Figure 11 shows that the cycle number increases from the start of the experiment and levels off after - 20 days. Figures 9 and 10 demonstrate that the reactor was operated at rather low stationary enzyme concentrations. Increasing the enzyme content in the reactor would give higher space-time yields. Productivities up to 1 kg/ liter-day have been realized, which is much higher than reported values for fixed bed reactors.

The optimal ratio of the enzyme activities depends on the kinetic constants, and concentrations are not necessary equal. It is one of the advantages of the enzyme-membrane reactor that the concentration of the enzymes involved in the process can be chosen and maintained at will. The system is amenable for modeling based on kinetic data and can therefore be optimized on rational grounds. Enzymes isolated from different organisms can be mixed and applied together in homogeneous phase. For this reason the enzyme-membrane reactor appears to have advantages compared to the utilization of whole cells, as long as the number of single reactions taking place remains fairly small and the enzymes employed are sufficiently stable in solution during operation. Results shown in Figs. 9 and 10 indicate that these conditions can be fulfilled. The average enzyme

[ 3 ] C O I M M O B I L I Z A T I O N OF NAD A N D D E H Y D R O G E N A S E S 21

activity consumption with respect to both enzymes was less than 300 U/ kg product over a period of 3 months of continuous operation.

A c k n o w l e d g m e n t

The coenzyme preparation was from A. F. Biickmann, and the enzymes were isolated by H. Schiitte and K. H. Kroner. The chemical engineering measurements at continuous operation were carried out by Mrs. U. Mackfeld. The engagement of all these co-workers is gratefully acknowledged.

[3] C o i m m o b i l i z e d S y s t e m o f N A D wi th D e h y d r o g e n a s e s

By YOSHIMITSU YAMAZAKI a n d HIDEKATSU MAEDA

Recycling of NAD is a prerequisite for the industrial application of dehydrogenases. The most effective way of realizing this is to maintain both NAD and the coupled dehydrogenases in one reactor system (bioreactor) and recycle NAD in situ. Several methods have been developed for this purpose: NAD bound to a water-soluble polymer and the dehydrogenases are either placed all together in an ultrafiltration appa- ratusf1-3 enclosed with a semipermeable membrane, 4,5 microencapsula- ted, 6 or immobilized in a collagen membrane. 7 Solid-phase coimmobiliza- tion of NAD and a dehydrogenase ~,9 and the covalent binding of an NAD derivative in or near the active site of dehydrogenases ~°,~ have also been reported.

l y . Yamazaki and H. Maeda, Agric. Biol. Chem. 50, 3213 (1986). S. Furukawa, N. Katayama, T. Iizuka, I. Urabe, and H. Okada, FEBS Lett. 121, 239 (1980).

3 R. Wichmann, C. Wandrey, A. F. Btickmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981).

4 p. Davies and K. Mosbach, Biochim. Biophys. Acta 370, 329 (1974). 5 A. Malinauskas and J. Kulys, Anal. Chim. Acta 98, 31 (1978). 6 j. Grunwald and T. M. S. Chang, J. Appl, Biochem. 1, 104 (1979). 7 Y. Morikawa, I. Karube, and S. Suzuki, Biochim. Biophys. Acta 523, 263 (1978). 8 S. Gestrelius, M.-O. M~nsson, and K. Mosbach, Fur. J. Biochem. 57, 529 (1975). 9 M. A. Mazid and K. J. Laidler, Biotechnol. Bioeng. 24, 2087 (1982).

10 M.-O. Mhnsson, P.-O. Larsson, and K. Mosbach, Eur. J. Biochem. 86, 455 (1978). I~ M.-O. M~nsson, N. Siegbahn, and K. Mosbach, Proc. Natl. Acad. Sci. U.S.A. 80, 1487

(1983).

Copyright © 1987 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 136 All rights of reproduction in any form reserved.

[methods in enzymology] immobilized enzymes and cells, part c volume 136 || [2] continuous enzymatic...

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