TOYOTA EXHIBIT 2038
`
`Reactive Surfaces Ltd. LLP v.
`Toyota Motor Corporation,
`IPR2016-01462
`
`

`

`26
`
`tivity of the S. solfataricus malic enzyme. In addition, we
`investigated the correlation between the hydrophobicity of
`the organic medium and the enzymatic behaviour: for this
`purpose, we used the logarithm of the partition coefficient for
`the water/organic-solvent mixture (log Pmix) as a quantitative
`measure of hydrophobicity.
`
`MATERIALS AND METHODS
`
`Cliemicm’s
`
`NADP+, Tris (Trizma reagent), urea and guanidine
`hydrochloride were from Sigma; acetone, 2-propanol, meth-
`anol and tetrahydrofuran from Merck; ethanol, n—butanol
`and dimethylformamide from Baker; L-malic acid, sodium
`deoxycholate and Triton X-100 from BDH chemicals. The
`Bio-Rad protein reagent and SDS were from Bio-Rad labora-
`tories. All other chemicals used were of the highest purity
`available.
`Malic enzyme was purified to homogeneity from the ex—
`treme thermoacidophilic archaebacterium Sulfolobus sulfa—
`taricus, strain MT-4, according to the published procedure
`[4]~
`
`Enzyme assay
`
`The oxidative decarboxylation of L—malate was followed
`with a Varian DMS-100 recording spectrophotometer by ob-
`serving the appearance of NADPH at 340 nm. The standard
`reaction mixture (final volume, 1 ml) contained 20 mM Tris/
`HCl, pH 8.0, 1 mM L-malate (adjusted to pH 7.0 with KOH),
`0.05 mM NADP+, 0.1 mM MnClz, 50 mM ammonium
`sulfate and enzyme (5 — 10 ug, an amount that caused an
`increase in absorbance in the range 0.02—0.15 min”); the
`assay temperature was 60 “C except where otherwise indicated.
`The pH was always adjusted at the indicated temperature and
`measured experimentally using model mixtures having the
`same composition. All the initial velocity determinations were
`averages of measurements performed in duplicate or tripli-
`cate. One unit of enzyme activity was defined as the amount
`of enzyme reducing 1 umol NADP+/min under the assay
`conditions.
`
`The protein concentration was determined by the Bio-Rad
`method [8] using bovine serum albumin as standard.
`
`Enzyme stability
`
`The stability of the malic activity in the presence of a given
`water-miscible organic solvent was examined by incubating,
`in stoppered glass tubes at 25"C and in some cases at 45 OC
`and 600C, a mixture containing the malic enzyme (about
`150 ug) and the solvent to be tested in 20 mM Tris/HCl,
`pH 8.0,
`in a final volume of 0.5 ml. Immediately after the
`addition of the solvent, and at convenient time intervals, 30—ul
`aliquots were removed from each incubation mixture after
`mixing and these were assayed for enzyme activity as already
`described. The stability of the malic activity in urea, guanidine
`hydrochloride and SDS was carried out at 25°C, essentially
`as described above for the stability in organic solvents. The
`reported results are expressed as percentages of residual ac—
`tivity as a function of the incubation time.
`
`Enzyme activity in non-aqueous media
`
`The effects of increasing concentrations of organic s01-
`vents (5—70%) or detergents (0.01 — 1.5%) on malic activity
`
`were investigated at 25‘7C and, in some cases, at 45 °C and
`60C in standard assay conditions, using stoppered cuvets to
`avoid evaporation. The organic solvents were added in place
`of an equivalent volume of buffer (by vol.) and addition of
`the solvents was, assumed to result in strictly additive changes.
`The average error in the determination of the initial reaction
`rate was i10% when the reaction mixture contained an
`organic solvent concentration in the range 60—70%. The
`slight changes in pH of the reaction mixture caused by the
`addition of the organic solvent [9] were ignored since the malic
`activity was found to be unchanged between pH 8.0 and 9.0
`at any temperature [4]. The malic activity values are expressed
`as a percent of the control (100%) performed in the absence
`of solvent or detergent.
`Log P was used as a parameter of hydrophobicity; P
`denotes the partition coefficient of a given solute in a standard
`water/octanol two-phase system. Log P values for organic
`solvents (log P50”) and for the water (log PW) were obtained
`from hydrophobic fragmental constants according to Rekker
`et al. [10] and Leo et a1. [11], Log P values for the water/
`organic—solvent mixtures (log Pmix) were calculated using the
`following semi-empirical formula [12]:
`
`log Pmix = (1-x) log P30“, + x log PW,
`
`where x is the water concentration (in mole fraction) [13] and
`log PW is the log P value for water, considered to be equal
`to —1.38.
`
`Circuiar dichroism
`
`Far-ultraviolet circular dichroism spectra were measured
`by a Jasco J-500A automatic recording spectropolarimeter at
`25 “C, with a temperature control system employing a Haake
`bath. Measurements were made with stoppered quartz cells of
`1-mm path length, An enzyme concentration of 0.1 mg/ml
`was utilized.
`
`RESULTS
`
`Stability Ofthe malic activity in chaotropic agents
`and water-miscible organic solvents
`
`Fig.1 shows the effects of urea, guanidine hydrochloride
`and SDS on the stability of the malic activity at 25"C, The
`enzyme retained almost 90% of the initial activity after 24 h
`of incubation in 6 M urea and had a half-life of 10 h in 7.5 M
`urea; the enzyme was completely stable after 24 h in 4 M urea
`(not shown). Guanidine hydrochloride was a more potent
`inactivator than urea, the half-life being 30 min in 6 M. No
`loss of activity was detected after 24 h of incubation in the
`presence of 0.05% SDS, whereas the enzyme half-life was 5 h
`‘ in 0.075% SDS; the enzyme lost all its activity within 2 h in
`0.1% SDS (not Shown).
`The stability study in the presence of various water-mis-
`cible organic solvents was carried out at 25 OC (Fig.2). When
`the enzyme was incubated in 10% (by vol.) 2-propanol, the
`time progress curve of the stability showed that the activity
`increased slightly reaching a maximum value after 6 h of incu-
`bation; when incubated in 30% 2-propanol, the enzyme ap-
`peared to inactivate with a half—life of 10 h. The enzyme had
`a half-life of 8 h in 50% ethanol, Whereas it kept 85% of its
`initial activity after 24 h in 15% ethanol or 50% methanol.
`No loss of activity was detected after 24 h of incubation in
`the presence of a concentration as high as 50% dimethyl-
`formamide. The enzyme had a half-life of 30 min in 50%
`
`

`

`27
`
`TIME (hours)
`12
`
`3
`
`24
`
`100
`
`75
`
`50
`
`ACTIVITY
`%RESIDUALMALIc
`
`
`25
`
`5
`
`15
`
`TIME (min)
`
`Fig.3. Effect of 50% methanol on the thermostabi/ity of the S.
`solfataricus malic enzyme. The enzyme was incubated in stoppered
`glass tubes at 25°C, 45°C and 600C in 20 mM Tris/HCl, pH 8.0.
`containing 50% methanol. At convenient time intervals, 30-111 ali—
`quots were removed from each incubation mixture and assayed for
`the residual malic activity in standard conditions. In the time scale
`(hours) at the top of the figure, the inactivation was carried out at
`25 “C and 45 "C; in the time scale (min) at the bottom the inactivation
`was carried out at 60C
`
`Table 1. Effects ofdetergents on the activity ofthe S. solfataricus malic
`enzyme
`The malic activity was assayed at 25‘“C in 20 mM Tris/HCI, pH 8.0,
`containing 1 mM L-malate, 0.05 mM NADP+, 0.1 mM MnClz,
`50 mM ammonium sulfate and the detergent to be tested at
`the
`indicated concentration. Each point represents the mean of multiple
`measurements
`
`Addition
`
`Concentration
`
`Relative
`activity
`
`TIME (hours)
`
`Fig. 1. Effects ofcommon protein denaturants on the stability of the S.
`solfataricus malic enzyme at 25°C. The enzyme was incubated in
`stoppered glass tubes at 25 0C in 20 mM Tris/HCl, pH 8.0,
`in the
`presence of the denaturing agent. At the times indicated. the residual
`malic activity was measured by the standard assay using 30-ul aliquots
`from each incubation mixture. (0) 0.05% SDS; (0) 0.075% SDS;
`(I) 6 M urea; (
`) 7.5 M urea; (A) 6 M guanidine hydrochloride
`
`
`
`
`ACTIVITY
`%RESIDUALMALIC
`
`
`0
`
`4
`
`10
`
`16
`
`24
`
`TIME (hours)
`
`"/0
`
`
`—
`
`0.02
`0.5
`1.5
`
`0.2
`0.5
`1.5
`
`0.05
`0.1
`0.3
`
`100
`
`100
`90
`75
`
`100
`100
`95
`
`85
`60
`0
`
`None
`
`Triton X-100
`
`Sodium deoxycholate
`
`Fig.2. bfieCts ofwater-miscible organic solvents on the stability of the
`S. solfataricus malic enzyme at 25°C. The conditions of incubation
`and assay of the residual activity were as in Fig.1. All the solvent
`percentages were by vol.:
`(0)
`10% 2—propanol; (I) 50%
`dimethylformamide; (A) 50% methanol; (
`) 15% ethanol; (0) 30%
`2—propanol; (A) 50% ethanol; (11') 50% tetrahydrofuran
`
`
`
`
`tetrahydrofuran. The effect of 50% methanol on the
`thermostability of the malic activity was also investigated
`(Fig. 3); the enzyme half-life was 16 h and 3 min at 45 0C and
`60 0C, respectively. When incubated at the same temperatures
`in the absence of the solvent, the enzyme retained all
`the
`activity over 12 h.
`
`SDS
`
`Effect of'a'etergents and water-miscible organic solvents
`on the malic activity
`
`Since the activity of some thermophilic enzymes has been
`reported to be stimulated by detergents [14}, we tested the
`effect of such compounds on the malic activity. Tablel
`summarizes the results obtained at 25 0C with increasing con-
`centrations of neutral (Triton X-100) and ionic (sodium
`deoxycholate and SDS) detergents in the assay medium. There
`was no activation over the whole range of concentration
`(0.01 —1.5%) for each detergent. Nevertheless, a correlation
`
`is likely between the hydrophilic/lipophilic balance (HLB) of
`the detergent, which is an inverse measure of hydrophobicity
`[15], and the effect exerted on the enzyme activity. In fact.
`the extremely hydrophobic detergents Triton X-100 (HLB =
`13.5) and sodium deoxycholate (HLB 2 16) appeared to
`affect in minimum extent the activity of the enzyme as com-
`pared to SDS (HLB = 40).
`Fig.4A depicts the effects of increasing concentrations of
`several water-miscible organic solvents on the malic activity
`at 25"C. All the solvents stimulated the initial rate of the
`
`they differed in the concen-
`malate conversion; however,
`tration required for maximal stimulation and the extent of
`
`ACTIVITY
`%‘RESIDUALMALIc
`
`0
`
`6
`
`12
`
`18
`
`24
`
`

`

`B
`
`a
`
`7!»E
`
`EE
`
`3'o
`Ei.
`
`i:
`E 2
`'—
`
`O< 2 2
`
`'E
`
`
`1
`I
`l
`4
`l
`I
`7.5
`8.0
`8.5
`9.0
`9.5
`50
`150
`
`250
`
`28
`
`§
`
`160
`
`ACTIVITY 8' 0
`%CONTROLMALIC
`
`50
`
`% SOLVENT
`
`°/o METHANOL
`
`pH
`
`KCI(mM)
`
`Fig.4. Effects of increasing concentrations of water-miscible organic
`solvents at 25”C (A) and methanol at 25°C, 450C and 60C (B) on
`the activity of the S. solfataricus malic enzyme. The malic activity was
`assayed at the designed temperature in 20 mM Tris/HCI, pH 8.0,
`containing 1 mM L-malate, 0.05 mM NADP, 0.1 mM MnClZ, 50 mM
`ammonium sulfate and the solvent to be tested, using stoppered
`cuvcts. All the solvent percentages were by vol.; each point represents
`the mean of multiple measurements. (A) Methanol; (0) ethanol;
`(Fl) acetone; (O) 2-propanol; (I) dimethylformamide
`
`Fig. 5. Dependence of the S. solfataricus malic activity on pH (A) and
`ionic strength (B) in the absence (0) and in the presence (A) of50%
`methanol in the reaction mixture at 25 “C. (A) The assay mixtures were
`standard except for the buffer employed: 20 mM Tris/HCl was used
`in the pH range 75—916, 20 mM glycine/NaOH was used for the
`point at pH 9.5. The pH values of the mixtures containing 50%
`methanol were corrected according to Bates [9]. (B) The standard
`mixtures contained concentrations of KCl in the range 0—0.25 M.
`The scale on the ordinate is valid for both A and B
`
`Table 2. Reversible enhancement of the activity of the S. solfataricus
`malic enzyme by methanol
`The enzyme was incubated at 25 0C in 20 M Tris/HCl, pH 8.0, 50 mM
`ammonium sulfate, containing 50% ethanol for 10 min, then diluted
`in standard assay mixture at 250C with or without 50% methanol
`
`Although not shown, no aggregation or disaggregation of the
`enzyme molecule was caused by 50% methanol, as determined
`by gel-filtration chromatography performed on a Superose 12
`(Pharmacia) at 25 0C, both in the absence and presence of the
`solvent.
`
`Incubation
`conditions
`
`Activity after dilution in
`
`0% methanol
`
`50% methanol
`
`umol ‘ min’1 - mg’1
`
`0% methanol
`50% methanol
`
`2.06
`2.04
`
`3.95
`4.02
`
`activation. The most effective solvent for the rate enhance-
`ment was methanol. The rate of malate conversion increased
`
`reaching a
`linearly with methanol concentration,
`almost
`maximum at 60% (by vol.) methanol, which corresponds to
`a 1 .1-fold rate enhancement. Stimulated levels of malic activity
`were still evident at a concentration as high as 70% methanol
`or ethanol. The other solvents showed a slighter activating
`effect at
`lower concentrations
`(acetone > 2-pr0panol
`z dimethylformamide); however, they caused inhibition at
`relatively high concentrations (not reported in Fig.4). Tetra-
`hydrofuran and n-butanol did not display any activating effect
`(data not shown). No reduction of NADP+ was observed in
`the presence of any solvent when the enzyme was excluded.
`The malate conversion proceeded linearly with respect to time,
`without an initial lag phase, in the standard aqueous medium
`as well as in the presence of an organic solvent.
`Stimulation of the malic activity by methanol was com—
`pletely reversible. The enzyme was incubated at 25°C in
`20 mM Tris/HCl buffer, pH 8.0, 50 mM ammonium sulfate,
`containing 50% methanol, for 10 min, then diluted into the
`standard reaction medium at 25 0C with or without 50% meth—
`
`anol; the enzyme activity returned to the control level when
`the solvent concentration was lowered to 0.3% (Table 2).
`
`Effect of temperature, pH and ionic strength; kinetic constants
`
`In order to shed some light on the role of organic solvents
`on the perturbation of the stucture/function relationship
`in the malic enzyme, we analyzed the dependence of the malic
`activity on temperature, pH and ionic strength in an organic
`medium. The solvent used was methanol since it had shown
`the highest activating effect. We tested the solvent effect on
`the catalysis at 45 DC and 60 OC, considering that the malic
`enzyme from S. solfatarieus is a thermophilic enzyme. The
`data in Fig.4B show the detection of activation by methanol
`at 25 “C, 45 "C and 60 0C. It is noteworthy that the extent of
`enzyme activation is inversely related to temperature. The
`other solvents tested (ethanol and 2-propanol) behaved in a
`similar manner (not shown).
`in agreement with previous findings [4], the malic activity
`in the standard aqueous medium did not vary with the pH
`ranging from 8.0—9.0; above pH 9.0 the activity rapidly de-
`creased. When the initial reaction rate was investigated over
`the same pH range in non-aqueous mixtures containing 50%
`methanol, a progressive increase of the activity up to pH 8.9
`was detected, although methanol stimulated the rate of malate
`conversion over the entire pH range tested (Fig.5A). In the
`presence of 50% methanol,
`the malic activity displayed a
`slight change in the dependence on the ionic strength increase
`in the reaction mixture (Fig. 5B).
`Linear Lineweaver-Burk plots were obtained both in the
`absence and presence of 50% methanol. The Michaelis con-
`stants for malate (71 uM) and NADP+ (12.5 uM) did not
`change significantly in methanol, while the Vmax for both
`substrates doubled (data not shown).
`
`

`

`)-
`
`29
`
`
`
`—1.15
`
`-1.05
`
`—1.35
`
`—1.25
`
`ta '
`
`92 E a
`
`‘D
`QinII
`t:
`aO
`"
`
`log P mixture
`
`Fig. 6. Influence aflog PM, on the stability of the S. solfataricus malic
`enzyme in organic solvents. The values of the residual malic activity
`at 1.5h (El), 4h (0) and 12h (A) of incubation in the water-
`miscible organic solvent were taken from Fig. 2 and plotted versus the
`correspondent values of log Pmix calculated as described in Materials
`and Methods. The log Pmi, values for 10% 2-propanol, 50%
`dimethylformamide, 15% ethanol, 30% 2-propanol, 50% methanol,
`50% ethanol and 50% tetrahydrofuran were —— 1.337, —1.308,
`—1.275, —1.227, —1.188, —1.099, —1.037, respectively
`
`We chose log P, the logarithm of the partition coefficient of
`a compound in a water/octanol
`two-phase system, as
`hydrophobicity parameter because it not only senses the dif-
`ferences in hydrophobicity between all common organic sol-
`vents, but well quantifies the hydrophobicity of mixtures
`differing in the water/organic-solvent ratio [23]. In this work,
`the log P values for organic solvents were obtained from
`hydrophobic fragmental constants [10, 11] and the log P value
`for each water/solvent mixture (log Pmix) was calculated
`taking into account the water concentration [12]. When the
`logarithms of the residual activity at 1.5 h, 4 h and 12 h of
`incubation in the presence of each solvent were plotted versus
`the corresponding log Pmix values, an inverse correlation was
`found (Fig.6). The higher its log P value, the more hydro-
`phobic a mixture was: so, the enzyme stability decreased with
`the increase of hydrophobicity of the incubation mixture.
`An opposite enzyme stability/hydrophobicity relationship has
`been reported in certain two-phase systems [24]. A rationale
`is that miscible solvents strip or distort the water molecules
`at the protein surface which are required for stability and,
`when in contact with the protein, they exert a denaturing effect
`depending directly on their hydrophobicity; on the contrary,
`stabilization of the water layer by the immiscible solvents, (the
`more hydrophobic they are) can result in an increased thermal
`and storage stability of the protein [25]. Fig.7 shows the
`logarithm of the solvent molar concentration which gave
`maximal stimulation of the enzyme activity (log C), versus the
`corresponding log Pmix value; the relative activity value for
`each assay mixture is also reported. Two curves were thus
`obtained showing a similar behaviour; in fact, as outlined in
`the insert, a plot of the log C values versus the relative activity
`values was almost linear, thus suggesting the existence of a
`direct correlation between medium hydrophobicity and en-
`zyme activation.
`Activation by water—miscible organic solvents is not un-
`known in thermophilic enzymes [18, 26, 27], but very little
`information is available about the effects of such solvents on
`the catalytic and structural properties of these enzymes. The
`organic solvents may affect different protein molecules in a
`variety of different ways; so, it seems unlikely that a general
`explanation of the activation phenomenon can be found and
`
`DISCUSSION
`
`The resistance of a number of thermophilic enzymes to
`chaotropic agents and detergents is well documented and is
`interesting both for structural implications and biotechnologi-
`cal potentials [16]. The reported stability, of the malic enzyme
`from the extreme thermoacidophilic archaebacterium S.
`solfataricus, to inactivation by urea, guanidine hydrochloride
`and detergents is unusual even when compared to that of
`other dehydrogenases from thermophilic sources [17, 18].
`In order to investigate correctly the effects of the water-
`miscible organic solvents on the behaviour of the malic en-
`zyme from S. solfataricus, two distinct phenomena were con-
`sidered: (a) the residual enzymatic activity in standard assay
`conditions after incubation for different lengths of time in a
`water/solvent mixture, termed stability; (b) the enzyme cata-
`lytic power when the solvent is incorporated into the reaction
`medium, termed activity.
`The malic enzyme from S. solfataricus was found to pos-
`sess a remarkable stability in certain water-miscible solvents:
`after 24 h of incubation at 25 OC, the enzyme did not lose its
`activity in 50% dimethylformamide and retained 85% of its
`initial activity in 50% methanol or 15% ethanol. Generally,
`for several mesophilic enzymes, inactivation is associated with
`denaturation [19], aggregation [20] or dissociation [21]. In the
`case of the S. .ro/fataricus malic enzyme, 50% methanol did
`not cause aggregation or dissociation of the enzyme molecule.
`However, tolerance towards the organic solvent was reduced
`at temperatures above 45 CC. According to reports on the
`enzymes from thermophilic sources, the S. solfataricus malic
`enzyme can function in the presence of a notable amount of
`water-miscible organic solvents and a number of solvents have
`even been found to enhance the activity of this enzyme (see
`Fig.4A). In any case, a bell-shaped curve was obtained, with
`the reaction rate at first stimulated and then decreased. In
`
`the case of alcohols, the activating effect followed the series
`methanol > ethanol > 2-propanol at the same concentration
`(by vol.). Since it is known that the denaturing power of
`alcohols on proteins is inversely related to the same series [22],
`we can hypothesize that at each solvent concentration we are
`measuring a compromise between the activation, the inhi-
`bition (due to the lateral alkyl chain of the alcohol) and,
`probably, other effects. Activation by methanol was immedi-
`ate and reversible, involving a slight change in the pH opti-
`mum of the malic activity and in the dependence of activity
`on ionic strength. An investigation of the kinetic parameters
`in methanol was made. Lineweaver-Burk analysis revealed
`that 50% methanol doubled the Vmax, whereas the Km for
`both NADPJr and L-malate was unmodified. The absence of
`a change in the Michaelis constants demonstrates that there
`are no inhibition effects and the enzyme retains its substrate
`specificity. Also, the efficiency of the reaction (expressed by
`kcm/Km ratio) was higher in methanol, with respect to the
`aqueous buffer, indicating that the stimulation is due to an
`increase of the turnover number of the enzyme.
`The conformational status of the enzyme in 50% methanol
`was investigated by far-ultraviolet circular diehroism. The CD
`spectrum of the enzyme in organic solvent did not change
`significantly with respect to that in water. By inference, the
`secondary structure of the malic enzyme in 50% methanol
`remains the same (or nearly the same) as in aqueous solutions.
`Since organic solvents change the solution polarity, we
`attempted to search for a correlation between the hydro-
`phobicity of the medium and the reported data of stability
`and activity of the malic enzyme in nonaqueous environment.
`
`

`

`30
`
`REFERENCES
`
`200
`
`100
`
`I50
`"/- ACTIVITY
`
`200
`
`
`
`
`
`8%CONTROLMALICACTIVITY(H) §
`
`
`
`—1.35
`
`—1.25
`
`-1.15
`
`IogP mixture
`
`Fig.7. Influence oflog PM, on the activation ofthe S. solfataricus malic
`enzyme by organic solvents. The values of activation extent (0) and
`log C (O), the logarithm of the solvent molar concentration required
`for maximal activation, were taken from Fig.4; log Pm values were
`calculated as described in Materials and Methods. The log Pmix values
`for 10% dimethylformamide, 10% 2-propanol, 30% acetone, 60%
`methanol and 50% ethanol were — 1.37, — 1.337, — 1.27, ~1.19,
`41.099, respectively. The inset shows a replot of the values of log C
`versus the activation extent
`
`the present study does not do so. However, there is hope that
`the accumulation of data will make it possible to answer
`questions regarding the reason why the thermophilic enzymes
`are stable in organic media.
`It has been found that the thermophilic proteins have rigid
`structures, at room temperature, compared to those of the
`mesophilic proteins; this concept arose from the high toler-
`ance against proteolysis of the thermophile enzymes com-
`pared with the mesophilic counterpart [18, 28], in addition to
`the results obtained using spectroscopic techniques [29] or
`parameters (flexibility indices) derived from refined three-
`dimensional structures [30]. The rigid, thermostable proteins
`reach the flexibility required for catalysis at higher tempera-
`tures. Of interest is the observation that at elevated tempera—
`tures the well known tolerance of thermophilic enzymes
`towards denaturants
`such as urea [31 —33], guanidine
`hydrochloride [33], detergents [31, 33] and organic solvents
`[31] (this paper) was greatly reduced. This finding suggests
`that structural rigidity is a prerequisite for general stability:
`thus, the more rigid the protein structure, the higher its overall
`resistance to denaturants.
`
`Finally, we would emphasize the biotechnological pros—
`pects of the S. solfataricus malic enzyme. The resistance to
`denaturants and, remarkably, to water—miscible organic sol-
`vents,
`in addition to its thermostability and dual cofactor
`specificity, render this thermophilic oxidoreductase suitable
`for use in the production of reduced NAD(P) + or in coenzyme
`regeneration systems in organic synthesis.
`
`Prof. A. Fontana and Prof. C. Grandi (University of Padua,
`Italy) and their coworkers are gratefully acknowledged for making
`available the data of circular dichroism. This work was partially
`supported by the Commission of the European Communities, con»
`tract BAP 0052.1.
`
`1.
`
`b.)
`
`00
`
`Kandler, O. & Zillig, W., eds (1986) Archaebacterio ’85, Proceed~
`ings of the EMBO workshop on archaebacteria, Munich, 23 —
`26 June, 1985. Gustav Fischer Verlag, Stuttgart, New York.
`. Fontana, A. (1988) Biophys. Chem. 29, 181 ~19}.
`. Rossi, M. (1987) Biofutur 53, 39~42.
`Bartolucci, S., Rella, R., Guagliardi, A., Raia, C. A.,
`Gambacorta, A., De Rosa, M. & Rossi, M. (1987) J. Biol.
`Chem. 262, 772557731.
`. Cacace, M. G., De Rosa, M. & Gambacorta, A. (1976) Biochemis-
`try I5, 1692 — 1696.
`. Ochoa, 5., Mehler, A. H. & Kornberg, A. (1947) J. Biol. Chem.
`167, 871 —872.
`Lamed, R. & Zeikus, J. G. (1981) Biochim. Biophys. Acta 660,
`251 —255.
`. Bradford, M. M. (1976) Anal. Biochem. 72, 248—254.
`Bates, R. G. (1973) Determination of pH: theory and practice,
`Wiley, New York.
`Rekker, R. F. & de Kort, H. M. (1979) Eur. J. Med. Chem. 14,
`479 — 488.
`Leo, A., Hansch, C. & Elkins, D. (1971) Chem. Rev. 71, 525—
`616.
`Rcslow, M., Adlercreutz, P. & Mattiasson, B. (1987) Biocatalysis
`in organic media ‘ studies in organic chemistry 29 (Laanc, C..
`Tramper, J. & Lilly, M. D., eds) pp.349—353, Elsevier Science
`Publishers, Amsterdam.
`Sorensen, J. M. & Arlt, W. (1979) Liquid—liquid equilibrium data
`collection. Binary systems. Chemistry data series, vol. 5. part 1,
`Dechema, Frankfurt.
`Norling, B. (1986) Biochem. Biophys. Res. Commun. 136, 899—
`905.
`Furth, A. J. (1980) Anal. Biochem. 109, 207—215.
`Hartley, B. S. & Payton, M. A. (1983) Biochem. Soc. Symp. 48,
`133 — 146.
`
`10.
`
`11.
`
`12.
`
`13.
`
`14.
`
`15.
`16.
`
`17.
`
`18.
`
`19.
`20.
`
`21.
`
`22.
`
`23.
`
`24.
`25.
`
`26.
`
`27.
`
`28.
`
`29.
`30.
`31.
`
`32.
`
`lijima, S., Saiki, T. & Beppu, T. (1980) Biochim. Biophys. Acta
`613, 1 #9.
`Veronese, F. M., Boccu, E., Schiavon, 0., Grandi, C. & Fontana.
`A. (1984) J. Appl. Biochem. 6, 39—47.
`Butler, L. G. (1979) Enzyme Microb. Technol. 1, 253fl259.
`Tanizawa, K. & Bender, M. L. (1974) J. Biol. Chem. 249, 2130*
`2134.
`Contaxis, C. C. & Reithel, F. J. (1981) J. Biol. Chem. 246, 677—
`685.
`Herskovits, T. T., Gadegbeku, B. & Jaillet, H. (1970) J. Biol.
`Chem. 245, 2588 a 2598.
`Laane, C., Boeren, S., Vos, K. & Veeger, C. (1987) Biotech. Bioeng.
`30, 81 — 87.
`Carrea, G. (1984) Trends Biotechnol. 2, 102— 106.
`Zaks, A. & Klibanov, A. M. (1988) J. Biol. Chem. 263, 3194—
`3201.
`Fujita, S., Oshima, T. & lmahori, K. (1976) Eur. J. Biochem. 64.
`57 — 68.
`Sundaram, T. K., Wright, I. P. & Wilkinson. A. E. (1980) Bio-
`chemistry 19, 2017—2022.
`Daniel, R. M., Cowan, D. A., Morgan, H. W. & Curran. M. P.
`(1982) Biochem. J. 207, 641 —644.
`Wagner, G. & Wiitrich, K. (1978) Nature 275, 247—248.
`Vihinen, M. (1987) Protein Eng. 1, 477~480.
`Suzuki, Y., Yuki, T., Kishigami, T. & Abe, S. (1976) Biochim.
`Biophys. Acla 445, 386—397.
`Veronese, F. M., Boccu, E. & Fontana, A. (1976) Biochemistrr
`15, 4026— 4033.
`. Cowan, D. A. & Daniel, R. M. (1982) Biochim. Biophys. Acta
`705, 293 A 305.
`
`