Mandelate racemase assayed bypolarimetryHartmut Stecher†, Albin Hermetter‡ and Kurt Faber†*†Institute of Organic Chemistry, ‡Institute of Biochemistry, Graz University of Technology, Stremayrgasse 16, A-8010Graz, Austria.

An accurate and convenient assay for mandelate racemase based on polarimetry was developed by making use of thetime-dependent decrease of the optical rotation of the substrate (D- or L-mandelate) at 589 nm (Na-D-line) during theenzyme-catalyzed racemization. In comparison to the indirect assay methods hitherto used, this method has thefollowing advantages: (i) it is faster and more accurate than the two-enzyme redox assay (which needs a membrane-bound protein fraction), (ii) it is also applicable to non-natural substrates other than mandelate, provided their specificoptical rotation is large enough and (iii) it does not rely on expensive equipment such as circular dicroism.

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Figure 1 Reaction catalysed by mandelate racemase(mandelate dehydrogenase reaction in dashed square).

IntroductionMandelate racemase [EC 5.1.2.2] is one of the few race-mases that are independent of a cofactor for catalyticactivity (Adams, 1976). The enzyme catalyzes the racemi-zation of mandelate by the reversible interconversion of theD- and L-enantiomer (Fig. 1) by the mechanistic aid of anessential Mg21 (or Mn21) ion required for catalytic activity(Petsko et al., 1993; Kenyon and Hegeman, 1979; Kenyonet al., 1995). Mandelate racemase was first isolated fromPseudomonas putida ATCC 12633 which is able to grow onmandelate as the sole source of carbon and energy(Hegeman et al., 1970; Fee et al., 1974). The presence ofthis enzyme enables the bacterium to allow both enantio-mers of mandelate to be degraded through the so-calledmandelate pathway via benzoate. The latter is furthermetabolized via the b-ketoadipate pathway, finally leadingto acetyl-CoA (Hegeman, 1966). Besides its natural sub-strate, mandelate racemase also accepts various p-substituted mandelate derivatives, in particular thosebearing an electron-withdrawing substituent, such as Brand Cl, on the aryl moiety are excellent substrates(Hegeman et al., 1970). Remarkably, also other substrateswhich are structurally related to mandelate to a muchlesser extent, such as a-hydroxy-b,g-unsaturated car-boxylic acids [for instance, vinyl- (Li et al., 1995) andpropargyl-glycolate (Landro et al., 1992)] were readilyaccepted and racemized at good rates. The latter substrate,however, causes inactivation. These observations suggestedthat the substrate-spectrum for this enzyme is much widerthan previously assumed and that also other (non-natural)b,g-unsaturated a-hydroxycarboxylic acids of syntheticvalue are likely to be accepted (U. Felfer and K. Faber,unpublished). During our studies directed towards thedynamic kinetic resolution of a-hydroxycarboxylic acids by

© 1998 Chapman & Hall

making use of an enzyme-catalyzed in-situ racemization ofthe substrate (Stecher and Faber, 1997) we recently devel-oped a large-scale production of mandelate racemase(Stecher et al., 1997). In this paper we describe an accurateand convenient assay for this enzyme.

Two different methods to assess the activity of mandelateracemase were described up to now:

(I) The standard assay which was employed most widely,makes use of a two-enzyme cascade consisting of mandelateracemase and an L-specific mandelate dehydrogenase (Fig.1, dashed frame) (Hegeman, 1970). Thus, whereas man-delate racemase catalyses the interconversion of both man-delate enantiomers, the L-enantiomer is selectively

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oxidized by mandelate dehydrogenase to furnish ben-zoylformate at the expense of the blue dye,2,6-dichlorophenol-indophenol (DCPIP). The reaction isspectrophotometrically monitored by the decolorization ofthe co-oxidant at its lmax at 600 nm. Several featuresrender this assay a relatively unreliable procedure: (i) sincemandelate dehydrogenase is a membrane-bound enzyme, ithas to be employed as particulate fraction, which leads to aturbid heterogeneous assay mixture impeded by light-scattering, which is far from ideal considering spectropho-tometic measurements. Thus, agitation such as stirring orshaking are required in order to avoid the gradual settlingof the particulate fraction from the soluble mandelateracemase in the cuvette, which leads to inhomogeneousdistribution of both enzymes.1 (ii) For accurate measure-ment of mandelate racemase activity, mandelate dehy-drogenase has to be employed in large excess to avoid ratelimitations in the second step. (iii) This assay is notapplicable to non-natural substrates other than mandelateas long as the acceptance of these substrates by mandelatedehydrogenase at a sufficient rate is verified in separateexperiments. (iv) Since the blue chromophor, which isdecolorized during reduction, can be reoxidized though theaction of components from the electron-transport chain inthe particulate fraction, a significant background reactionis observed. (v) As the absorbancy of DCPIP is ratherhigh2, the chromophor has to be employed in very smallquantities. In addition, the absorbance is also pH-dependent (Armstrong, 1964).

(II) Alternatively, circular dichroism (CD) measured at 262nm as a direct kinetic probe of mandelate racemase activityhas been suggested (Sharp et al., 1979). This assay issignificantly more sensitive and allows the equilibrium ofracemization to be approached from either D- or L-mandelate with equal facility. However, the CD-spectrophotometer required does not belong to thestandard laboratory equipment and is expensive. In addi-tion, the instrument has to be modified to follow CD versustime (Sharp et al., 1979).

These considerations along with the notion that a direct

1 Attempts to avoid the undesired gradual settling of the partic-ulate fraction from the soluble mandelate racemase by enzymeimmobilization led only to limited improvement: Thus, filterpaper strips were soaked with a concentrated suspension of theparticulate fraction and dried. For measurement, the strips weremounted sideways on the walls of the cuvette without obstruct-ing the lightpath. Although the reproducibility was slightlyenhanced, the additional manipulations rendered the whole pro-cedure rather tedious.2 At a pH of 7.0, the molar absorbancy coefficient is 20.6·106 at600 nm. Thus, the oxidation of 1 mmol of L-mandelate tobenzoyl formate is equivalent to a decrease of 6.72 absorbancyunits.

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enzyme assay is always preferable to a coupled assay(McClure, 1969), led us to the development of a new assaybased on polarimetry. Although two isolated trials werereported using the optical rotation of mandelate at thesodium D line to follow a single time-course for mandelateracemase (Hegeman et al., 1970; Weil-Malherbe, 1966),the limits and the accuracy of this method were notinvestigated. On the other hand, polarimetry has beensuccessfully employed for the determination of the kineticsof other racemase reactions, such as proline racemase(Cardinale and Abeles, 1968) and hydroxyproline2-epimerase (Finlay and Adams, 1970).

Materials and methodsAll polarimetric measurements were performed on a PerkinElmer Polarimeter 341 using a cuvette of 10 cm lengthand 1.5 ml total volume. Data were recorded at 20°C andwere transferred from the polatimeter via a serial RS-232Cport to a computer using the software ‘‘Terminal’’ (Micro-soft). This allowed the continuous recording of opticalrotation versus time. The activity was measured in aqueousHEPES buffer (50 mM, pH 7.5) containing MgCl2 (3.3mM). The standard aqueous substrate solution containedD-mandelate (13.1 mM), its pH was adjusted to 7.5 using1M NaOH. A partially purified lyophilized preparation ofmandelate racemase with a protein content of ,25%(Bradford) was obtained as described recently (Stecher etal., 1997). Samples of crude lyophilized mandelate race-mase (50 mg) were dissolved in HEPES buffer (3 ml) andshaken. After centrifugation (10,000g, 3 min), the clearsupernatant was used as a stock solution showing a proteincontent of 4.17 mg/ml. For measurements, the reactionwas initiated by addition of an aliquot of the enzyme stocksolution (50–200 ml, depending on activity) to a solutionof substrate (2 ml) and mixed well. This mixture was thenimmediately transferred to the cuvette. After a shortcalibration time, the data were recorded on a tab-separatedfile with a recording interval of 10 sec.

Results and discussionAccuracyAlthough the literature value for the specific optical rota-tion of D-(–)-mandelate (aD

20) shows a value of 2152(13.1 mM in water 5 0.2 g/100 ml), the recorded initialvalue in the assay mixture was lower due to the presence ofother species (buffer components, salts and protein).Depending on the concentrations used, the value readbetween 280 and 290. However, since for kinetic meas-urements only the decrease of optical rotation with time{d[a]/dt} is required, this fact is without consequence, aslong as the absolute value is in a range which is highenough to ensure good accuracy of the polarimetricmeasurement.

Figure 2 Time-dependent decrease of optical rotation (A). The pseudolinear range used for the determination ofenzyme activity is zoomed (B).

Mandelate racemase assayed by polarimetry

Fig. 2 shows a typical plot of optical rotation versus time,starting with a high concentration of D-mandelate assubstrate (65.7 mM). The pseudolinearity of the initial ratewithin the first five minutes is excellent (r 5 0,996). Fromthe slope of the initial rate, the decline of optical rotationversus time was obtained as k 5 5.71. From this value, thespecific activity of the enzyme, corresponding to n[a]D

20

g–1 min–1 was obtained as follows:

Specific Activity 5

k [Da/min]

Enzyme Solution [mL] · Enzyme Concentration [g/mL]

SensitivityThe limits with respect to substrate concentration in theassay buffer were checked by measurement of the opticalrotation over a period of about five minutes in the absenceof enzyme.3 When the [a]D

20 was plotted versus time for alow (0.6 mM) and a high (3.3 mM) substrate concentration(data not shown), it appeared that the blank-value wasunstable below a concentration of ,3.5 mM, but higherconcentrations (up to 20 mM) gave a sufficiently stablebaseline with a relative mean deviation of ,4%. Thedependence of enzyme concentration (in the absence ofsubstrate) on the baseline-stability showed the opposite

3 The following concentrations (mM) were used: 0.2, 0.4, 0.6,0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.3, 6.6, 9.9, 13.1, 16.4, 19.7.

trend.4 Whereas the optical rotation showed significantfluctuation at higher enzyme amounts (caused by light-scattering emerging from remaining particles from thecrude enzyme preparation), low concentrations were foundshow no adverse effects. In general, aliquots of #100 mlenzyme stock solution (corresponding to a protein contentof #209 mg/ml), gave a stable baseline with a meanrelative deviation of ,3%.

Determination of the KM -value and enzymeinhibitionWhen the reaction rate {D[a]D

20 / time} was recorded withincreasing substrate concentrations aiming at the determi-nation of the KM-value, a non-Michaelis-Menten behaviourwas observed (insert in Fig. 3). The decrease of the velocityof the reaction with increasing substrate concentrationssuggests inhibition of the enzyme by the substrate, inparticular at concentrations beyond 13 mM. Interestingly,although the CD-assay measurements were performed atsimilar substrate concentrations (6 mM), inhibition by thesubstrate was not mentioned (Sharp et al., 1979). From ourdata it can be concluded that KM should be below 3.3 mM.However, due to technical reasons (i.e. the inaccuracy ofoptical rotation values at very low substrate concentrations)KM-measurements could not be extended below thisvalue.

4 The following enzyme aliquots were measured in HEPES assay-buffer (3ml): 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350ml, 400 ml.

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Figure 3 Dependence of enzyme activity on substrate concentration (A). Enzyme inhibition at elevated substrateconcentrations shown in B.

Figure 4 Dependence of enzyme activity on enzymecontent. Measurements were carried out under substratesaturation.

H. Stecher et al.

Enzyme saturationUnder substrate saturation (13.1 mM) enzyme activitylinearly depended on enzyme content. From the slope ofFig. 4 it can be concluded that no deviation from satura-tion is observed up to 400 mg protein/ml, which renders asufficiently wide operational window where linearity isconserved. This linearity levelled off at a protein concentra-tion of about 1mg/ml. From the inverted test – i.e.variation of substrate concentration at a fixed amount ofenzyme (400 ml aliquot of standard stock solution, 834 mgprotein/ml) – it was shown that linearity was not reachedbelow a substrate concentration of about 13 mM and thathigher concentrations of up to ,20 mM led into a linearrange.

Correlation of optical rotation with enantiomericcomposition and determination of unitsIn order to exclude non-linear effects (Kagan et al., 1996)which might impair the correlation between the recordedoptical rotation and the enantiomeric composition, the[a]D

20 of D-mandelate was recorded from racemate toenantiomeric purity at 10%-intervals (data not shown).Perfect correlation within experimental error ensured theabsence of non-linear effects.

As a consequence, the value of the decline in opticalrotation versus time {d[a]D

20/dt} could be correlated to thespecific enzyme activity expressed as the international unit(I.U. 5 mM/min·g). This is explained by of the followingexample: A 10% drop in the e.e. of the substrate isindicated by a decline of the optical rotation of 11.61.Since a decrease of the e.e. by 10% (during a given period

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of time) corresponds to an amount of 5% of substrate beingconverted into its mirror image enantiomer,5 the amount ofsubstrate converted versus time can be calculated from theknown substrate concentration.

SummaryAn accurate and convenient assay for mandelate racemase[EC 5.1.2.2] from Pseudomonas putida ATCC 12633 was

5 In order to reduce an e.e. of 100% (enantiomeric composition100:0) to 90% (95:5), 5% of the substrate molecules have to beinverted.

Mandelate racemase assayed by polarimetry

developed based on simple polarimetric measurement ofthe decline of the optical rotation versus time. In contrast tothe assay methods hitherto employed, this method can beperformed with standard laboratory equipment and is alsoapplicable to non-natural substrates provided that theyexhibit a sufficiently high absolute optical rotation. Inorder to obtain most accurate and reproducible results, thesubstrate and enzyme concentrations should be within arange of 13–20 mM and aliquots of lyophilized crudeenzyme preparation (Stecher et al., 1997) corresponding toa protein content of 100–400 mg/ml, respectively.

AcknowledgementsWe would like to express our cordial thanks to U. Felfer(Graz) for protein determinations. This project was per-formed within the Spezialforschungsbereich Biokatalyseand funding by the Fonds zur Forderung der wissenschaft-lichen Forschung (projects no. F115 and F107) is grate-fully acknowledged.

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Received: 6 January 1998Revisions requested: 19 January 1998

Revisions received: 30 January 1998Accepted: 31 January 1998

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