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SCIENCE

2005;

71

: 924–930

Blackwell Science, LtdOxford, UKFISFisheries Science0919-92682005 Blackwell Science Asia Pty Ltd714924930Original ArticleAlanine racemase activity in the microalgaT Yokoyama

et al.

*Corresponding author: Tel: 81-192-44-1915. Fax: 81-192-44-3930. Email: nagahisa@kitasato-u.ac.jp

Received 17 September 2004. Accepted 22 March 2005.

Alanine racemase activity in the microalga

Thalassiosira

sp.

T

akehiko

YOKOYAMA,

1

Y

umiko

TANAKA,

1

M

inoru

SATO,

2

N

obuhiro

KAN-NO,

1

T

oshiki

NAKANO,

2

T

oshiyasu

YAMAGUCHI

2

AND E

izoh

NAGAHISA

1

*

1

School of Fisheries Sciences, Kitasato University, Ofunato, Iwate 022-0101 and

2

Graduate School of Agriculture, Tohoku University, Sendai 981-8555, Japan

ABSTRACT:

In this paper, the authors report the detection of alanine racemase activity in themarine diatom

Thalassiosira

sp. Since the

Thalassiosira

sp. was cultured under germ-free conditions,it appeared that D-alanine was not derived from bacteria but was produced through catalysis by algalalanine racemase. The rate of conversion of L-alanine to D-alanine was approximately the same asthat for the reverse reaction, and the enzyme catalyzed the equilibration of the D- and L-forms. Thecrude enzyme preparation obtained from the cells at the stationary phase of the growth cycle had anoptimal pH of approximately 9.5. The Lineweaver–Burk analysis showed that the

K

m

for D- and L-ala-nine was 16.5 mM and 29.4 mM, respectively. It appears that the enzyme is highly specific for D- orL-alanine because it does not catalyze the racemization of other amino acids. In addition, after gel fil-tration, the enzyme did not require exogenous pyridoxal 5

′

-phosphate (PLP) for its activity, however,the effects of several chemicals suggest that the enzyme may be PLP-dependent. The enzyme ismore similar to that found in invertebrates when compared with that found in bacteria. This is the firstreport on the occurrence of alanine racemase activity in the microalga

Thalassiosira

sp.

KEY WORDS:

alanine racemase, D-alanine, D-amino acid, diatom, microalga,

Thalassiosira

.

INTRODUCTION

D-Amino acids are found in various animals

1

andplants

2,3

as well as in algae.

4–6

Recently, the authorspublished the first report on the distribution of freeD-aspartate and D-alanine in microalgae.

7

Amongseveral D-amino acids, D-alanine was the mostabundant in several types of macro- and microal-gae.

6,7

The biochemical and physiological func-tions of D-amino acids have received increasingattention in various research fields. It has beenhypothesized that in marine invertebrates, D-aspartate and D-alanine are involved in neuraltransmission

8,9

and osmoregulation,

10–12

respec-tively. Recently, it was reported that D-aspartateand aspartate racemase were involved in anaero-bic energy metabolism in

Scapharca broughtonii

.

13

However, the role of D-amino acids in plants,including algae, remains unclear. Ogawa

et al

. sug-gested that combined or free D-aspartate and D-alanine might participate in the development ofpea seedlings.

3

In a previous paper,

4

the authors

suggested that free D-aspartate might play animportant role during the early stages of growth ofthe macroalga

Hizikia fusiformis

.To clarify the physiological function of D-amino

acids in algae, the authors previously studied theirlevels in germ-free microalgae cultured under arti-ficial conditions in the laboratory.

7

It was foundthat the contents of free D-aspartate and D-alanineand the D/(D + L) ratios in microalgae increasedfrom the exponential growth phase to the station-ary phase and then decreased to the decline phase.These results support the idea that free D-aspar-tate and D-alanine participate in the growth ofmicroalgae. However, the origin of D-amino acidsin algae remains unclear.

Microalgae are fundamentally autotrophs,therefore, it can be surmised that D-amino acidsare produced by the microalgae themselves andare not taken up from exogenous sources. Thus,several enzyme activities involved in the biosyn-thesis of D-alanine were surveyed, which is gener-ally the most abundant D-amino acid in algae. Inthis paper, described for the first time, is the occur-rence of alanine racemase [EC 5.1.1.1.] activity inthe diatom

Thalassiosira

sp. along with some of itsproperties.

Alanine racemase activity in the microalga

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925

MATERIALS AND METHODS

Materials

Thalassiosira

sp. was sampled from Okirai Bay inIwate Prefecture, Japan, in May 1996. The diatomcells were isolated by the capillary method, andsingle cells were inoculated into each medium.Axenic strains were isolated by the method ofKotaki

et al

.

14

The strains were confirmed to begerm-free by performing bacterial growth tests onseveral types of agar media (Marine Broth 2216[DIFCO], Yeast Extract Glucose, sterility testmedium

15

) by incubation at 20

°

C for 1 month. The

Thalassiosira

sp. cells were cultured germ-free in50 mL of T1/seawater medium

16

at 15

°

C and illu-minated using cool white fluorescent lamps at60–70

μ

E with a 16 h light/8 h dark cycle. Each day,the cell number was visually determined using aThoma's hemocytometer under a microscope.

Preparation of crude enzyme

Thalassiosira

sp. was cultured in 5 L of T1 mediumat 15

°

C. When cell growth reached the stationaryphase (after

∼

10 days), the cells were collected bycentrifugation at 3000

×

g

for 10 min. The resultingcell pellet was rinsed with 5 volumes of fresh T1medium and centrifuged again. Using an ultra-sonic homogenizer (UD-200, Tomy Seiko, Tokyo,Japan), the pellet was then homogenized with 3volumes of 50 mM Tris-HCl buffer, pH 7.6, contain-ing 5 mM EDTA and 10 mM 2-mercaptoethanol.The homogenate was centrifuged at 10 000

×

g

for30 min at 0

°

C. The resulting precipitate was againextracted under the same conditions, and thisprocess was repeated three times. The resultingsupernatants were combined and fractionatedwith ammonium sulfate. The precipitate formedbetween 30 and 80% saturated ammonium sulfateand was dissolved in a minimal volume of 50 mMTris-HCl buffer, pH 7.6, and passed through a 0.22-

μ

m pore Millex-GS filter (Millipore, Billerica, USA).The filtrate was then passed through a ToyopearlHW-55F gel chromatography column(22

×

900 mm; Tosoh, Tokyo, Japan) and eluted at20 mL/h with 20 mM Tris-HCl, pH 7.6, containing0.2 M NaCl. Gel chromatography was used toexclude low-molecular-weight materials, such aspyridoxal 5

′

-phosphate (PLP), and to partiallypurify the protein. After gel chromatography, thealanine racemase activity was measured by the col-orimetric assay (see below), and the active frac-tions were combined and used as a crude enzymepreparation. The crude enzyme preparation wasstored at 0

°

C and used within 3 days.

Detection of alanine racemase activity by high-performance liquid chromatography

The alanine racemase activity was assessed in a0.4-mL reaction mixture containing 0.2 mL crudeenzyme preparation, 0.2 mM D- or L-alanine, and20 mM borate-NaOH buffer, pH 9.5. An enzymesolution heated at 100

°

C for 3 min was used as thenegative control. After incubating the mixture for0–8 h at 30

°

C, the reaction was stopped by adding0.4 mL of 1 M perchloric acid. The solution wascentrifuged at 3000

×

g

for 10 min, and the resultingsupernatant was neutralized with potassiumhydroxide and centrifuged again at 3000

×

g

for10 min. The supernatant was passed through a0.20-

μ

m Millex-GN filter (Millipore) and analyzedby high-performance liquid chromatography(HPLC) by using the method of Nimura andKinoshita

17

with some alterations.

6

First, thesample solution was converted to fluorescentderivatives by using

o

-phthalaldehyde (OPA),

N

-acetyl-L-cysteine, and sodium borate by themethod of Aswad.

18

The fluorescent derivativeswere immediately injected into a reversed-phasecolumn (TSK-ODS 80T, 4.6

×

250 mm, Tosoh). TheHPLC system (Jasco LC-800, Tokyo, Japan)consisted of an 880-PU intelligent HPLC pump, aFP-1520 intelligent fluorescence detector, and anintegrator. The following solvents were used: Sol-vent A, 50 mM sodium acetate buffer (pH 5.6); Sol-vent B, methanol. The fluorescent derivatives wereeluted at 40

°

C over a 30-min period with a gradientof 20 to 28% solvent B in solvent A delivered at1.0 mL/min. The excitation and emission wave-lengths for the detection of the fluorescent deriva-tives were 348 and 450 nm, respectively. A total of 1enzyme unit was defined as the amount of D- or L-alanine (

μ

mol) produced per minute per milligramprotein of crude enzyme preparation at 30

°

C.

Colorimetric assay of enzyme activity

The alanine racemase activity was measured by thecolorimetric method of Shibata

et al

.

19

with somealterations. In brief, the reaction was performed ina 0.5-mL mixture containing 0.1 mL crude enzymepreparation, 100 mM L-alanine, 100 mM Tris-HCl,pH 9.0, 0.2 U of porcine kidney D-amino acid oxi-dase (Biozyme Laboratories, Blaenavon, UK), and100 U of catalase (Sigma, St. Louis, USA). The reac-tion was started by the addition of the enzymesolution at 37

°

C. After incubation for 30 min, thereaction was stopped by adding 0.2 mL of 25%(w/v) trichloroacetic acid. After centrifugation at1600

×

g

for 15 min, 0.5 mL of the supernatant wasmixed with 0.1 mL of 0.1% (w/v) 2,4-dinitrophenyl-

926

FISHERIES SCIENCE

T Yokoyama

et al.

hydrazine in 2 M HCl and incubated at 40

°

C for15 min. The solution was then mixed with 0.4 mLof 3.75 M NaOH. The absorbance of the 2,4-dinitrophenylhydrazone derivative was measuredat 445 nm. A total of 1 enzyme unit was defined asthe amount of D-alanine (

μ

mol) produced perminute per milligram protein of crude enzymepreparation at 37

°

C.

Effects of various chemicals on alanine racemase activity

It is known that alanine racemases require PLP as acofactor for the catalysis and that PLP-dependentenzymes, including microbial alanine racemases,are inhibited by aminooxyacetic acid, hydroxy-lamine, phenylhydrazine and sodium borohy-dride. These chemicals were used for testing theeffects of various chemicals on alanine racemaseactivity. The reaction mixture contained 100 mML-alanine, 100 mM Tris-HCl, pH 9.0, 0.2 U of D-amino acid oxidase, 100 U of catalase, 0.1 mL crudeenzyme preparation, and the indicated chemicalsin a final volume of 0.5 mL; it was incubated at37

°

C for 30 min. The alanine racemase activity wasdetermined by the colorimetric assay describedabove. The activity was expressed as a percentageof the control activity.

RESULTS AND DISCUSSION

Demonstration of alanine racemase activity in a crude enzyme preparation from

Thalassiosira

sp.

The alanine racemase activity was directly assayedin the crude enzyme preparation (Table 1) from the

Thalassiosira

sp. cells by labeling D- and L-alaninefluorescently and then separating the fluorescentderivatives by reversed-phase HPLC. This methodpermits direct comparison of the amount ofproducts before and after the alanine racemasereaction. Figure 1 shows the change in thechromatograms of the D- and L-alanine derivativesat 0 and 8 h after incubation. When L-alanine wasused as the substrate, the peak of the D-alanine

derivative appeared at 8 h after incubation(Fig. 1a), whereas no peak of the D-alanine deriva-tive was observed at 8 h in the negative control(data not shown for negative control). These resultsdemonstrate that the D-alanine is produced fromL-alanine by alanine racemase. Similarly, it wasfound that L-alanine was produced when D-alanine was the substrate (Fig. 1b). These dataclearly demonstrate the occurrence of alanineracemase activity in the microalga

Thalassiosira

sp.Although many enzymes are unstable at room

temperature, the

Thalassiosira

sp. alanine race-mase maintained its activity for at least 8 h at 30

°

C(Fig. 2). After an 8-h incubation, the enzyme activ-ity was 89% of the initial value. This shows that thealanine racemase from

Thalassiosira

sp. is rela-tively stable at 30

°

C.The authors tried to detect the activity of D-

alanine aminotransferase [EC 2.6.1.21.], which isanother enzyme that can produce D-alanine. D-Alanine aminotransferase has been known as theenzyme that converts pyruvate and D-glutamate toD-alanine and 2-oxoglutarate. Using HPLC meth-ods, the production of D-alanine by using super-natant of cell homogenate, 100 mM pyruvate, and100 mM D-glutamate as a substrate was studied,but the activity could not be detected. The activityof D-amino acid oxidase [EC 1.4.3.3.], which isinvolved in decomposition of neutral D-aminoacids, was also attempted to be detected. UsingHPLC methods, the decomposition of D-alaninewas studied by using supernatant of the cell homo-genate, 100 mM D-alanine as a substrate, and20

μ

M flavin adenine dinucleotide, but the activitycould not be detected.

Optimal pH of alanine racemase from

Thalassiosira

sp.

The pH dependence of the alanine racemase activ-ity in the

Thalassiosira

sp. is shown in Fig. 3.Maximal activity was found at approximatelypH 9.5. At physiological pH (7.0 and 7.5), the activ-ity was 45 to 80% of the activity at pH 9.5. This opti-mal alkaline pH is similar to that reported for thealanine racemases from the bivalve mollusk

Cor-

Table 1

Partial purification of alanine racemase from

Thalassiosira

sp.

StepTotal activities

(units)Total protein

(mg)Specific activities

(units/mg)Purification

foldYield(%)

Supernatant of homogenate 0.93 177 0.0053 1 10030%-80% (NH

4

)

2

SO

4

0.84 23 0.04 7 90Toyopearl HW-55F 0.74 7 0.11 20 80

Alanine racemase activity in the microalga

FISHERIES SCIENCE

927

bicula japonica

(pH 9.5)

20

and from crustaceans(pH 8.5).

12

Maximum velocity and substrate affinity of alanine racemase from

Thalassiosira

sp.

The

K

m

and

V

max

for alanine racemase from the

Thalassiosira

sp. were determined using the HPLC

method. The

K

m

and

V

max

values of alanine race-mase from the

Thalassiosira

sp. were calculatedfrom a Lineweaver–Burk plot (Fig. 4). The apparent

K

m

for D-alanine and L-alanine was 16.5 mM and

Fig. 1

Alanine racemase activityin a crude enzyme preparationfrom the microalga

Thalassiosira

sp. Racemase activity wasassessed in a crude enzyme prep-aration after 0 and 8 h at 30

°

C byusing 0.2 mM (a) L-alanine or(b) D-alanine as the substrate.The alanine enantiomers werederivatized with

o

-phthalalde-hyde, and the fluorescent deri-vatives were separated byreversed-phase high-perfor-mance liquid chromatography.The small peak number 2 at 0 h(b) was the unknown peak thatwas possibly derived from thederivatization reagent (sodiumborate). 1, D-alanine derivatives;2, L-alanine derivatives.

Fig. 2

Stability of alanine racemase from

Thalassiosira

sp. at 30

°

C. The crude enzyme preparation was preincu-bated at 30

°

C for 0–8 h. Using the colorimetric enzymeassay, a 0.1-mL portion was used to determine alanineracemase activity.

Fig. 3

The effect of pH on the activity of alanine race-mase from

Thalassiosira sp. The activity of the crudeenzyme preparation was determined by the colorimetricassay, in the presence of one of the following buffers: �,50 mM phosphate-NaOH (pH 6.0–8.0); �, 50 mM Tris-HCl (pH 7.5–9.0); �, 50 mM borate-NaOH (pH 8.5–10.0);�, 50 mM sodium hydrogen carbonate-NaOH (pH 9.5–11.0); or �, 50 mM sodium phosphate-NaOH (pH 11.0–12.0). The reactions were carried out for 30 min at 37°C.The activity is expressed as a percentage of the maximalactivity.

928 FISHERIES SCIENCE T Yokoyama et al.

29.4 mM, and the Vmax was 0.08 μmol/min per mgprotein and 0.16 μmol/min per mg protein, respec-tively. This Km value is close to that reported for thebivalve mollusk C. japonica (9.2 mM and 22.6 mM,respectively)20 compared with crustaceans. InC. japonica, the concentration of D- and L-alaninein the tissues varies from 5 to 100 mM dependingon the external salinity,10 and based on this Km

value, alanine racemase appears to function in theregulation of the organism's osmolyte concentra-tion. Currently, it is unclear whether the enzymealso functions to control the osmolyte concentra-tion in Thalassiosira sp.

Substrate specificity of Thalassiosira sp. alanine racemase

The HPLC condition was modified as described ina previous report.7 The crude enzyme preparationfrom the Thalassiosira sp. did not catalyze the race-mization of L-arginine, L-aspartate, L-glutamine,L-isoleucine, L-leucine, L-serine, L-threonine, L-tyrosine, L-valine, or their optical isomers (data notshown). The authors could not examine the race-mization of cysteine, histidine, lysine, methionine,phenylalanine, proline and tryptophan becauseenantiomeric separation of their derivatives was

not possible by the analytic method. These resultsindicate that alanine racemase from Thalassiosirasp. is highly specific for catalyzing the equilibriumbetween D- and L-alanine.

Figure 5 shows the time courses of the alanineracemase reactions. The production of D- and L-alanine was detected by the reversed-phase HPLCmethod. When L-alanine was used as the substrate,the peak of D-alanine appeared after 2 h of incuba-tion. The content of D-alanine increased depend-ing on the incubation time and was accompaniedby a parallel decrease in the L-alanine content. Theracemase activity was almost the same in both cat-alytic directions (i.e. L to D vs D to L; Fig. 5a,b).Racemization reached equilibrium 8 h afterincubation.

Effects of various chemicals on alanine racemase from Thalassiosira sp.

Table 2 shows the effects of various chemicals onthe alanine racemase activity of the crude enzymepreparation from the Thalassiosira sp. It was foundthat aminooxyacetic acid (0.1 mM), hydroxylamine(1 mM), phenylhydrazine (1 mM), and sodiumborohydride (1 mM) strongly inhibited the enzymeactivity (reduced to 98, 92, 98, and 96% of the con-trol, respectively). These results are in agreementwith the characteristics of alanine racemase fromother eukaryotes.19,20 PLP is known to be a cofactorof bacterial alanine racemase and is known to con-tribute to the stability of its activity. However, the

Fig. 4 Kinetic analysis of alanine racemase activity incrude enzyme preparation from Thalassiosira sp. Theactivity of the crude enzyme preparation was deter-mined using the high-performance liquid chromatogra-phy (HPLC) method. The assay mixture contained20 mM borate-NaOH buffer, pH 9.5, different concentra-tions (5–50 mM) of (�) D-alanine or (�) L-alanine,0.086 mg of protein and was incubated at 30°C for 2 h. D-Alanine or L-alanine produced was determined by HPLCanalysis. The Lineweaver–Burk analysis was used todetermine the Km and Vmax. The double-reciprocal(1/velocity vs 1/[alanine]) replot is shown.

Fig. 5 Time courses of Thalassiosira sp. alanine race-mase reactions. Catalysis in both directions (L to D and Dto L) by alanine racemase from Thalassiosira sp. wasassessed by determining the production of the productand disappearance of the substrate. The reactions werecarried out at 30°C and pH 9.5 by using (a) D-alanine or(b) L-alanine as the substrate. The levels of (�) D-alanineand (�) L-alanine were determined using o-phthalalde-hyde derivatization, followed by enantiomeric separa-tion on reversed-phase high-performance liquidchromatography.

Alanine racemase activity in the microalga FISHERIES SCIENCE 929

alanine racemase from the Thalassiosira sp. didnot require exogenous PLP (Table 2). Despite this,the Thalassiosira sp. enzyme could still be PLP-dependent because this enzyme was inhibited bythe chemicals that inactivate PLP-dependentenzymes.20 These results suggest that PLP may bindmore tightly to the Thalassiosira sp. enzyme thanto the bacterial enzymes. Thus, alanine racemasefrom Thalassiosira sp. resembles those from inver-tebrates, such as the bivalve mollusk C. japonica20

and crustaceans,19 rather than the bacterialenzymes.

In bacteria, alanine racemase has been known tobe involved in the formation of D-alanine, which isincorporated into the peptidoglycan layer of thecell wall. The alanine racemase in eukaryotes hasbeen chiefly studied in bivalve mollusks and crus-taceans and has recently been purified from theseorganisms.19–22 However, it is not currently knownwhether alanine racemase occurs in algae.

In the present study, the occurrence of alanineracemase activity in the marine diatom Thalassio-sira sp. was demonstrated. Since the Thalassiosirasp. was cultured under germ-free conditions, D-alanine in the alga does not appear to be derivedfrom bacteria, but rather produced by the alga withalanine racemase. As D-alanine has been found inseveral other microalgal species, alanine racemasecould be widely distributed in microalgae. SinceThalassiosira sp. has the ability to biosynthesize D-alanine, this amino acid is likely to play a positivephysiological role, however, the determination ofthe physiological function of D-alanine and alanineracemase in algae requires further investigation.

ACKNOWLEDGMENTS

This work was supported in part by a Grant-in-Aidfor Scientific Research from the Ministry of Educa-tion, Culture, Sports, Science and Technology ofJapan, and a Kitasato University Research Grant forYoung Researchers.

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Table 2 Effect of various chemicals on the activity ofalanine racemase from Thalassiosira sp.

ChemicalConcentration

(mM)Relative activity

(%)

None 100Aminooxyacetic acid 0.01 14

0.1 21 0

Hydroxylamine 0.01 880.1 461 8

Phenylhydrazine 1 210 0

Sodium borohydride 1 410 0

PLP 0.02 1050.025 1010.05 970.1 101

930 FISHERIES SCIENCE T Yokoyama et al.

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19. Shibata K, Shirasuna K, Motegi K, Kera Y, Abe H, Yamada R.Purification and properties of alanine racemase from cray-fish Procambarus clarkii. Comp. Biochem. Physiol. 2000;126B: 599–608.

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