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Journal of Molecular Catalysis B: Enzymatic 59 (2009) 237–242

Contents lists available at ScienceDirect

Journal of Molecular Catalysis B: Enzymatic

journa l homepage: www.e lsev ier .com/ locate /molcatb

ene cloning, purification, and characterization of �-methylserine aldolase fromosea sp. AJ110407 and its applicability for the enzymatic synthesis of-methyl-l-serine and �-ethyl-l-serine

iroyuki Nozaki ∗, Shinji Kuroda, Kunihiko Watanabe, Kenzo Yokozekiminoscience Laboratories, Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan

r t i c l e i n f o

rticle history:eceived 7 April 2008eceived in revised form 7 June 2008ccepted 7 June 2008vailable online 17 June 2008

a b s t r a c t

The gene encoding �-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysisrevealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0%similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene wasexpressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molec-ular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer.

′

eywords:-Methylserine aldolase-Methyl-l-serine-Ethyl-l-serineoseayridoxal 5′-phosphate

The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5 -phosphate per mole of the subunit. The Vmax value was 1.40 �mol min−1 mg−1, and the Km value was1.5 mM for the reaction wherein formaldehyde was released from �-methyl-l-serine. This enzyme couldalso catalyze the reverse reaction, i.e., the synthesis of �-methyl-l-serine from l-alanine and formalde-hyde. This activity was inhibited in the excess of formaldehyde; however, �-methyl-l-serine was efficientlyproduced from l-alanine in the presence of formaldehyde. This method was also applicable for producing

amin

mpt�

2

2

�-ethyl-l-serine from l-2-

. Introduction

�-Methylserine hydroxymethyltransferase (EC 2.1.2.3) catalyzeshe transfer of the hydroxymethyl group between �-methyl-l-erine and d-alanine via tetrahydrofolate (THF) [1–6]. In contrast,-methylserine aldolase, which was first characterized by ourroup [7], can catalyze the interconversion between �-methyl-l-erine and l-alanine in the absence of THF (Fig. 1). THF is relativelynstable and expensive and is therefore rather impractical for

ndustrial use.In recent years, �-alkyl-�-amino acids and peptides containing

hese residues have been attracting increasing attention for phar-

aceutical use. The stereospecific transfer of the hydroxymethyl

roup to an �-amino acid seems to be one of the most effectiveethods for the production of �-alkyl-l-serine.

Abbreviations: PLP, pyridoxal 5′-phosphate; EDTA, N,N,N′ ,N′-ethylenediamine-etraacetic acid; THF, tetrahydrofolate or tetrahydropteroylglutamate; IP-G, isopropyl-�-d-thiogalactopyranoside.∗ Corresponding author. Present address: Fine Chemical & Pharmaceutical Indus-

rialization Center, Ajinomoto Co. Inc., 1730 Hinaga-cho, Yokkaichi 515-0885, Japan.el.: +81 593 46 0121; fax: +81 593 46 0127.

E-mail address: hiroyuki nozaki@ajinomoto.com (H. Nozaki).

f(hofIafcH2(w

381-1177/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.molcatb.2008.06.007

obutyric acid.© 2008 Elsevier B.V. All rights reserved.

This article describes the cloning of the gene encoding �-ethylserine aldolase from Bosea sp. AJ110407 and the subsequent

urification and characterization of the recombinant enzyme. Fur-her, we report the enzymatic synthesis of �-methyl-l-serine and-ethyl-l-serine.

. Materials and methods

.1. Materials

�-Methyl-l-serine and �-methyl-d-serine were purchasedrom Acros Organics Inc. (Geel, Belgium). �-Methyl-dl-serine,S)-2-amino-1-propanol, (R)-2-amino-1-propanol, (S)-�-ydroxymethyltyrosine, and (R)-�-hydroxymethyltyrosine werebtained from Sigma Chemical Co. Ltd. (MO, USA). PLP andormaldehyde solution were purchased from Nacalai Tesquenc. (Kyoto, Japan) and the l and d forms of 2-aminobutyriccid, from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Aormaldehyde test kit was obtained from Wako Pure Chemi-

al Industries Ltd. (Osaka, Japan). HiLoad 26/10 Q sepharose,iLoad 16/10 phenyl sepharose, and HiLoad 16/60 Superdex00 pg columns and gel filtration low-molecular-weightLMW) and high-molecular-weight (HMW) calibration kitsere obtained from Amersham Bioscience Corp. (NJ, USA). The

238 H. Nozaki et al. / Journal of Molecular Cataly

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ig. 1. The interconversion between l-alanine and �-methyl-l-serine with theydroxymethyl transfer by �-methylserine aldolase.

utrient broth used was obtained from BD Biosciences (CA,SA).

.2. Microorganisms and culture conditions

The Bosea sp. strain AJ110407, which we previously describeds an �-methylserine aldolase producer [7], was isolated from soil.his strain was maintained on the nutrient broth plate.

.3. Enzyme assay

The activity of �-methylserine aldolase was measured by the fol-owing protocol. Its activity in releasing formaldehyde was assayedn a reaction mixture (total volume, 0.1 ml) containing 50 mMotassium phosphate buffer (pH 7.4), 10 mM �-methyl-l-serineupplemented with 0.1 mM PLP, and an appropriate concentrationf the enzyme. Following incubation at 30 ◦C for 10 min, the reactionas arrested by the addition of an alkaline solution (5N NaOH), and

he formaldehyde released was quantified using the formaldehydeest kit according to the instructions provided in the kit manual.ne unit of enzyme activity was defined as the amount of enzyme

equired to catalyze the release of one micromole of the product perinute under the abovementioned conditions. The reaction mix-

ure used for the assay of the �-methyl-l-serine synthesis activityf the enzyme contained 100 mM potassium phosphate buffer (pH.4), 0.1 mM PLP, 5–100 mM l-alanine, and 1–50 mM formaldehyden a final volume of 0.2 ml. The mixture was incubated at 30 ◦C formin. The reaction was arrested by thoroughly mixing the solu-

ion with 0.2 ml ice-cold CuSO4 at a concentration of 20 mM. The-methyl-l-serine released was then analyzed by performing high-erformance liquid chromatography (HPLC) as described below.

.4. Cloning of the gene encoding ˛-methylserine aldolase

A 1.3-kb DNA fragment was amplified using pSKA 04098 [7], har-oring the gene encoding �-methylserine aldolase from Ralstoniap. AJ11040, as the template and specifically synthesized oligonu-leotide primers (CGG AAT TCG AGA GGA ACT GAG CAT GTT GAAGC-3′ and 5′-AAC TGC AGT TAG CGC AGG AAA TGC AGC TTG TTG-′). The genomic DNA extracted from the Bosea sp. AJ110407 wasigested with PstI; the digested fragments with a length of 5–6 kbere collected and ligated to the PstI recognition site of pUC118.

. coli cells of the JM109 strain were transformed with this plas-id, and the library of clones obtained was screened by colony

ybridization, using the probe described above. The positive clonesarrying the pUCB2-R1 plasmid were used for further analysis.

To construct the expression plasmid, the gene encoding �-ethylserine aldolase was amplified with pUCB2-R1 DNA as the

emplate by using the primers (5′-CCG AAT TCG GAG GAT GGG GCAGA CGG C-3′) and (5′-AAC TGC AGT CAG CGC ATG AAA TGC AGC-3′). The amplified product was then digested with EcoRI/PstI and

atpi1

sis B: Enzymatic 59 (2009) 237–242

nserted into pUC18 that had been digested with the same enzymesn order to create the pUCBHMT. The nucleotide sequence of theolymerase chain reaction (PCR) product was determined by usingDNA sequencer (ABI-3100, Applied Biosystems, CA, USA) to con-rm that no error had occurred during amplification

.5. Purification of ˛-methylserine aldolase

E. coli cells of the JM109 strain carrying the pUCBHMTJM109/pUCBHMT cells) were precultured in 100 ml Luria-BertaniLB) medium containing 100 �g/ml ampicillin Na at 30 ◦C for 24 h.ollowing this, the cells were inoculated into 1-l LB mediumontaining 100 �g/ml ampicillin Na and 0.1 mM isopropyl-1-thio--d-galactoside (IPTG). The cells were grown at 37 ◦C for 16 h,arvested by centrifugation (8000 × g for 10 min), and washedwice with 25 mM Tris–HCl buffer (pH 7.4) containing 20 �M PLPnd 1 mM EDTA (buffer A). The cell suspension in buffer A was soni-ated with an Insonator 201 instrument (Kubota, Tokyo, Japan) andentrifuged at 12,000 × g for 20 min. The supernatant was ultracen-rifuged at 200,000 × g for 30 min to remove the insoluble fraction,nd the resultant supernatant was used as the cell-free extract. Allhe procedures were carried out at 4 ◦C or on ice.

The cell-free extract was applied to a Hi-Load 26/10 Q sepharoseolumn that had been equilibrated with buffer A. The enzyme wasluted with NaCl in a linear concentration gradient of 0–1 M.

The active fractions were collected and dialyzed against 25 mMotassium phosphate buffer (pH 7.0) containing 20 �M PLP andmM EDTA (buffer B). The dialyzed solution was mixed with anqual volume of buffer B supplemented with 2 M ammonium sul-ate, applied to a Hiload 16/10 phenyl sepharose column that hadeen equilibrated with buffer B containing 1 M ammonium sul-ate, and eluted with ammonium sulfate in a linear concentrationradient of 1–0 M.

The purified enzyme was dialyzed against buffer B and stored at◦C for approximately 1 month. During storage for a longer dura-

ion (3 months), an equal volume of glycerol was added, and thenzyme solution was preserved at −20 ◦C.

.6. Protein analysis

The protein concentrations were determined by the Bradfordethod [8], using bovine serum albumin as the standard. Sodium

odecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)as performed, wherein the samples were run on a 10–20% poly-

crylamide gel (Daiichi Pure Chemicals, Tokyo, Japan) along with arecision protein marker (Bio-Rad Laboratories, CA, USA). The rela-ive molecular mass of the native enzyme was determined by usingHiLoad 16/60 Superdex 200 pg column and gel filtration LMW andMW calibration kits.

.7. PLP content

After the purified enzyme had been extensively dialyzed against5 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTAnd 20 �M PLP, the PLP concentrations inside and outside the dial-sis bag were determined using phenylhydrazine [9].

.8. Enzymatic synthesis of ˛-methyl-l-serine

JM109/pUCBHMT cells were cultured in the manner described

bove. The cells were collected from 200 ml culture medium by cen-rifugation (8000 × g for 10 min) and washed twice with 100 mMotassium phosphate buffer (pH 7.4). They were then suspended

n 20 ml of the same buffer. The reaction mixture (total volume,00 ml) contained 100 mM potassium phosphate buffer (pH 7.4),

H. Nozaki et al. / Journal of Molecular Catalysis B: Enzymatic 59 (2009) 237–242 239

F s indics dolasey hydroh

0tT

2

etr(tu

TctKtf

2

ig. 2. Primary structure alignment. The presumed PLP-binding lysine-residue ip. AJ110407 (GenBank accession no. AB426472); Ral MSALD, �-methylserine almethyltransferase from S. pomeroyi (AAV96754); Par MSALD, �-methylserineydroxymethyltransferase from E. coli (AAA23912).

.1 mM PLP, 150 mM l-alanine, and the cell suspension. To this solu-ion, 50 ml of 300 mM formaldehyde was added at a rate of 2 ml/h.he reaction was performed at 30 ◦C with moderate stirring.

.9. Enzymatic synthesis of ˛-ethyl-l-serine and its isolation

The reaction was performed in the manner described above,xcept that l-2-aminobutyric acid was used as the substrate in

his case. The reaction was performed at 30 ◦C for 25 h, with stir-ing. Following this, the cells were separated by centrifugation8000 × g for 10 min), and 50 ml of the supernatant was appliedo a Mega Bond Elut SCX column (Varian Inc., CA, USA). The col-mn was washed with 100 ml water, and then eluted with 0.5N HCl.

noC

ated with asterisk. Bosea MSALD, �-methylserine aldolase isolated from Boseafrom Ralstonia sp. AJ110405 (AB426471); S. pomeroyi, putative serine hydrox-

xymethyltransferase from Paracoccus sp. AJ110402 (AB426468); E. coli, serine

he eluted fractions containing �-ethyl-l-serine were collected andoncentrated under reduced pressure. The resultant solution wasreated with diethylaminoethyl (DEAE) cellulose (Whatman JapanK, Tokyo, Japan) to adjust the pH to 4.0–6.0. Following evaporation,

he crude crystals of �-ethyl-l-serine obtained were recrystallizedrom aqueous acetone.

.10. HPLC analysis

�-Methyl-l-serine, �-methyl-d-serine, the l and d forms of ala-ine, and the l and d forms of 2-aminobutyric acid were analyzedn a Sumichiral OA-6100 HPLC column (4.6 mm × 150 mm; Sumikahemical Analysis Service Ltd., Osaka, Japan). The analysis was per-

2 Catalysis B: Enzymatic 59 (2009) 237–242

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40 H. Nozaki et al. / Journal of Molecular

ormed at 30 ◦C and at a wavelength of 215 nm, using 0.5 mM CuSO4s the mobile phase.

. Results and discussion

.1. Cloning and expression of the gene encoding ˛-methylserineldolase and purification of the recombinant enzyme

We obtained pUCB2-R1 from the gene library by performingcreening with the DNA fragment corresponding to �-methylserineldolase isolated from a Ralstonia sp. Sequence analysis performedor the 5173 bp insert in pUCB2-R1 revealed the presence of threepen reading frames (GenBank accession no. AB426472). One ofhese frames was found to encompass 1320 bp, corresponding to40 amino acids. Further, the deduced amino acid sequence was5.0% similar to the sequence of �-methylserine aldolase isolatedrom the Ralstonia sp. AJ110405 [7], 62.9% to that of the putativeerine hydroxymethyltransferase from Silicibacter pomeroyi [10],6.4% to that of �-methylserine hydroxymethyltransferase fromaracoccus sp. AJ110402 [6], and 28.9% to that of serine hydrox-methyltransferase from E. coli (Fig. 2).

The enzyme activity of formaldehyde releasing in the cell-freextract obtained from the JM109/pUCBHMT cell was 0.28 U mg−1.he typical procedure by which the enzyme was purified from theell-free extract is summarized in Table 1. �-Methylserine aldolaserom Bosea sp. AJ110407 was purified 4.7-fold by performing col-mn chromatography. SDS–PAGE revealed that the purified enzymeas homogeneous in its composition (Fig. 3).

The apparent native molecular mass of the �-methylserineldolase recombinant was approximately 78 kDa, as determined byel filtration.

.2. Substrate specificity and stability of the enzyme

The Vmax value was 1.40 U mg−1 and the Km value was 1.5 mMor the reaction wherein formaldehyde was released from �-

ethyl-l-serine. Further, HPLC analysis revealed that l-alanineas formed as a product of the enzymatic reaction on �-ethyl-l-serine. No enzyme activity in releasing formaldehydeas noted in the reactions with of �-methyl-d-serine, l-serine,-serine, (S)-2-amino-1-propanol, (R)-2-amino-1-propanol, (S)--hydroxymethyltyrosine, (R)-�-hydroxymethyltyrosine, �-iso-utyl-dl-serine, �-iso-propyl-dl-serine, and �-benzyl-dl-serine.ompared to this enzyme purified from the Ralstonia sp. [7], theffinity of the enzyme investigated in the present study toward �-ethyl-l-serine in a formaldehyde-releasing reaction was higher;

owever, the specific activities of the two enzymes were almostdentical (Vmax, 1.48 U mg−1; Km, 11.9 mM).

The maximum enzyme activity was observed at 50 ◦C and pH.0 in releasing formaldehyde when �-methyl-l-serine was useds the substrate. The enzyme was stable in the storage buffer wheneated for 30 min up to a temperature of 40 ◦C.

The �-methyl-l-serine synthesis activity of the enzyme coulde detected using l-alanine and formaldehyde as the substrates,ut no activity was noted when d-alanine was used. The specificctivity was 61.6 U mg−1 of protein when 100 mM l-alanine and0 mM formaldehyde were used as the substrates; however, thisctivity was found to be inhibited in the presence of formaldehydet a concentration of more than 10 mM (Fig. 4). Such inhibition has

een reported previously in the case of the enzyme isolated fromhe Ralstonia sp. [7].

Serine hydroxymethyltransferase can catalyze the intercon-esion between glycine and l-serine via tetrahydrofolate, andhe folate-independent hydroxymethyl transfer activity from

fiawo

ig. 3. SDS–PAGE of purified �-methylserine aldolase. Lane 1, standard proteinarker; lane 2, purified �-methylserine aldolase.

-serine was demonstrated by the mutated serine hydrox-methyltransferase [11]. And, interconversion between d-alaninend �-methyl-l-serine was catalyzed by �-methylserine hydrox-methyltransferase [1–6]. �-Methylserine aldolase from Bosea sp.J110407 can neither act on l-serine nor did it catalyze the hydrox-methyl transfer reaction with d-alanine, and it can catalyze theeaction in the absence of tetrahydrofolate, indicating that thisnzyme is clearly different from the folate-dependent enzymes. Inact, we have also examined the hydroxymethyl transferase activ-

ty from �-methyl-l-serine to THF in the purified �-methylserineldolase from Bosea sp. AJ110407, but the THF-dependent activityas not be discriminated because of the spontaneous formationf 5,10-methylenetetrahydrofolate from formaldehyde and THF in

H. Nozaki et al. / Journal of Molecular Catalysis B: Enzymatic 59 (2009) 237–242 241

Table 1Purification of the recombinant �-methylserine aldolase

Fraction Total protein (mg) Specific activity (U mg−1) Total activity (U) Yield (%)

Cell-free extract 625 0.277 173 100QP

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itformed after the 25-h reaction revealed one major peak, which waspresumed to have been yielded by �-ethyl-l-serine (HPLC anal-ysis revealed 14.3 mmol of the product to be �-methyl-l-serine),and the presence of 0.10 mmol residual l-2-aminobutyric acid and

sepharose 116 1.29henyl sepharose 89.0 1.30

he activity of �-methylserine aldolase was measured with �-methyl-l-serine as th

he enzyme assay [12,13]. By amino acid sequence alignment, itppears to be different in affinity to THF between �-methylserineldolase and serine hydroxymethyltransferase, since Glu57, Leu127,nd Asn347 of the residues relating THF-binding in serine hydrox-methyltransferase from E. coli [14] are corresponded to Leu80,al150, Ile371, respectively (Fig. 2). Further detailed functionalnalyses of the three-dimensional structure will help us to resolvehe reaction mechanism.

.3. Inihibitors

Treatment with the sulfhydryl reagent N-ethylmaleimide at aoncentration of 1 mM reduced the enzyme activity in releasingormaldehyde to 76%. The enzyme activity was also slightly affectedy 1 mM iodoacetate amide (activity reduction to 93%) and 1 mModoacetic acid (85%).

Divalent cations such as those released by FeSO4, MgSO4, oriCl2 at a concentration of 1 mM did not affect the activity of �-ethylserine aldolase. However, the enzyme activity was inhibited

y CaCl2 (activity reduction to 77%), CoCl2 (85%), CuCl2 (18%), ornCl2 (71%), each at a concentration of 1 mM.

.4. PLP content and absorption spectrum

The PLP content of the enzyme was calculated as.8–0.9 mol/mol of the enzyme subunit. The enzyme exhib-

ted absorption maxima at wavelengths of 280 and 429 nm at pH.4, with an A280/A429 value of approximately 4.7 (Fig. 5). Treatmentith 1 mM hydroxylamine resulted in a loss of the enzyme activity,

ith the disappearance of the peak at 429 nm. Following redialysis

gainst the buffer in the absence of hydroxylamine, 65.1% of thenzyme activity was recovered. The reduction of the enzyme byodium borohydride [15] resulted in a loss of the enzyme activity,ith the disappearance of the absorption maximum at 429 nm

ig. 4. Inhibition of the �-methyl-l-serine synthesis activity of the enzyme byormaldehyde. The enzyme activity was assayed using 1–20 mM formaldehyde and(©), 10 (�), 20 (�), 50 (♦), and 100 mM (×) l-alanine, as described in Section 2.

FseEc

149 86.1116 67.0

strate, as described in Section 2.

nd an increase in the absorbance at 340 nm. The addition of PLPid not restore this lost activity.

This result was consistent with previous reports on otherLP-related enzymes including serine hydroxymethyltransferase16,17]. This suggests that sodium borohydride reduces theldimine linkage in the internal Schiff base. Based on the observedrimary structure alignment, we consider that Lys258 is probablyesponsible for the formation of the internal Schiff base (Fig. 2).

.5. Enzymatic synthesis of ˛-methyl-l-serine and-ethyl-l-serine

�-Methyl-l-serine was produced from l-alanine by theM109/pUCBHMT whole cells with formaldehyde feeding. When5.2 mmol of formaldehyde was fed, 14.1 mmol �-methyl-l-serineas obtained from 14.9 mmol l-alanine within 25 h. In the solu-

ion, 0.05 mmol residual l-alanine and 0.31 mmol d-alanine wereetected, but �-methyl-d-serine was absent (Fig. 6A). The effectiveroduction of �-methyl-l-serine was achieved in the absence ofHF, although the conversion of �-methyl-l-serine from d-alaniney E. coli cells expressed the gene encoding �-methylserine hydrox-methyltransferase was dependent on THF, and the conversionield from d-alanine was 64% [6].

Subsequently, we used l-2-aminobutyric acid (14.9 mmol)nstead of l-alanine as the substrate, and the reaction was allowedo progress in the manner described above. HPLC analysis per-

ig. 5. Absorption spectrum of purified �-methylserine aldolase. The absorptionpectra were obtained on a Beckman DU800 spectrophotometer. The purifiednzyme (0.5 mg/ml) in 25 mM potassium phosphate buffer (pH 7.4) containing 1 mMDTA was used. Curve A, native enzyme; curve B, hydroxylamine-treated enzyme;urve C, sodium borohydride-reduced enzyme.

242 H. Nozaki et al. / Journal of Molecular Cataly

Fig. 6. Production of �-methyl-l-serine (A) and �-ethyl-l-serine (B) with E. coli cellsexpressed the gene encoding �-methylserine aldolase. The reaction was preformedwms

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ith the feeding of formaldehyde as described in Section 2. (A) l-Alanine (©); �-ethyl-l-serine (�); d-alanine (�). (B) l-aba, l-2-aminobutyric acid (©); �-ethyl-l-

erine (�); d-aba, d-2-aminobutyric acid (�).

.30 mmol d-2-aminobutyric acid (Fig. 6B). �-Ethyl-l-serine waseparated from 2-aminobutyric acid by performing chromatogra-hy and was then isolated in the crystalline form by using therocedure described in Section 2. The yield from the reaction solu-ion was 55%, and the values of the other parameters were as

D D

ollows: [˛]20 , −3.4 (c = 1 in 5N HCl); [˛]20 , −4.5(c = 10 in 5N HCl)DIP-370, Jasco Corp., Tokyo, Japan); [M+H]+,134.2; [M−H]−, 132.2TSQ700, ESI–MS, Thermo Fisher Scientific, MA, USA); 1H NMRD2O), ı = 0.95 (tt, 3H), 1.84 (qt, 2H), 3.71, 3.99 (m, 2H) (AVANCE00, Brucker AXS, Brucker, Germany). The specific rotation of the

[

[

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sis B: Enzymatic 59 (2009) 237–242

rude �-ethyl-l-serine crystals extracted from the reaction solutionas almost identical to the value reported previously [18,19].

This enzyme seemed to catalyze the racemization of l-alaninend l-2-aminobutyric acid, since the d-form of the correspondingmino acid was detected at the end of the reaction; such catalysisas been reported for other PLP-related enzymes [17,20,21].

The stereo-specific hydroxymethyltransfer to an �-amino acidsn the �-methylserine aldolase system seems to be one of the

ost effective methods in the production of optically pure �-lkyl-l-serine, and it might be applicable to the preparation of-alkyl-�-amino acids with various kinds of aldehyde compound.

n contrast, it remains still unclear what role this enzyme playsn the metabolism, so it could be elucidated by the more detailunctional analyses.

. Conclusion

The gene encoding �-methylserine aldolase from the Bosea sp.J110407 was cloned, and the recombinant enzyme was purifiednd characterized. Further, we constructed the whole-cell cataly-is method, and the enzymatic synthesis of �-methyl-l-serine and-ethyl-l-serine was achieved with high efficiency and high enan-

ioselectivity.

cknowledgements

We thank Mayuko Yoda for providing technical support, Yukikomezawa for assisting with the N-terminal amino acid sequencenalysis, and Naoko Hirose for helping with the specific opticalotation analysis.

eferences

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