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Enzyme and Microbial Technology 41 (2007) 407–412

Pilot-scale production of d-p-hydroxyphenylglycine fromdl-5-p-hydroxyphenylhydantoin by Burkholderia cepacia JS-02

Min Jiang a, Longan Shang b,c,∗, Ping Wei a, Ronghua Yu a, Ning Shen a,Pingkai Ouyang a,∗∗, Ho Nam Chang c

a College of Pharmacy and Life Science, Nanjing University of Technology, Nanjing 210009, Chinab College of Biological and Chemical Engineering, Ningbo Institute of Technology,

Zhejiang University, NingBo 315100, Chinac Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong,

Yuseong-gu, Taejon 305-701, Republic of Korea

Received 20 October 2006; received in revised form 10 February 2007; accepted 21 February 2007

bstract

In a 50 L pilot scale reactor d-p-hydroxyphenylglycine (d-HPG) is produced enzymatically from dl-5-p-hydroxyphenylhydantoin (dl-HPH)ith the resting cells of Burkholderia cepacia JS-02, requiring only corn steep liquor as a nitrogen source instead of the expensive yeast extractr peptone required by other strains. Both the fermentation process for preparing resting cells and the bioconversion were optimized in 5 L benchcale reactors. The cells showed the highest hydantoinase and carbamoylase activities (0.640 and 0.304 U/mL-borth, respectively) at a fermentationf 18 h when Co2+ ions and dl-5-methylthioethyl hydantoin as an inducer were used. The optimal temperature and initial pH for bioconversionere 40 ◦C and 9, respectively. However, starting from the initial pH 9, pH dropped rapidly to near 7, at which level both key enzymes showed

onsiderable activity. A pilot-scale bioconversion was carried out in a 50 L reactor with a productivity of 0.68 g/L h.

Unlike conventional processes, this process using B. cepacia JS-02 can utilize inexpensive nitrogen and carbon sources for the production of the

esting cells that contain the key enzymes. Also, it showed a high specific productivity during bioconversion without the use of a buffer solution.n economic analysis of this process showed that these advantages could lower production costs effectively.2007 Published by Elsevier Inc.

e; d-p

5Ntcbic

eywords: dl-5-p-Hydroxyphenylhydantoin; d-Carbamoylase; d-Hydantoinas

. Introduction

Optically active d-amino acids are widely used in phar-aceutical industries as intermediates for the synthesis of

emi-synthetic antibiotics, peptide hormones, pyrethroids, andesticides. Among them, d-p-hydroxyphenylglycine (d-HPG)as markedly increased its commercial value since it can also

e used as a precursor of synthetic cephalosporins [1].

d-Amino acids have been commercially produced by awo-stage chemo-enzymatic process. In this process, the dl-

∗ Corresponding author at: College of Biological and Chemical Engineering,ingbo Institute of Technology, Zhejiang University, NingBo 315100, China.el.: +86 574 8822 9546; fax: +86 574 8813 4062.

∗∗ Corresponding author.E-mail addresses: lashang@nit.zju.edu.cn (L. Shang), yhljt99@yahoo.com

P. Ouyang).

ahqacEpasc

141-0229/$ – see front matter © 2007 Published by Elsevier Inc.oi:10.1016/j.enzmictec.2007.02.010

-Hydroxyphenylglycine; Burkholderia cepacia JS-02

-substituted hydantoin is first asymmetrically hydrolyzed to-carbamoyl-d-amino acid by a d-specific hydantoinase, and

hen converted to a homologous d-amino acid through ahemical process [2,3]. Compared to the chemical conversion,ioconversion with d-carbamoylase is more appealing becauset can be conducted under mild conditions with a low toxi-ity. Several microorganisms that express both hydantoinasend carbamoylase have been isolated [4–6]. The specificity ofydantoinase and carbamolyase for d-configuration allows auantitative conversion of dl-hydantoin into the homologous d-mino acids. This ensures that the production of d-amino acidsan be simply accomplished in a one-step process. Recombinantscherichia coli could be used to express the two enzymes for

roducing the d-amino acid from dl-hydantoin [7–11]. Gener-lly, peptone and/or yeast extract have been used as nitrogenources in fermentations for preparing the resting cells thatontain both key enzymes. Additionally, a large amount of the

4 robial Technology 41 (2007) 407–412

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Table 1Effect of metal ions on the activities of hydantoinase and carbamoylase in thecase of dl-5-methylthioethyl hydantoin used as the inducer

Metal ions(0.8 mM)

Hydantoinase activity(U/mL broth)

Carbamoylase activity(U/mL broth)

None 0.532 0.187Fe2+ 0.623 0.278Mn2+ 0.568 0.225Zn2+ 0.604 0.257CC

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08 M. Jiang et al. / Enzyme and Mic

esting cells were also needed in the bioconversion processes. Tour knowledge, there was no report on the large-scale bioprocessevelopment of d-HPG production.

In this paper, Burkholderia cepacia JS-02, a newsolated strain, was used for the bioconversion of dl-5-p-ydroxyphenylhydantoin (dl-HPH) into d-HPG in both benchnd pilot-plant scales. Several key parameters in the fer-entation and bioconversion processes were optimized. In

rder to reduce the production cost, resting cells were pre-ared with inexpensive nitrogen and carbon sources. Anconomic analysis of this process was done using the BioProesigner.

. Materials and methods

.1. Chemicals

Five-substituted hydantoin and other hydantoin derivatives were preparedrom the corresponding aldehydes or amino acids. d-p-Hydroxyphenylglycineas purchased from Janssen Chimica (Pantin, France). All chemicals used weref the best analytical grade available.

.2. Microorganism and culture media

B. cepacia JS-02 isolated from a soil sample was used in this study. The cul-ure media per liter contains: 20 g sucrose, 25 mL corn steep liquid, 2 g KH2PO4,g NaCl, 0.025 g MgSO4·7H2O, and 8 mM inducers (Table 2). Several otherarbon and nitrogen sources such as glucose, fructose, peptone, and yeast extractere also investigated for optimizing the culture media.

.3. Preparation of resting cells

Seed culture was prepared in a 500 mL flask containing 50 mL cultureedium with 5% inoculants by incubating at 30 ◦C for 16 h. Flask culturesere performed as the same procedures as the seed culture, but the incubation

ime was prolonged from 16 to 24 h. Aerobic fermentation was done in a 5-Lermentor (Marubishi, Japan) containing 3 L culture medium for 16–18 h. Dif-erent aeration rates (0.2–0.6 vvm), temperatures (25–35 ◦C), and agitation rates250–600 rpm) were employed in the process optimization. At the conclusion ofxponential phase, the cells were harvested by centrifugation and washed twicesing cold water. Based on the results of the 5 L fermentor, scaling up was donen a 50-L fermentor.

.4. Bioconversion with resting cells

Five litres of DL-HPH solution (25 g/L) was mixed with the harvested cellsrom 3 L culture broth (about 125 g wet cells), and the initial pH was adjustedith NaOH. After stripping off the oxygen with nitrogen for stabilizing hydan-

oin and enzymes, the reaction mixture was maintained at a certain temperaturefrom 25 to 44 ◦C) for 30–40 h with a moderate agitation to determine the optimalonditions for bioconversion. A 50-L bioconversion process was also carried outt the optimal conditions found in the 5-L scale process. The intermediate prod-ct, N-carbamyl-d-hydroxyphenylglycine (N-C-d-HPG), was separated usinghe method previously reported by Lee [12].

.5. Enzyme assay

A predetermined amount of resting cells were incubated with 1% dl-5-

PH or N-C-d-HPG in 100 mL Na2HPO4–NaH2PO4 buffer solution (0.1 mol/L,H 8.0) for 30 min at 35 ◦C, with gentle shaking. Aliquots of samples wereithdrawn to determine the concentrations of N-C-d-HPG and d-HPG. Specific

nzyme activity, defined as �mol of product made per minute by resting cellsarvested from per milliliter culture broth, was measured.

uosc

u2+ 0.073 0.041o2+ 0.640 0.304

.6. Analytical methods

Cell concentrations during the fermentation were monitored by measuringhe absorbance at 640 nm (A640). In the bioconversion process, the concentra-ions of d-HPG, N-C-d-HPG and dl-5-HPH were determined at 210 nm withhigh performance liquid chromatography equipped with a Kromasil C18 col-mn (4.6 mm × 250 mm). The mobile phase consisted of H2O/CH3CN/H3PO4

80/20/0.02, v/v/v), and was injected at 1.0 mL/min. In order to determine theptical purity of d-HPG, optical rotation values of d-HPG was measured usingolarimeter (Jasco model P-1020, Japan) at 1% concentration of d-HPG inmol/L HC1.

.7. Process economic evaluation

A comparative economic analysis of the two processes, one proposed inhis work and another by Chao [8], was done using the software tool BioProesigner (Intelligen Inc., USA). In this work, sucrose (US$ 0.40 kg−1) and

orn steep liquid (US$ 0.13 kg−1) were used in an 18-h fermentation with aield of 0.33 g-biomass/g-sucrose, and bioconversion was carried out in 31 hith a yield of 0.94 g-(d-HPG)/g-(d-HPH). In Chao’s work [8], glycerol (US$.5 kg−1), tryptone (US$ 2.0 kg−1), and yeast extract (US$ 2.7 kg−1) were usedn a 10-h fermentation, and the 15-h bioconversion was performed with a yieldf 0.97 g-(d-PHG)/g-(d-HPH). The purification method used in this work waspplied for both economic evaluations.

. Results and discussion

.1. Preparation of resting cells

The optimization of the culture medium used for preparinghe hydantoinase and carbamolyase with high activities from cul-ivation of B. cepacia JS-02 was first investigated in flask culturest 30 ◦C. Various materials, such as glucose, sucrose, fructose,orn steep liquid, peptone, and yeast extract, were tested indi-idually as potential carbon and nitrogen sources in the cultureedium. The highest activity of two key enzymes were gainedhen sucrose (20 g/L) and corn steep liquid (2.5 vol.%) wassed. It was also found that increasing the concentrations ofucrose and corn steep liquid did not improve the cell growthnd the target enzyme activity.

Based on the optimal culture medium, effects of the initialedium pH and metal ions on the enzyme activity of resting cellsere investigated. It was found that the best initial pH was 7.2,

nd that the enzyme activity of resting cells could decrease by

p to l8% when the initial pH was in the range of 6–8. The effectf metal ions (0.8 mM) on the enzyme activity of resting cells ishown in Table 1. It was found that Fe2+, Mn2+, Zn2+ and Co2+

ould stimulate the enzyme formation to some extent, but Co2+

M. Jiang et al. / Enzyme and Microbial Technology 41 (2007) 407–412 409

Table 2Effects of inducers on the activities of hydantoinase and carbamoylase in thecase of only Co2+ used as the metal ions

Compounds (8 mM) Hydantoinase activity(U/mL broth)

Carbamoylase activity(U/mL broth)

None 0.058 0.028dl-5-Methylthioethyl-

hydantoin0.640 0.304

dl-5-Benzylhydantoin 0.151 0.037Hydantoin 0.166 0.035dl-5-Hydroxy-

phenyldantoin0.137 0.032

dl-5-Methylhydantoin 0.157 0.110Uracil 0.228 0.068DA

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tvdacafIyield was 85%. Fig. 4 shows the time profiles of d-HPG, N-C-d-HPG and dl-HPH concentrations, and pH. During the reaction,

ihydrouracil 0.269 0.049llantoin 0.284 0.053

as the best. Only Cu2+ exhibited an inhibitory effect on theormation of enzymes. For Agrobacterium sp. and Arthrobacterrystallopoiets AM2, it has been reported that employingydantoin derivatives or their structural analogues can inducehe formation of hydantoinase and carbamoylase and improveheir activities [6,13]. Here, the possible induction effects ofydantoin derivatives and their structural analogues on the for-ation of hydantoinase and carbamoylase in B. cepacia JS-02ere investigated. As shown in Table 2, dl-5-methylthioethylydantoin enhanced the activities of both enzymes remarkablys did the others to lesser extents, and was chosen as thenducer for the preparation of resting cells in batch culture of. cepacia JS-02.

Cultivation of B. cepacia JS-02 in a 5-L fementator wasnvestigated by adjusting important operation parameters suchs aeration rate and agitation rate; these were tested at intervalsn the ranges of 0.2–0.6 vvm and 250–600 rpm, respectively. Theest result was obtained when the cultivation was done with aneration rate of 0.5 vvm and agitation rate of 400 rpm, as shown

n Fig. 1. Clearly, the enzyme activities of resting cells increasedapidly in the exponential phase, and peaked at the end of expo-ential phase, and then decreased. Therefore, it is best to harvest

ig. 1. Time profiles of cell concentration (�), hydantoinase activity (�), andarbamoylase activity (�) in 5-L scale fermentation with the optimal aerationate (0.5 vvm) and agitation rate (400 rpm).

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ig. 2. Effect of temperature on the d-HPG yield in a bioconversion withoutuffer solution at an initial pH of 9.0.

ells at the end of exponential phase, and about 35–40 g wet cellser liter would be produced in 18 h fermentation.

.2. Bioconversion

Generally, bioconversion is performed in a buffer solutiono maintain the pH constant [6,8,9,13]. Here, a simple biocon-ersion process that does not require any buffer solution waseveloped for d-HPG production to reduce the production costss much as possible. At first, only the initial pH of the bio-onversion broth was adjusted. The influences of the initial pHnd temperature are shown in Figs. 2 and 3, respectively. It wasound that the optimal temperature and pH were 40 ◦C and 9.n a 40 h reaction, 92% of HPH was converted and the molar

-HPG and N-C-d-HPG concentrations initially increased veryuickly. After 12–15 h, N-C-d-HPG concentration peaked, and

ig. 3. Effect of initial pH on d-HPG yield in a bioconversion without bufferolution at a temperature of 40 ◦C.

410 M. Jiang et al. / Enzyme and Microbial Technology 41 (2007) 407–412

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hen began to decrease, whereas d-HPG concentration did noteach its peak until after 38–40 h. The concentration of dl-HPHhroughout the process was about 2 g/L because of its poor sol-bility in water. Clearly, the pH in the bioconversion processhanged dramatically from its optimal initial value of 9 to itsowest value of 6.5. The sudden decrease of pH at the beginningas attributed to the fast formation of N-C-d-HPG. In order to

ee whether or not the very low pH would affect the bioconver-ion process, we determined the activity of two key enzymes in aH range of 6–10, as shown in Fig. 5. Carbamoylase activity wasigh in a fairly limited pH range of 7.0–7.5, while hydantoinasectivity was high in a wide range of pH, from 8.0 to 9.0, andt was still adequately high even at pH levels of 7.0–8.0. Con-idering these characteristics of the two enzymes, we allowedhe pH to drop from 9.0 to 7.2 and then maintained it at 7.2 in

he bioconversion process with 3N NaOH solution. By applyinghis strategy, 99% of dl-HPH was converted, and the molar yieldncreased to 94%. Additionally, the reaction time decreased to0 h from 40 h.

ig. 5. Hydantoinase activity (�) and carbamoylase activity (�) of resting cellsn different pH levels at a temperature of 40 ◦C. Ta

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The reaction broth was decoloured using activated carbon,nd then concentrated. d-HPG was crystallized and washedith methanol twice, and then recrystallized again. Finally, theptical purity of d-HPG reached 99.2% and [�]23/d = −159.0◦1c, 1 mol/L HCl), which was found to be strictly d-specific14].

.3. Scaling up

Based on the optimal results found in the small-scale process,caling up experiment was done in a 50-L fermentor and a 50-

bioconversion reactor. In 16–18 h fermentation, about 38 get cells per liter were produced. By mixing the substrate and

esting cells in the same ratio as mentioned above, the conversionatio and the molar yield reached 99% and 94%, respectively,n 30.5 h. These results are identical to those obtained in themall-scale process.

There are several microorganisms that can be used to pro-uce hydantoinase and carbamoylase for d-HPG production,s shown in Table 3. The highest productivity (3.24 g/L h) waschieved when a large amount of resting cells (19.2 g dry cellser liter) was used in the bioconversion. In this study, however,nly 25 g wet cells per liter were used in the bioconversion.his shows that the two key enzymes produced from B. cepaciaS-02 have high activities. Also, because our new process usednly cheap nitrogen and carbon sources in fermentation processnd did not require any buffer solution in the bioconversion,he production cost was lowered. Fig. 6 shows the compari-on of production costs in the two processes, ours and Chao’s8], in different scales. When the annual output was 1000 tons,he production cost of this new process decreased by 30%

−1

from US$ 17.48 to US$ 12.25 kg ) as compared to Chao’sork [8], and the annual materials cost decreased by more

han 40%. Because of its low production cost, this new processas already been successfully setup in Nanjing Tianchen Bio-

ig. 6. d-HPG production cost in different production scales. Symbols: (�) thisork; (�) Chao’s work [8].

[

[

[

l Technology 41 (2007) 407–412 411

hemical Engineering Co. Ltd., China, with an annual output of0 tons.

. Conclusion

This study shows that the new isolated B. cepacia JS-02 cane used to produce the hydantoinase and carbamoylase with highctivity. This new proposed process for d-HPG production usesnly cheap nitrogen and carbon sources for preparing restingells, and bioconversion can be performed under a low usagef resting cells without any buffer solution. All these make theroduction cost of D-HPG lower effectively.

cknowledgements

We thank Chunyan Tu of Key Lab of Industrial biotechnologyiangsu Province, China, for assistance with HPLC analyses andanjing Tianchen Biochemical Engineering Co. Ltd., China, forelp in scaling up. Special thanks also to the BK21 project inorea for its support. This work was supported by the Nationalasic Research Program of China (2003CB716004) and theational Natural Science Foundation of China (20336010).

eferences

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[2] Takahasi S, Ohashi T, Kii Y, Kumagai H, Yamada H. Microbial transfor-mation of hydantoins to N-carbamoyl-d-amino acids. J Ferment Technol1991;57:328–32.

[3] Gokhale DV, Bastawde KB, Patil SG, Kalkote UR, Joshi RR, Joshi RA,et al. Chemoenzymatic synthesis of d-phenylglycine using hydantoinaseof Pseudomonas desmolyticum resting cells. Enzyme Microb Technol1996;18:353–7.

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[5] Kenzo Y, Shigeru N. Optimal conditions for the enzymatic production ofd-amino acids from the corresponding 5-substituted hydantoins. Agric BiolChem 1987;54:715–9.

[6] Serge R, Nicolas C, Eric O. d-p-Hydroxyphenylglycine production fromdl-5-p-hydroxyphenylhydantoin by Agrobacterium sp. Appl MicrobiolBiotechnol 1990;33:382–8.

[7] Park JH, Kim GJ, Kim HS. Production of d-amino acid using wholecells of recombinant Escherichia coli with separately and coexpressedd-hydantoinase and N-carbamoylase. Biotechnol Prog 2000;16:564–70.

[8] Chao YP, Fu HY, Lo TE, Chen PT, Wang JJ. One-step production of d-p-hydroxyphenylglycine by recombinant Escherichia coli strains. BiotechnolProg 1999;15:1039–45.

[9] Grifantini R, Galli G, Carpani G, Pratesi C, Franscotti G, Grandi G. Effi-cient conversion of 5-substituted hydantoins to d-�-amino acids usingrecombinant Escherichia coli strains. Microbiology 1998;144:947–54.

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d-Amino acid production by genetically engineered Escherichia coli. Ger.Offen. 2004. 17 pp. CODEN: GWXXBX DE 10251184 Al.

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13] Moller A, Syldatk C, Schulze M, Wagner F. Stereo- and substrate-specificity of a d-hydantoinase and a d-N-carbamyl-amino acidamidohydrolase of Arthrobacter crystallopoietes AM2. Enzyme MicrobTechnol 1988;10:618–25.

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14] Shigeki Y, Chikara H, Ichiro C. Preparation of d-p-hydroxyphenylglycine:optical resolution of dl-p-hydroxyphenylglycine by prefer-ential crystallization procedure. Agric Biol Chem 1978;42:1521–6.