Bioresource Technology 133 (2013) 635637

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Bioresource Technology
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Short Communication

Enzymatic synthesis of S-phenyl-L-cysteine from keratin hydrolysis industries wastewater with tryptophan synthase
Lisheng Xu a,b, Zhiyuan Wang a, Pingting Mao a, Junzhong Liu a, Hongjuan Zhang a, Qian Liu a, Qing-Cai Jiao a,
a b

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing 210093, PR China Department of Chemistry and Life Science, Suzhou University, Suzhou 234000, PR China

h i g h l i g h t s
" Preparation of S-phenyl-L-cysteine by keratin acid hydrolysis wastewater is reported. " Tryptophan synthase could efciently convert KHW to S-phenyl-L-cysteine. " L-Serine of KHW conversion rate reached 97.1%. " Final S-phenyl-L-cysteine concentration reached 38.6 g l
1

a r t i c l e

i n f o

a b s t r a c t
An economical method for production of S-phenyl-L-cysteine from keratin acid hydrolysis wastewater (KHW) containing L-serine was developed by recombinant tryptophan synthase. This study provides us with an alternative KHW utilization strategy to synthesize S-phenyl-L-cysteine. Tryptophan synthase could efciently convert L-serine contained in KHW to S-phenyl-L-cysteine at pH 9.0, 40 C and Trion X-100 of 0.02%. In a scale up study, L-serine conversion rate reach 97.1% with a nal S-phenyl-L-cysteine concentration of 38.6 g l1. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 17 January 2013 Received in revised form 31 January 2013 Accepted 1 February 2013 Available online 9 February 2013 Keywords: S-Phenyl-L-cysteine Keratin acid hydrolysis wastewater Tryptophan synthase Enzymatic synthesis

1. Introduction S-Phenyl-L-cysteine has been found to have utility as a structure of an HIV protease inhibitor, an anti-AIDS drug, or as an amino acid of a constituent of a hydroxyethylamine dipeptide isostere of aspartic protease inhibitors (Fukuda et al., 1996). There is accordingly a desire for the development of a process for the economical and high-purity production of S-phenyl-L-cysteine. The enzymatic method using tryptophan synthase has merits in terms of both high yields and high purity of the product. Tryptophan synthase, which is mainly found in bacteria and plants, catalyzes S-phenylL-cysteine synthesis from L-serine and thiophenol (Panke et al., 2002; Stark and Stockar, 2003; Foresti et al., 2007; Hui et al., 2011). In recent years, an interesting alternative to high cost of the substrates is the usage of wastewater, which involves low investment and low operating cost (Albuquerque et al., 2007; Bengtsson
Corresponding author. Tel./fax: +86 025 83594376.
E-mail address: jiaoqingcai@yahoo.com.cn (Q.-C. Jiao). 0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.02.011

et al., 2008). For example, Polyhydroxyalkanoates production using Pseudomonas otitidis, a newly isolated strain from PHA producing bioreactor was investigated using acidogenic efuents from biohydrogen reactor at different organic loading rates (Venkateswar et al., 2012). Keratin hydrolysis industries wastewater is a rich resource of amino acids, including serine, glutamic acid, aspartic acid, etc. There are a lot of KHW in China, and most KHW was disposed by condensing into low value-added fertilizer or amino acids feed additives. Recycling of keratin hydrolysis industries wastewater has enormous economic importance. In continuation to our interest in amino acids (Xu et al., 2011, 2012; Zhao et al., 2010), and we reported on the utilization of hair acid hydrolysis wastewater for Ltryptophan production (Zhao et al., 2011). Tryptophan synthase could efciently convert L-serine contained in HHW to L-tryptophan, but little is known about the utilization of keratin hydrolysis industries wastewater for production of S-phenyl-L-cysteine. In the present research, we used recombinant tryptophan synthase (E.C. 4.2.1.20) to synthesize S-phenyl-L-cysteine from KHW. Key factors

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such as temperature, pH, salinity and surfactant were investigated. At the same time, bioconversion efciency and S-phenyl-L-cysteine yield are determined under the optimal conditions.

2.3. TSase activity assay By measuring the S-phenyl-L-cysteine synthesized from thiophenol and KHW containing L-serine, TSase activity was determined with amino acid analyzer (Hitachi L-8900, Japan). The reaction was carried out in 10 ml containing KHW, 0.21 g thiophenol, 30 ll PLP, 30 ll Trion X-100 and 0.1 g TSase cell at 37 C for 1 h. One enzyme unit (U) was dened as the amount of TSase that synthesize 1 lmol of S-phenyl-L-cysteine per min from L-serine and thiophenol. The specic activity is dened as unit/g of TSase.

2. Methods 2.1. Chemicals The KHW, including feather and hair acid hydrolysis wastewater, used as a raw material was provided with Shine Star (Hubei) Biological Engineering Co. Ltd. (Hubei, China). The organic contents depend on the species of initial feather and hair used in the amino acids production, but most KHW contain ammonium chloride, pigment, suspended solids and about eight amino acids. Physicochemical characteristics of KHW are shown below: 13.54 1.03 g l1 aspartic acid, 11.62 1.11 g l1 threonine, 20.11 1.61 g l1 serine, 15.46 1.54 g l1 glutamic acid, 19.01 1.43 g l1 proline, 7.87 0.09 g l1 glycine, 8.54 0.95 g l1 alanine, 7.34 0.31 g l1 valine, 24.02 1.35 g l1 ammonium chloride, 0.93 0.03 g l1 suspended solids, 17.4 1.15% transmittance, 100 15 chroma and pH 2.45 0.11. Lactose and pyridoxal-50-phosphate (PLP) were from Sigma (St. Louis, MO, USA). All other chemicals and reagents used in this work were of analytical grade.

2.4. Rapid detection of S-phenyl-L-cysteine by TLC The reaction mixture was adjusted to pH 0.5 by 50% (m/v) HCl and centrifuged for 10 min at 6000 rpm to analyze the product by thin layer chromatography (TLC). Each reaction mixture was applied 1 ll onto a Silicagel GF254 aluminum sheet (6.0 10.0 cm) purchased from Shanghai Chemical Co. Ltd. (Shanghai, China) and chromatography separation in a mobile phase containing n-butylalcohol:acetic acid:H2O (3:1:0.5 v/v). The bands were detected by spraying TLC plate with 0.5% (m/v) ninhydrin followed by baking at 180 C for 5 min.

2.5. Scale up fermentation and bioconversion 2.2. Microorganisms and shake ask fermentation The gene encoding tryptophan synthase (TSase) was cloned from Escherichia coli k-12 MG1655. The E. coli strain BL21(DE3) carrying the recombinant plasmid pET28a-trpBA(DM206), was constructed in our laboratory (Xu et al., 2011). A loopful of strain was used to inoculate a 100 ml Erlenmeyer ask containing 20 ml of LB broth. The ask was incubated on a rotary shaker at 220 rpm and 30 C for 10 h. Erlenmeyer asks containing 100 ml of sterilized fermentation media containing: 1% (m/v) peptone, 3% (m/v) corn plasm, 1% (m/v) lactose and inoculated with 2% (v/v) of preculture prepared as above. These asks were incubated in a shaker-incubator for 15 h at 30 C, 220 rpm. Fermented broth was collected at the end of fermentation process and centrifuged at 6000 rpm, 4 C for 15 min to obtain wet cells, and then these wet cells were re-suspended and centrifuged twice in 0.5 mM TrisHCl (pH 7.0) for further research as described in the following sections. The preliminary experiment of TSase fermentation was investigated in shake asks of varied volumes (0.51.0 l). Finally, fermentation was carried out in a stirred tank 20 l fermentor (working volume: 14 l, East biotech., Jiangsu, China) which was charged with media 14 l containing 5% (m/v) corn plasm, 1.5% (m/v) peptone, 1.5% (m/v) lactose and (0.1% v/v) polypropylene glycol (PPG, Sigma) solution as an anti-foam agent. The media temperature was maintained at 30 C, fermentation pH was controlled automatically at 7 0.2. At the end of fermentation process (18 h), fermentation broth was centrifuged at 6000 rpm, 4 C for 15 min to obtain wet cells. The wet cells directly mixed with KHW under the optimum reaction conditions for bioconversion in 40 l reactor (ZF-20, Jiangsu, China) under a nitrogen gas atmosphere. Nitrogen can prevent thiophenol oxidation. The reaction was carried out in 40 l reactor containing 20.11 g l1 L-serine, 21.15 g l1 thiophenol, PLP (1%, m/ v), Trion X-100 (0.02%, m/v) and tryptophan synthase at pH 9 and 40 C.

Fig. 1. TLC analysis of reaction system under different bioconversion conditions. (A) (from left to right): Std: a standard 1% S-phenyl-L-cysteine; SB: substrate blank; pH 6.0; 7.0; 8.0; 9.0; 10.0. (B) (from left to right): Std; SB; 30; 35; 40; 45; 50 C. (C) (from left to right): Std; SB; 0.005%; 0.01%; 0.015%; 0.02%; 0.025% (m/v). (D) (from left to right): Std; SB; 30, 60, 90, 120 to 150 (g l1).

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200

637

S-phenyl-L-cysteine, L-serine (mmol/l)

150

100

mixture to adjust its pH to 0.5. The activated carbon was added, followed by heating at 50 C for 3 h. During the heating, the reaction mixture was sparged with air at the average aeration rate of 1.0 l/h. Filtration was then conducted and the ltrate was adjusted to pH 3 with NaOH. After cooling the ltrate to 10 C, ltration was conducted so that crystals of S-phenyl-L-cysteine were separated. After drying the crystals, 779 g of S-phenyl-L-cysteine were obtained. S-Phenyl-L-cysteine, mp 178183 C (decomp), specic  rotation a20 D 73  75 (c = 1, 1.5 M H2SO4). 4. Conclusion

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0 0 2 4 6 8 10 12 14 16

Time (h)
Fig. 2. Change of the concentrations of S-phenyl-L-cysteine and L-serine. The concentrations of S-phenyl-L-cysteine (j) and L-serine (h) were measured under different time. The reaction was carried out in 40 l reactor containing 20.11 g l1 Lserine, 21.15 g l1 thiophenol, PLP (1%, m/v), Trion X-100 (0.02%, m/v) and tryptophan synthase at pH 9 and 40 C.

Based on the present research of enzymatic synthesis of S-phenyl-L-cysteine from keratin acid hydrolysis wastewater, enzymatic bioconversion really could be as an effective and relatively low cost method for affording S-phenyl-L-cysteine from HHW. Through scaling up fermentation, L-serine conversion rate reach 97.1% with a nal S-phenyl-L-cysteine concentration of 38.6 g l1. The results also open up a economical research eld for utilizing industrial keratin acid hydrolysis wastewater. Acknowledgements This work was supported by the National Technology-Innovation Fund (02CJ-13-01-16) and the Open Fund of State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University, P.R. China. References
Albuquerque, M.G.E., Eiroa, M., Torres, C., Nunes, B.R., Reis, M.A.M., 2007. Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses. J. Biotechnol. 130, 411421. Bengtsson, S., Werker, A., Christensson, M., Welander, T., 2008. Production of polyhydroxyalkanoates by activated sludge treating a paper mill wastewater. Bioresour. Technol. 99, 509516. Foresti, M.L., Pedernera, M., Bucala, V., Ferreira, M.L., 2007. Multiple effects of water on solvent-free enzymatic esterications. Enzyme Microb. Technol. 41, 6270. Fukuda, K., Fukuhara, N., Mitsui, T.C., 1996. Production of S-phenyl-L-cysteine. PCT Patent Specication Japan Patent 9084592A. Hui, S.N., Tan, C.P., Chen, S.K., Mohd, N.M., Arbakariya, A., Tau, C.L., 2011. Primary capture of cyclodextrin glycosyltransferase derived from Bacillus cereus by aqueous two phase system. Sep. Purif. Technol. 81, 318324. Panke, S., Held, M., Wubbolts, M.G., Wiltholt, B., Schmid, A., 2002. Pilot-scale production of (S)-styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase. Biotechnol. Bioeng. 80, 3341. Stark, D., Stockar, U., 2003. In situ production removal (ISPR) in whole cell biotechnology during the last twenty years. Adv. Biochem. Eng. Biotechnol. 80, 150175. Venkateswar, R.M., Nikhil, G.N., Venkata, S.M., Swamy, Y.V., Sarma, P.N., 2012. Pseudomonas otitidis as a potential biocatalyst for polyhydroxyalkanoates (PHA) synthesis using synthetic wastewater and acidogenic efuents. Bioresour. Technol. 123, 471479. Xu, L.S., Wang, Z.Y., Liu, J.Z., Mao, P.T., Zhang, H.J., Gao, J., Liu, Q., Jiao, Q.C., 2012. Effects of polyhydroxy compounds on enzymatic synthesis of L-tryptophan catalyzed by tryptophan synthase. Catal. Lett. 142, 282286. Xu, L.S., Liu, J.Z., Wang, Z.Y., Zhang, H.J., Liu, W., Liu, Q., Jiao, Q.C., 2011. Enzymatic synthesis of L-tryptophan catalyzed by tryptophan synthase in a water/organic solvent biphase system. Chin. J. Catal. 32, 14051410. Zhao, G.H., Li, H., Liu, W., Zhan, W.G., Zhang, F., Liu, Q., Jiao, Q.C., 2010. Preparation of optically active b-hydroxy-a-amino acid by immobilized Escherichia coli cells with serine hydroxymethyl transferase activity. Amino Acids 40, 215220. Zhao, G.H., Liu, J.Z., Dong, K., Zhang, F., Zhang, H.J., Liu, Q., Jiao, Q.C., 2011. Enzymatic synthesis of L-tryptophan from hair acid hydrolysis industries wastewater with tryptophan synthase. Bioresour. Technol. 102, 35543557.

3. Results and discussion 3.1. Temperature, pH, surfactant effect and ammonium chloride Enzyme activity can be affected by environmental factors, such as pH, temperature and surfactant, etc. pH can change the dissociation of groups on the enzyme active site and affect the enzyme substrate binding. Enzymatic reactions require appropriate pH environment. In this study, the optimal initial pH and temperature was 9.0 and 40 C, respectively (Fig. 1A and B). During the enzymatic reaction, surfactants can increase the cell permeability. It is conducive to the substrate and the product out of the cell. The effect of surfactant including Trion X-100 was examined at 40 C.Trion X-100 leads to the emergence of the highest relative activity at 0.02%. The results of TLC were showed in Fig. 1C. During the feather and hair acid hydrolysis, feather and hair were rstly hydrolyzed by hydrochloric acid, and then adjusted pH to 35 by ammonia liquid. Wastewater contains a larger number of ammonium chloride. The effect of ammonium chloride ranging from 30, 60, 90, 120 to 150 (g l1) on enzymatic activity showed that TSase activity was slightly independent on salt concentration (Fig. 1D). 3.2. Scale up fermentation and bioconversion, isolation and identication of S-phenyl-L-cysteine Fermentation broth was centrifuged at 6000 rpm, 4 C for 15 min to obtain 712 g wet cells. The wet cells directly mixed with KHW under the optimum reaction conditions of pH 9.0, 40 C, Trion X-100 0.02%. In the process of bioconversion, the reactor was stirred at 170 rpm under a nitrogen gas atmosphere. The concentration of L-serine reduced gradually, and the concentration of S-phenyl-L-cysteine increased in 12 h. The results were showed in Fig. 2. After 12 h, hydrochloric acid was added to the reaction