1. Ling Li and Nam Soo Han* Department of Food Science &Technology, Chungbuk National University, Cheongju 361-763, Korea E-mail : liling1113@hanmail.net Production of D- Phenyllactic Acid using Leuconostoc mesenteroides ATCC8293 Whole Cells involving Highly Active D -Lactate Dehydrogenase Abstract 4. Inhibition of cell growth by PLA 3. L. mesenteroides ATCC8293 produced D- PLA 7. PLA production by growing cells of L. mesenteroides ATCC8293 Aims Introduction Conclusions 5. Kinetic parameters of ldh responsible for the conversion of PPA into PLA. Results 1. High PPA conversion yields of Leuconostoc spp. 8. Bioconversion of PPA to PLA using resting cells D- Phenyllactic acid (PLA) is a potential antibacterial and antifungal compound. Leuconostoc mesenteroides ATCC8293 were used to produce high levels of D- PLA from phenylpyruvic acid (PPA). When cultured with PPA (≤50 mM), the growing cells produced 35 mM D- PLA and the conversion yields were between 75.2% and 83.3% after 24 h, permitting a stereoselective transformation process with high initial substrate concentration. Higher conversion yields were observed at pH 6.0–7.0. The D- lactate dehydrogenase encoded by LEUM_1756 of L. mesenteroides ATCC8293 was responsible for the PLA conversion; for PPA, the K m and k cat values of the recombinant enzyme expressed in E. coli were 15. 4 mM and 5645 s -1, respectively. By using the resting cells, the reaction time for full conversion of PPA to PLA was shortened to 3 h. Conclusively, high-level D - PLA production from inexpensive PPA was first carried out by using Leuconostoc growing and resting cells. In L. mesenteroides ATCC8293 pathway No.Leuconostoc spp.Source Culture condition Cell density Yield (%) 1L. lactisKCTC3528 MRS/28 ℃ 1.4877.9 2 L. mesenteroides B-512F NRRL B-512F MRS/28 ℃ 1.5978.9 3L. mesenteroidesKCTC3719 MRS/28 ℃ 1.5077.5 4 L. mesenteroides subsp. dextranicum KCTC3530 MRS/28 ℃ 0.9477.3 5 L. mesenteroides subsp. mesentero ides ATCC8293 MRS/30 ℃ 1.5080.0 6 L. mesenteroides subsp. mesentero ides KCTC3100 MRS/28 ℃ 1.4678.9 7 L. mesenteroides S7 Our lab MRS/28 ℃ 1.4781.3 8 L. mesenteroides S13 Our lab MRS/28 ℃ 1.5777.4 9 L. mesenteroides B-742C NRRL MRS/28 ℃ 1.3376.1 10L. citreum HJ-P4KACC91035 MRS/28 ℃ 1.6176.5 11L. citreumKCTC3526 MRS/30 ℃ 1.5678.5 12L. citreum 75Our lab MRS/28 ℃ 1.6077.3 13L. citreum 95Our lab MRS/28 ℃ 1.4780.7 14L. citreum D3Our lab MRS/28 ℃ 1.5679.8 15L. citreum 87Our lab MRS/30 ℃ 1.5278.8 16L. citreum 74Our lab MRS/30 ℃ 1.4676.7 17L. citreum 61Our lab MRS/30 ℃ 1.8079.0 18L. citreum 63Our lab MRS/30 ℃ 1.5374.7 19L. citreum 64Our lab MRS/30 ℃ 1.1676.4 20L. fallaxKCTC3537 MRS/30 ℃ 1.8676.0 21L. fructosumKCTC3544 MRS/30 ℃ 1.5879.6 Table 1. Leuconostoc spp. used in this study and comparison of PLA yields and cell densities Fig. 1. Effects of PPA concentrations on (A) PLA production and (B) PLA yields by L. mesenteroides ATCC8293. A, PLA production at various PPA concentrations ( ▨ ); B, PLA yields ( ▨ ) and cell density ( ● ). Fig. 2. HPLC analysis of PLA product in 50 mM Tris-HCl buffer (pH 6.0) containing 30 mM PPA using L. mesenteroides ATCC8293. (A) D-PLA and L-PLA standards; (B) reaction product at 0 h as control ; (C) reaction product after 4 h as sample. 2. Utilization of PPA in various concentrations SubstrateK m (mM) V max (µM/min) k cat (S -1 ) k cat /K m (M -1 S -1 ) PPA15.4555.565646 3.67 ⅹ 10 5 Pyruvate0.58476.192900 4.99 ⅹ 10 6 Table 2. Kinetic parameters of the D -ldh enzyme (LEUM_1756) for pyruvate and PPA Fig. 5. Monitoring PLA production by growing cells of L. mesenteroides ATCC 8293 in MRS, 30 mM MRS-PPA, or 40 mM MRS-PPA medium for 24 h. Fig. 6. PPA consumption ( △ ) and PLA production ( ▲ ) in 50 mM Tris-HCl buffer (pH 6.0) containing 30 mM PPA, using 0.16 g/mL of L. mesenteroides ATCC8293 resting cell. 6. Optimal pH condition for PLA production Fig. 4. Effect of initial pH on PLA production during the growth of L. mesenteroides ATCC8293. Fig. 3. Comparison of the growth rates of L. mesenteroides ATCC 8293 in MRS media containing various concentrations of PLA. We have found that L. mesenteroides ATCC8293 can efficiently convert inexpensive PPA to optically pure D- PLA which has higher antibacterial activity than L- PLA. Both the growing and resting cells produced high concentration of PLA at relatively higher PPA concentration, permitting a stereoselective transformation at high initial substrate concentration. The D- lactate dehydrogenase was responsible for the PLA conversion and the enzyme encoded by LEUM_1756 of L. mesenteroides ATCC8293 showed higher values of catalytic efficiencies. A B References Li, L., Eom, H.J., Park, J-M., Seo, E., Ahn, J.E., Kim, T-J., Kim, J.H., Han, N.S., 2012. Characterization of the major dehydrogenase related to D-lactic acid synthesis in Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293. Enzyme Microb. Technol. 51, 274-279. Jin, Q., Yoon, H.S., Han, N.S., Lee, J., Han, J.S., 2006. Effect of lactic acid bacteria on D- and L-lactic acid contents of Kimchi. Food Sci. Biotechnol. 15, 948-953.