Jinlong Huang1,2, Yun Zhang1,3, Yunfeng Hu1,3,4*

Functional Characterization of a Marine Bacillus Esterase and Its Utilization in the Stereo-Selective Production of D-Methyl Lactate

Jinlong Huang1,2, Yun Zhang1,3, Yunfeng Hu1,3,4*

1 Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, People's Republic of China

2 University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China

3 Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, People's Republic of China

4 South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou, People's Republic of China

*Corresponding author: , Tel: 86-20-89024092

Supplementary Table 1. Kinetic analysis of esterase BSE01701 towards various p-NP esters.

P-NP esters / Km (mM) / Vmax (U mg−1) / kcat (s−1) / kcat/Km (s−1 mM−1)
C2 / 0.82 ± 0.03 / 222 ± 16 / 370 ± 27 / 454 ± 27
C4 / 0.50 ± 0.10 / 626 ± 39 / 1044 ± 65 / 2028 ± 305
C6 / 1.24 ± 0.04 / 197 ± 7 / 328 ± 11 / 264 ± 18
C8 / 1.45 ± 0.04 / 93 ± 5 / 155 ± 8 / 107 ± 9

Supplementary Table 2. Effect of heptane concentration on the kinetic resolution of racemic methyl lactate by esterase BSE01701.

Heptane concentration
(v/v, %) / ees (%)a / c (%)b / Ec
0 / 49.77 / 38.25 / 14.98
10 / 71.05 / 51.66 / 10.34
20 / 78.19 / 54.35 / 11.17
30 / 89.15 / 57.12 / 14.65
40 / 93.05 / 58.91 / 15.35
50 / 96.69 / 60.19 / 17.70
60 / 98.19 / 60.99 / 19.25
70 / 90.49 / 56.74 / 16.49
80 / 50.83 / 41.40 / 10.08

Reaction conditions: 500-μL enzymatic reactions containing 50 mM racemic methyl lactate, 0.064 mg/mL (40 U/mL) enzyme, different heptanes concentrations, and 100 mM Tris-HCl buffer (pH 9.0), at 35 °C, 30 min, and 200 rpm. a ees was determined by chiral GC. b conversion of racemic methyl lactate (c), c=D0+L0-D-LD0+L0, where D0 and L0 represent the concentrations of D- and L-methyl lactate before the reaction, and D and L represent the concentrations of D- and L-methyl lactate after the reaction. c Enantioselectivity (E), E = ln1-c1-eesln1-c1+ees.

Supplementary Table 3. Effect of triton concentration on the kinetic resolution of racemic methyl lactate by esterase BSE01701.

Triton concentrations (w/v, %) / ees (%) / c (%) / E
0 / 74.67 / 57.48 / 7.49
0.05 / 89.05 / 63.10 / 8.91
0.10 / 88.87 / 62.81 / 9.02
0.20 / 88.11 / 63.34 / 8.43
0.30 / 89.55 / 64.26 / 8.44
0.40 / 89.53 / 64.33 / 8.40
0.50 / 89.51 / 62.07 / 9.77

Reaction conditions: 500-μL enzymatic reactions containing 50 mM racemic methyl lactate, 0.064 mg/mL (40 U/mL) enzyme, triton of different concentrations, and 100 mM Tris-HCl buffer (pH 9.0), at 35 °C, 30 min, and 200 rpm.

Supplementary Table 4. Effect of a combination of heptane and triton on the kinetic resolution of racemic methyl lactate by esterase BSE01701.

Additives / concentration / ees (%) / c (%) / E
Heptane / 60% (v/v) / 96.71 / 59.23 / 19.54
Triton / 0.05% (w/v) / 89.79 / 64.72 / 8.29
Heptane + Triton / 60% + 0.05% / 94.87 / 58.48 / 18.17

Reaction conditions: 500-μL enzymatic reactions containing 50 mM racemic methyl lactate, 0.064 mg/mL (40 U/mL) enzyme, heptanes, triton or mixtures, and 100 mM Tris-HCl buffer (pH 9.0), at 35 °C, 30 min, and 200 rpm.

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