Supplementary Information for Biotechnology Letters [BILE-D-13-00914R1]

Enantioselectivehydrolysis ofracemicstyrene oxide and its substituted derivativesusing newly isolated Sphingopyxis sp. BSNA05exhibiting a novel epoxidehydrolaseactivity

Jung-Hee Woo1,*, and EunYeol Lee2,*

1Gyeongbuk Institute for Marine Bio-Industry (GIMB),Uljin 767-813, Gyeongbuk, Republic of Korea

2Department of Chemical Engineering, Kyung Hee University,

Gyeonggi-do 446-701, Republic of Korea

*To whom correspondence should be addressed

Tel: 82-54-780-3454, Fax: 82-54-780-3469, E-mail:

Tel: 82-31-201-3839, Fax: 82-31-204-8114, E-mail:

Supplementary Table1.Parameters of enantioselective hydrolysis of racemicstyrene oxide (SO) by the strain 8-NA-5.

Epoxide / Time (h) / ee (%) / ca / Eb / Yield (%)c / Abs. conf.d
4 mMSOe / 13 / 86.46 / 0.8039 / 3.9 / 21.8 / S
4 mMSOf / 7 / 99.99 / 0.7606 / 12.2 / 20.6 / S

a. The extent of conversion (c) [c ={1−(ERs+ESs/ERso+ESso)}], where the initial concentrations of (R)- and (S)-epoxidesare denoted as ERso and ESso, respectively, and the concentrations of the remaining (R)- and (S)-epoxidesare denoted as ERs and ESs, respectively.

b. The enantiomeric ratio (E-value) was derived from the extent of conversion (c) and the enantiomeric excess of the remaining substrate enantiomers (ees) [E= In{(1−c) (1−ees)}/In{(1−c) (1+ees)}]

c. Yield of the remaining epoxide

d. Absolute configuration of the remaining epoxide

e. Using whole cells (dry weight, 5 mg)

f. Using whole cells (dry weight, 10 mg)

Supplementary Table 2.EnantioselectiveEHase activity of Sphingopyxis sp. BSNA05toward styrene oxide and its derivatives.

Strains / Hydrolysis rate
(x 10-2) mg/hour
SO / 2CSO / 3CSO / 4CSO
(R) / (S) / (R) / (S) / (R) / (S) / (R) / (S)
Sphingopyxis sp.
BSNA05
(8-NA-5) / 1.48 / 0.50 / 1.76 / 0.51 / 0.87 / 0.08 / 0.97 / 0.34

Isolation of microbial consortium and single colonies from PAH–based enrichment culture

A 0.5 g mud sample from the oil-spilled foreshore was suspended in 5 mL MSM with PAHs (naphthalene, phenanthrene, pyrene and benzopyrene) at 25 oC for 50 days. Then, 1 mL of the enriched culture was re-suspended in fresh MSM with PAHs for two weeks. A microbial consortium was obtained from the enriched culture, and then many single colonies possessing PAH-degrading activity were isolated from the consortium. Microbial strains possessing EHase activity were identified from the isolated colonies based on GC analysis for kinetic resolution of racemic styrene oxide using whole cells.

Marine broth medium (Difco) and mineral salt medium (MSM) (4 g NaNO3 l-1; 1.5 g KH2PO4 l-1; 0.005 g FeCl36H2O l-1; 0.2 g MgSO4 l-1; 0.01 g CaCl2H2O l-1; 0.5 g Na2HPO4 l-1; pH 7.2) were used in this study. Microorganisms were enriched using polycyclic aromatic hydrocarbon (PAH) and isolated from the samples of the oil-spilled foreshore of South Korea (Yeosu, Byeonsan, Gunsan and Taean).

Phylogenetic analysis of microbial strains using 16S rRNA sequencing

Genomic DNA of the isolated strain was isolated using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). The 16S rRNA was amplified using 16S rRNA primers, 27F (5'-AGA GTT TGA TCM TGG CTC AG-3'; Escherichia coli nucleotide 8~27) and 1518R (5'-AAG GAG GTG ATC CAN CCR CA-3'; E. coli nucleotide 1541~1522), from genomic DNA. PCR of 16S rRNA was conducted using PCR buffer containing 50 mMTris-HCl (pH 9.0), 0.1 % TritonX-100, 1.5 mM MgCl2, 0.2 mMdNTPs, 0.2 μM primers, 2.5 U Taq DNA polymerase (Promega), and 2.5 - 250 ng template in 50 μL. DNA amplification was conducted with a PE 2400 thermal cycler (PE Applied Biosystem, Foster City, California, USA). PCR conditions were as follows: initial denaturation at 94 oC for 5 min and 35 cycles of denaturation (94oC, 1 min), annealing (60oC, 1 min), extension (72 oC, 2 min), and then 7 min incubation at 72 oC. PCR products were separated using a DNA Purification System (Wizard PCR Preps DNA Purification System, Promega), and then were ligated into pGEM-T Easy Vector (Promega). The recombinant plasmids were transformed into E. coli JM109 host. The 16S rDNA sequence was determined using an automatic sequencer (ABI Prism 377 DNA Sequencer, Perkin Elmer, Waltham, MA, USA). Bioinformatic analysis of 16S rDNA was conducted using SIMILARITY-RANK of the Ribosomal Database Project (RDP), Basic Local Alignment Search Tool (BLAST) of the National Center Biotechnology Information (NCBI) and Phylogenetic Interference Package (PHYLIP, version 3.57c). Sequence similarity was analyzed using the DNADIST program, and a phylogenetic tree was constructed using the FITCH program.

Supplementary Fig. 1.Time-course of chiral GC analysis during enantioselective hydrolysis of 4 mMracemic styrene oxide by Sphingopyxis sp. BSNA05.

Supplementary Fig. 2.16S rRNA sequence-based phylogenetic tree of the strain 8-NA-5possessing epoxidehydrolase activity. The numbers within brackets indicate accession numbers from the GenBank data library.

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