Bioconversion of P-Coumaric Acid to P-Hydroxystyreneusing Phenolic Acid Decarboxylase

Bioconversion of p-Coumaric Acid to p-Hydroxystyreneusing Phenolic Acid Decarboxylase from B. amyloliquefaciens in Biphasic Reaction System

Da-HyeJunga, WonjiChoia,Kwon-Young Choia, EunokJunga, HyungdonYunb, Romas J. Kazlauskasa,c, Byung-Gee Kima*

aSchool of Chemical and Biological Engineering, Seoul National University, Seoul, #151-742, Republic of Korea

bSchool of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk #712-749, Republic of Korea

cUniversity of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics, 1479 Gortner Avenue, 174 Gortner Lab, St. Paul, MN 55108, USA

* To whom correspondence should be addressed.

Corresponding author:

Address School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea

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Supplementary Data

Supplementary data 1Oligonucleotides for amplification of eight PAD representative genes

PADs a
(Accession No.) / Oligonucleotides for amplification b
BAPAD
(YP_003921894.1) / forward / 5’-AAA GGATCC ATGGAAAACTTTATC-3’
backward / 5’-AAA AAGCTT TTTTAATTTTCCCG-3’
BCPAD
(YP_004859545.1) / forward / 5’-AAT GGATCC ATG AAA ACA TTG AAG-3’
backward / 5’-TTC AAGCTT TGA AAA TTT TAG TTT -3’
CiPAD
(ZP_06355613.1) / forward / 5’-ATA GGATCC ATG AGC GCG TTT G AT -3’
backward / 5’-TTC AAGCTT GCG TAA ATT TTG CGG-3’
LFPAD
(ZP_03945087.1) / forward / 5’-AAA GGATCC ATGGCACAATTAAAC-3’
backward / 5’-AAA AAGCTT TTTTTGACGACGGTA-3’
LLPAD
(NP_268087.1) / forward / 5’-AAA GGATCC ATGAAAACATTTAAA-3’
backward / 5’-AAA CTCGAG ATGCTTAATCATTTTA-3’
LPPAD
(ZP_07078975.1) / forward / 5’-GTA GGATCC ATGACAAAAACTTTTAAA-3’
backward / 5’-GAA AAGCTTCTTATTTAA ACGATGGTA-3’
LSPAD
(YP_396314.1) / forward / 5’-AAA GGATCC ATGACAAAACAATTTA-3’
backward / 5’-AAA AAGCTT ACGATTAATCTTCTTGTA-3’
PPPAD
(YP_804027.1) / forward / 5’-TAA GGATCC ATGGAAAAAACTTTTA-3’
backward / 5’-TAA AAGCTT TTTGTTAATGCGTTTG-3’

aBAPAD = phenolic acid decarboxylase from B. amyloliquefaciens(KCTC 1660); BCPAD B. coagulans(KCTC 3625); CiPADCitrobacter sp.(KACC 16595); LFPAD = L. fermentum(KCTC 3112);LLPAD = L. lactis(KCTC 2013);LPPAD = L. plantarum(KACC 11451);LSPAD= L. sakei(KCTC 3598)and PPPAD = P. pentosaceus(KCTC 3507).

bUnderline indicates the restriction enzyme recognition site. Restriction sites are BamH1 for forward and HindIII, Sac1 for backward primers.

Supplementary data 2 Variation of activity of purified phenolic acid decarboxylase from Bacillus amyloliquefaciens with pH (a) and temperature (1 h incubation) (b). The optimum pH was determined by incubating BAPAD at pH ranges from 3 to 11 for 1 hr. Citric acid-sodium citrate buffer (50 mM) was used for pH 3-5, phosphate buffer (50 mM) for pH 5.8-8, boric acid buffer (50 mM) for pH 9-11. And the optimum temperature was measured by at different temperature from 10 to 70 C for 1 hr. Decarboxylation reaction of pCA was measured in 50 mM phosphate buffer (Final volume 100μl, pH 7.0) containing 1.25 mMpCA and 1 μM BAPAD at 30 C. The decarboxylase was most active at pH 5.8 and 37 °C.

Supplementary data 3 Substrate specificities of BAPAD.Decarboxylation rate of BAPAD toward cinnamic acid derivatives (p-coumaric, m-coumaric, o-coumaric, ferulic, caffeic, sinapinic and cinnamic acid) were measured at pH 5.8 and 30 °C by adding1 μM of purified

BAPADin 50 mM phosphate buffer (Final volume 100μl, pH 7.0) containing 1.25 mM each substrate.

Substrate / Product / Relative specific activity (%)a
cinnamate / ND / ND
p-coumaric acid
(4-hydroxycinnamate) / p-hydroxystyrene / 100
o-coumaric acid / ND / ND
m-coumaric acid / ND / ND
caffeic acid
(3,4-dihydroxycinnamate) / 3,4-dihydroxystyrene / 65
ferulic acid
(3-methoxy-4-hydroxycinnamate) / 3-methoxy-4-hydroxystyrene / 71
sinapinic acid
(3,4-dimethoxy-4-hydroxycinnamate) / 3,5-methoxy-4-hydroxystyrene / <1.0

a Specific activity values given as a percentage of the total specific activity measured with pCA (100%)

b 100% specific activity of BAPAD toward p-coumaric acid: 370 μmol min-1 mg-1

c ND (not determined) refers to measurements below the sensitivity of the assay

Supplementary data 4Initial decarboxylation rate of pCA using recombinant E.coli BL21(DE3) harboring BAPAD in the presence of various pHS concentrations. The sampling and reaction condition was elaborated in “Material and Method” section.

Supplementary data 5Conversion yields versus residence time of the substrate(pCA) in PBR equipped with a solvent extraction unit. The recirculationflow rates at an inlet were varied from 50to 400 ml/hr.Immobilized 0.49 g DCW of recombinant E.coli cells were packed in PBR reactor (glass column, Size 16 mm × 40 cm, 50 mL working volume) temperature-controlled at 37°C, the substrate solution (50 mMpCA, 100 mMTris-buffer pH7.6, 10 mM CaCl2) was fed upwardly from the bottom of the reactor.