Supplementary Data File

Supplementary data file:

Metagenomic Mining of FeruloylEsterases from Termite Enteric Flora

KonananiRashamuse, Tina Ronneburg,Walter Sanyika, KgamaMathiba, Edwin Mmutlane, and Dean Brady (2013)

Journal of applied Microbiology and Biotechnology (AMAB-D-13-00073),

CSIR Biosciences, MeiringNaudé Road; Brummeria; Pretoria; South Africa0001, E-mail: .

TABLE S1: A pair-wise sequence identity matrix of the seven feruloyl esterase amino acids sequences and the previously published.

References

FAE (ADU25538.1):

Cheng,F., Sheng,J., Cai,T., Jin,J., Liu,W., Lin,Y., Du,Y., Zhang,M.andShen,L (2012)A Protease-Insensitive Feruloyl Esterase from China Holstein Cow Rumen Metagenomic Library: Expression, Characterization, and Utilization in Ferulic Acid Release from Wheat Straw. J.Agric. Food Chem 60: 2546-2553

FAE (AEI54552.1)

Chandrasekharaiah,M., Thulasi,A., Vijayarani,K., Kumar,D.P., Santosh,S.S., Palanivel,C., Jose,V.L. and Sampath,K.T.(2012) Expression and biochemical characterization of two novel feruloylesterases derived from fecal samples of Rusaunicolor and Equusburchelli . Gene 500: 134-139

FAE (AFD03596.1)

Vieites,J.M., Ghazi,A., Beloqui,A., Polaina,J., Andreu,J.M., Golyshina,O.V., Nechitaylo,T.Y., Waliczek,A., Yakimov,M.M., Golyshin,P.N. and Ferrer,M.(2010) Inter-conversion of catalytic abilities in a bifunctional carboxyl/feruloyl-esterase from earthworm gut metagenome. J MicrobBiotechnol 3: 48-58

FAE (JQ303344)

Ferrer M, Ghazi A, Beloqui A, Vieites M, Corte P, Navarro J, Nechitaylo T, Guazzaroni M, Polaina J, Waliczek A, Chernikova T, RevaO,Golyshina O and Golyshi P. (2012) Functional Metagenomics Unveils a Multifunctional GlycosylHydrolase from the Family 43 Catalysing the Breakdown of Plant Polymers in the Calf Rumen. PLOS ONE

TableS2: A summary of FAEs purification

Steps / Total
Activity
(U) / Total Protein
(mg) / Specific activity
(U/mg) / Yield
(%) / Purification (-fold)
(cumulative)
Crude extract
FAE1 / 7243.4 / 170.0 / 42.6 ± 4.6 / 100.0 / 1.0
FAE2 / 1309.3 / 51.5 / 25.4 ±1.0 / 100.0 / 1.0
FAE4 / 1647.8 / 121.5 / 13.6 ± 3.1 / 100.0 / 1.0
FAE5 / 55.7 / 8.9 / 6.3 ± 0.1 / 100.0 / 1.0
FAE6 / 573.3 / 8.0 / 72.2 ± 0.7 / 100.0 / 1.0
FAE7 / 64.8 / 6.6 / 9.9 ± 0.1 / 100.0 / 1.0
(NH4)2SO4fractionation
FAE1 / 6343.1 / 141.9 / 44.7 ± 2.4 / 87.6 / 1.1
FAE2 / 1379.1 / 37.2 / 37.3 ±4.4 / 105.0 / 1.5
FAE4 / 1432.8 / 58.8 / 24.4 ± 3.3 / 87.0 / 1.8
FAE5 / 59.1 / 6.3 / 9.4 ± 0.1 / 106.2 / 1.5
FAE6 / 456.8 / 4.6 / 99.7 ± 0.9 / 79.7 / 1.4
FAE7 / 70.8 / 6.1 / 11.6 ± 0.1 / 109.3 / 1.2
Phenyl-Sepharose
FAE1 / 2871.6 / 4.2 / 683.7 ± 9.4 / 39.6 / 16.1
FAE2 / 1140.6 / 13.2 / 86.2 ±… / 87.0 / 3.4
FAE4 / 847.6 / 5.4 / 157.0 ± 5.0 / 59.2 / 11.6
FAE5 / 18.4 / 0.8 / 24.4 ± 0.7 / 33.1 / 3.9
FAE6 / 84.7 / 0.7 / 125.4 ± 1.8 / 14.8 / 1.7
FAE7 / 16.2 / 0.9 / 17.9 ± 0.3 / 25.1 / 1.8
Sephacryl S-300
FAE1 / 621.3 / 0.9 / 714.2 ± 26.7 / 8.6 / 16.8
FAE2 / 288.6 / 1.1 / 272.3 ± 10.1 / 22.0 / 10.7
FAE4 / 193.3 / 0.7 / 268.5 ± 7.1 / 22.8 / 19.8
FAE5 / 1.9 / 0.1 / 31.3 ± 2.8 / 3.4 / 5.0
FAE6 / 45.3 / 0.3 / 174.4 ± 3.0 / 7.9 / 2.4
FAE7 / 1.8 / 0.1 / 24.7 ± 0.2 / 2.8 / 2.5

Table S3:Determination of FAEs tertiary structure using Size exclusion chromatography

Enzyme / Mw
(kDa)
Subunit a / Mw
(kDa)
Native b / Globular State
Fae1 / 31.4 / 73.0 / Dimeric
Fae2 / 29.8 / 75.8 / Dimeric
Fae4 / 31.1 / 77.4 / Dimeric
Fae5 / 30.1 / 96.2 / Trimeric
Fae6 / 30.1 / 85.5 / Dimeric
Fae7 / 29.6 / 79.4 / Dimeric

a= Molecular mass subunits were predicted from amino acid sequences and were further confirmed by SDS-PAGE analyses

b=The native molecular mass were determined using Superdex 200 GL 10/300.

Table S4: Hydrolysis of p-nitrophenyl fatty acid esters by FAEs

Substrates / FAE1 / FAE 2 / FAE 4 / FAE 5 / FAE 6 / FAE 7
Relative rates of hydrolysis (%)
p-NP Acetate
(C3) / 31.6
(±1.4) / 100.0
(±3.4) / 15.1
(±1.3) / 18.1
(±3.2) / 26.3
(±2.8) / 52.5
(±5.4)
p-NP Butyrate
(C4) / 48.9
(±2.3) / 88.0
(±1.6) / 100.0
(±0.2) / 100.0
(±0.7) / 95.8
(±2.3) / 100.0
(±1.5)
p-NP Valerate
(C5) / 100.0
(±3.1) / 78.0
(±0.2) / 98.8
(±3.6) / 64.6
(±1.2) / 100.0
(±0.6) / 73.6
(±4.0)
p-NP Octanoate
(C8) / 24.8
(±3.6) / 26.9
(±4.8) / 25.2
(±14.9) / 17.4
(±1.3) / 79.7
(±2.4) / 31.7
(±3.3)
p-NP Decanoate
(C12) / 21.2
(±4.4) / 6.1
(±1.1) / 8.9
(±9.6) / 5.5
(±1.0) / 13.3
(±2.0) / 8.6
(±1.6)
p-NP Palmitate
(C16) / 0 / 0 / 0 / 0 / 0 / 0

All experiments were performed in triplicates and the values represent the means ± standard error from the triplicate values measured.

Figure S1: A Multiple sequence alignment of the seven termite derived FAEs. Conserved regions corresponding to the catalytic triad residue are boxed.The nucleotide sequences of the seven faegenes were submitted to GenBank, under the following accession numbers: FAE1 (KC493563), FAE2 (KC493564), FAE3 (KC493565), FAE4 (KC493566), FAE5 (KC493567), FAE6 (KC493568) and FAE7 (KC493569).

Figure S2: SDS-PAGE (12%) electrophoretogram showing the expression profile of the E.coliBL21(DE3) soluble fraction harbouring the following expression constructs. Lane M: Marker (kDa), Lane 1: soluble crude fraction of E. coli (BL21/ pET20b (+)), Lane 2: BL21/p20FAE1, Lane 3: BL21/p20FAE2, Lane 4: BL21/p20FAE3, Lane 5: BL21/p20FAE4, Lane 6: BL21/p20FAE5, Lane 7: BL21/p20FAE6, and Lane 8: BL21/p20FAE7

Figure S3: SDS-PAGE (12%) electrophoretogram showing the FAE fractions after the hydrophobic interaction and size exclusion chromatography. Lane M: Marker (kDa), Lane 1: FAE1, Lane 2: FAE2, Lane 3: FAE4, Lane 4: FAE5, Lane 5: FAE6, and, Lane 6: FAE7

Figure S4:(A) Temperature optima profiles of the six FAEs isolated from the termite hindgut.(B) Thermostability profile of FAE4over an 8h period. All experiments were performed in triplicates and the values represent the means from the triplicate values measured.

Figure S5: (A) pH optima profiles of the six FAEs isolated from the termite hindgut. All experiments were performed in triplicates and the values represent the means from the triplicate values measured.