Supplementary Material
Title: A Novel High-Throughput and Quantitative Method
Based on Visible Color Shifts for Screening Bacillus subtilis
THY-15 for Surfactin Production
Journal of Industrial Microbiology & Biotechnology
Huan Yang, Huimin Yu* and Zhongyao Shen
Key Laboratory for Industrial Biocatalysis of the Ministry of Education, Department
of Chemical Engineering, Tsinghua University, Beijing, 100084, China
* Corresponding author:
Telephone: 86-10-62795492, Fax: 86-10-62770304
E-mail: yuhm@tsinghua.edu.cn
Fig. S1 RP-HPLC chromatograms of four purified surfactin isoforms
(a) Surfactin mixture produced by THY-15.
(b) ~ (e) Verification of purified fraction 1-4 by RP-HPLC.
Fig. S2 MALDI-TOF mass spectra of C13 surfactin precursors in fraction 1
(a) Surfactin isoform ions, [M+H] + at m/z 1008, [M+Na]+ at m/z 1030 and [M+K] + at m/z 1046.
(b) MALDI-TOF-MS/MS of [M+Na]+ precursor at m/z 1030 in fraction 1.
(c) The molecular structure of surfactin isoform in fraction 1, containing a C13-β-hydroxy fatty acid
chain and a Glu-Leu-Leu-Val-Asp-Leu-Leu peptide.
Fig. S3 MALDI-TOF mass spectra of C14 surfactin precursors in fraction 2
(a) Surfactin isoform ions, [M+H] + at m/z 1022, [M+Na]+ at m/z 1044 and [M+K] + at m/z 1060.
(b) MALDI-TOF-MS/MS of [M+Na]+ precursor at m/z 1044 in fraction 2.
(c) The molecular structure of surfactin isoform in fraction 2, containing a C14-β-hydroxy fatty acid
chain and a Glu-Leu-Leu-Val-Asp-Leu-Leu peptide.
MALDI-TOF mass spectra of isoforms in HPLC fraction 1, 2 and 4 mainly showed the [M+Na]+ ions
at m/z 1030.6, 1044.6 and 1058.6, respectively, suggesting that there is only a difference of 14 Da
(-CH2-). The MS/MS also exhibited the difference of 14 Da (-CH2-) in the series of b+ fragment ions.
They were proved to be homologues with the same Glu-Leu-Leu-Val-Asp-Leu-Leu peptide but varied
in C13, C14 and C15 fatty acid chains.
Fig. S4 MALDI-TOF mass spectra of C14 surfactin precursors in fraction 3
(a) Surfactin isoform ions, [M+H] + at m/z 1008, [M+Na]+ at m/z 1030 and [M+K] + at m/z 1046.
(b) MALDI-TOF-MS/MS of [M+Na]+ precursor at m/z 1030 in fraction 3.
(c) The molecular structure of surfactin isoform in fraction 3, containing a C14-β-hydroxy fatty acid
chain and a Glu-Val-Leu-Leu-Asp-Leu-Val peptide.
Another peptide sequence slightly different from the typical surfactin was discovered in fraction 3 (Fig.
S4).
The
significant
ion
series
at
m/z
1030→931→818(-H2O,800)→703→590
and
693→594→481→368→253, represented the sequence of Val-Leu-Asp-Leu from the C-terminus and
Val-Leu-Leu-Asp in the middle, suggesting the precursor ion possessed of a peptide sequence of
Glu-Val-Leu-Leu-Asp-Leu-Val and a C14 fatty acid.
In Fig. 5C of the manuscript, the first and second peaks are both Asp. As shown in the following
chromatograms of standard amino acids, sometimes the fraction of Asp would be broken into two
peaks at different loading amount.
In other literatures, this phenomenon was also reported (Liu et al. 2009). The last peak might be the
remaining PITC (since it was found in the negative control), as described in the following
chromatograms in the same literature (Liu et al. 2009).
(A) Standard amino acids; (B) negative control; (C) samples from of the hydrolyte of N1
Reference
Liu X-Y, Yang S-Z, Mu B-Z (2009) Production and characterization of a C-15-surfactin-O-methyl ester
by a
lipopeptide
producing
strain Bacillus
44(10):1144-1151 doi:10.1016/j.procbio.2009.06.014
subtilis HSO121.
Process Biochemistry