Efficient Preparation of Streptochlorin from Marine Streptomyces sp. SYYLWHS-1-4 by Combination of Response Surface Methodology and High-Speed Counter-Current Chromatography
Abstract
:1. Introduction
2. Results
2.1. Optimization of Culture Conditions by RSM
2.1.1. Determination of the Best Time in Flask Fermentation
2.1.2. Plackett-Burman Design and Identification of Significant Variables that Affect Streptochlorin Production
2.1.3. Optimization by Box-Behnken Design
2.1.4. Validation of the Optimized Condition
2.2. Preparation of Streptochlorin by HSCCC
2.2.1. UPLC Analysis of the Crude Sample
2.2.2. Selection of Suitable Two-Phase Solvent System
2.2.3. HSCCC Separation
2.2.4. UPLC Analysis and Identification of Peak Fraction
2.2.5. Comparison of Two Chromatographic Systems for Preparative Separation of Streptochlorin
3. Discussion
4. Materials and Methods
4.1. Microorganism and Fermentation Conditions
4.2. Experimental Design and RSM Optimization
4.3. Production of Streptochlorin from the Streptomyces Strain SYYLWHS-1-4 by a Jar Fermenter
4.4. UPLC Analysis, Preparative HPLC Separation and Structure Identification
4.5. Preparation of Crude Sample
4.6. Evaluation of Partition Coefficient Value
4.7. Preparation of Solvent System and Sample Solution for HSCCC Separation
4.8. HSCCC Separation
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
RSM | Response surface methodology |
HSCCC | High-speed counter-current chromatography |
PBD | Plackett-Burman design |
BBD | Box-Bohnken design |
UPLC | Ultra Performance Liquid Chromatography |
HPLC | High Performance Liquid Chromatography |
ESI-MS | Electro Spray Ionization-Mass Spectrometry |
NMR | Nuclear Magnetic Resonance |
HRESIMS | High Resolution Electrospray Ionization Mass Spectroscopy |
TMS | Tetramethylsilane |
DKPs | Diketopiperazines |
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Variable | Code | Low Level | High Level | Coefficient | t-Value | p-Value |
---|---|---|---|---|---|---|
Intercept | 1.0930 | 17.44 | 0.000 | |||
Yeast extract (g/L) | X1 | 2 | 3 | −0.3460 | −5.52 | 0.000 |
soluble starch (g/L) | X2 | 10 | 15 | −0.1520 | −2.42 | 0.036 |
MgSO4 (g/L) | X3 | 0.5 | 0.75 | −0.0950 | −1.52 | 0.161 |
K2HPO4 (g/L) | X4 | 0.5 | 0.75 | −0.1980 | −3.16 | 0.010 |
CaCl2 (g/L) | X5 | 2 | 3 | 0.0370 | 0.59 | 0.568 |
Initial pH value | X6 | 6 | 8 | 0.0100 | 0.16 | 0.876 |
Medium volume (%) | X7 | 40 | 60 | −0.0330 | −0.53 | 0.610 |
Temperature (°C) | X8 | 25 | 30 | −0.0840 | −1.34 | 0.210 |
Marine salt (g/L) | X9 | 20 | 30 | 0.1130 | 1.80 | 0.102 |
Variables | Code | Level | ||
---|---|---|---|---|
−1 | 0 | 1 | ||
Yeast extract (g/L) | X1 | 1 | 1.5 | 2 |
Soluble starch (g/L) | X2 | 5 | 7.5 | 10 |
K2HPO4 (g/L) | X4 | 0.15 | 0.3 | 0.45 |
Source | SS | df | MS | F Value | p-Value |
---|---|---|---|---|---|
Model | 11.2888 | 9 | 1.25431 | 49.54 | <0.001 |
Residual | 0.1266 | 5 | 0.02532 | ||
Lack of Fit | 0.1162 | 3 | 0.03874 | 7.48 | 0.120 |
Pure Error | 0.0104 | 2 | 0.00518 | ||
Cor Total | 11.4154 | 14 |
Variables | Coefficient | p-Value |
---|---|---|
Intercept | 2.9040 | <0.001 |
X1 | 0.7703 | <0.001 |
X2 | 0.5307 | <0.001 |
X4 | 0.4665 | <0.001 |
X12 | −0.4688 | 0.002 |
X22 | −0.5895 | 0.001 |
X42 | −0.1994 | 0.006 |
X1 × X2 | 0.1315 | 0.500 |
X1 × X4 | −0.2668 | 0.162 |
X2 × X4 | −0.2163 | 0.153 |
Petroleum Ether–Ethyl Acetate–Methanol–Water | KD |
---|---|
5:5:5:5 | 6.47 |
8:2:5:5 | 3.47 |
9:1:5:5 | 1.33 |
9:0.8:5:5 | 1.05 |
10:0:5:5 | 0.12 |
HSCCC | Preparative HPLC | |
---|---|---|
Stationary phase | Upper phase petroleum ether–ethyl acetate–methanol–water (9:0.8:5:5, v/v/v/v) | YMC C18 column 250 mm × 20 mm ID 5 μm |
Mobile phase | Lower phase | Methanol–water (65:35, v/v) |
Sample capacity per run (mg) | 300 | 40 |
Run time (min) | 55 | 20 |
Organic solvent consumption (L/mg) | 0.048 | 0.15 |
Productivity (mg/min) | 0.3 | 0.06 |
Purity of isolated compound | 97% | 98% |
Sample recovery | 91% | 48% |
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Li, L.; He, S.; Ding, L.; Yuan, Y.; Zhu, P.; Epstein, S.; Fan, J.; Wu, X.; Yan, X. Efficient Preparation of Streptochlorin from Marine Streptomyces sp. SYYLWHS-1-4 by Combination of Response Surface Methodology and High-Speed Counter-Current Chromatography. Molecules 2016, 21, 693. https://doi.org/10.3390/molecules21060693
Li L, He S, Ding L, Yuan Y, Zhu P, Epstein S, Fan J, Wu X, Yan X. Efficient Preparation of Streptochlorin from Marine Streptomyces sp. SYYLWHS-1-4 by Combination of Response Surface Methodology and High-Speed Counter-Current Chromatography. Molecules. 2016; 21(6):693. https://doi.org/10.3390/molecules21060693
Chicago/Turabian StyleLi, Lin, Shan He, Lijian Ding, Ye Yuan, Peng Zhu, Slava Epstein, Jianzhong Fan, Xiaokai Wu, and Xiaojun Yan. 2016. "Efficient Preparation of Streptochlorin from Marine Streptomyces sp. SYYLWHS-1-4 by Combination of Response Surface Methodology and High-Speed Counter-Current Chromatography" Molecules 21, no. 6: 693. https://doi.org/10.3390/molecules21060693