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1 46 5 Vol.46, No OCEANOLOGIA ET LIMNOLOGIA SINICA Sep., 2015 (Bacillus amyloliquefacien) Macrolactin A * ( ) (Bacillus amyloliquefaciens)esb-2 Macrolactin A,,, Macrolactin A Macrolactin A, : Macrolactin A, 95%, ; Macrolactin A,, 1%, 18.5mg/L, 2 Macrolactin A ; ; Macrolactin A; Q936 doi: /hyhz (Tulp et al, 2004),, (Haefner, 2003;, 2006) Macrolactin Scripps Fenical 24 (He et al, 2012b), 19 (Gustafson et al, 1989; Jaruchoktaweechai et al, 2000) Macrolatin A 24, 1 Macrolactin A, B16-F10, Macrolactin A (Herpes simplex) HIV T (Kim et al, 1997;, 2008), Macrolactin A,,,,, Macrolatin A,, Macrolactin (Bacillus amyloliquefaciens) ESB-2 (He et al, 2012a), Macrolatin A,, Macrolatin A (He et al, 2012a), Macrolactin A (Yang et al, 2009),, (Kim et al, 2005) Macrolactin A ; (high speed countercurrent chromatography, HSCCC) *, ,, linhoudy@163.com :,,,, wuzufang@nbu.edu.cn : , :

2 (Ito, 1991),, (Owen et al, 1997; Li et al, 2001), (, 2003), (Dalhoff et al, 2003; Nishivama et al, 2000) Macrolactin A,,,, Macrolactin A, 1 Macrolactin A Fig.1 The chemical structure of Macrolactin A (Bacillus amyloliquefaciens)esb-2, E, 5.0g 1.0g 15.0g 35.0g 0.1g 1000mL, ESB-2 ;, 5.0g 1.0g 35.0g 0.1g 1000mL, ESB C, Pa 20min : TAUTO HSCCC-TBE300B (, 2.6mm, 280mL, 20mL), SHP DC-0506, AKTAprime plus, ; Waters e2695, YMC- Pack ODS-A(150 mm 4.6 mm, 5 μm), Waters ; SPX, ; GL- 21MC, ; UV-3300 ; LDZX-50KBS, ; SW-CJ-2D ; HGC-12D ; PHS-3C ph ; RE-52C (, CNW ), ADS-30, (, ) ESB-2 24h, 100mL 250mL,, 30 C 150r/min 24h,, 5%,, 30 C 150r/min 48h, Macrolactin A,, 45 C, 1mL, 12000r/min 15min, , 1%,, %,, 5%, 40%, 30 C, 150r/min, 48h,,,, 0. 45μm, HPLC Macrolactin A, Macrolactin A Macrolactin A Macrolactin A : 30L, (8000 r/min, 10min) ADS-30,,,,,,, Macrolactin A, : V( ) : V( ) : V( ).. V( ) , ;,, 20 min ; [ V( ) : V( ) = ]

3 5 : (Bacillus amyloliquefacien) Macrolactin A 1073 (, 2010), 30mL/min,, 800 r/min, 10mL/min, 280nm, : HPLC, YMC-Pack ODS-A (150 mm 4.6 mm, 5 μm), 280nm, 30 C, ; 60min 10% 90%, 10min, 0.8mL/min, 10μL Macrolactin A,, 1mg/mL, mg/L,, : Macrolactin A, YMC-Pack ODS-A (150 mm 4.6 mm, 5 μm), 280nm, 30 C, (A) : (B) ; 60min (A) 10% 90%, 0.8mL/min, 10μL ph (, 2014), ESB-2 5%,,, 2h 5mL OD ; ph ph; Macrolactin A Macrolactin A HSCCC V( ) : V( ) : V( ) : V( ) , 1.2.5, 10mL, HSCCC, HSCCC ( 2), 48%, HPLC, HPLC A Macrolactin A, HPLC, 95.8%, HPLC 3 2 Macrolactin A HSCCC Fig.2 HSCCC chromatogram of the crude extract of Macrolactin A 3 HSCCC Macrolactin A (a) (b) Fig.3 HPLC and UV of Macrolactin A by HSCCC ADS-30,,, 23% HSCCC, HSCCC, HSCCC,, 30s; K 0.5<K<2 (Cao et al, 2011) HSCCC, 48%, 350mg, HSCCC Macrolactin A 50.4mg,,, HSCCC Macrolactin A 1mg/mL, mg/L,

4 , Macrolactin A Y=2.9E+6X 33234, R 2 =0.998,,, (, 2013) ESB-2, 1%,, 30 C, 24h ( 4), 2216E,, Fig.4 4 ESB-2 Effect of carbon sourceon thenumber of totalcolony of ESB-2,,,, ESB-2,, Macrolactin A ESB-2 Macrolactin A, 7, 1% (W/V),,, Macrolactin A 1 1, Macrolactin A,,, Macrolactin A Macrolactin A (, 2010), (, 2011);,,, Macrolactin A 2216E, SAS,,,,, 表 1 碳源对 ESB-2 菌株生物量及 Macrolactin A 产量的影响 Tab.1 Effects of carbon source on biomass and Macrolactin A production by ESB-2 ph (10 8 cfu/ml) Macrolactin A (mg/l) ±1.0 e* 9.034±0.451 d Glucose ±0.50 d ±0.131 b Maltose ±1.80 b ±0.220 a Lactos ±3.11 c 5.095±0.110 de Starch ±5.17 a 9.322±0.153 d α-cyclodextrin ±1.32 e 0.665±0.114 f Sucrose ±1.32 ef ±0.321 c Glycerol ±0.57 f 1.545±0.211 e * ±, SAS (P<0.05)

5 5 : (Bacillus amyloliquefacien) Macrolactin A Macrolactin A ( g/l),, Macrolactin A, 5 5, Macrolactin A,,, 10 g/l, MA, 10 g/l, Macrolactin A, 10 g/l, Macrolactin A, g/l, 2.08,, 1% 5 Macrolactin A Fig.5 Effects of maltose concentration on Macrolactin A production, Macrolactin A,, Macrolactin A,, Macrolactin A,,, (, 2013) 2.2.4, ESB-2 (0 48h), 6,, ph,, 0 12h,,, Macrolactin A, ph 12 24h,, ph,, Macrolactin A 24 48h, ph,,, (, 2008), 24h,, (, 2013),,,, Fig 6 6 ESB-2 The fermentation curves of shaking flask culture

6 ,, ADS-30 Macrolactin A, 95.8%,,, ESB-2,, 1%, 2d mg/l, 2, Macrolactin A 7d,, Macrolactin A 3.5,,,,, , 8(4): 1 4,,, (Bacillus amyloliquefaciens) Macrolactin B., 44(6): , :, :,,, β-., 36(10): 66 70,, , 18(6): ,,, , 34(14): 57 62,,, , 20(5): , :,,, , 28(1): 68 72,,, Macrolactins X-2 PKS., 35(9): Cao X L, Wang Q E, Li Y et al, Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography. Journal of Chromatography B, 879(7 8): Dalhoff A, Nasu T, Okamoto K, Beta-lactamase stability of faropenem. Chemotherapy, 49(5): Gustafson K, Roman M, Fenical W, The macrolactins, a novel class of antiviral and cytotoxic macrolides from a deep-sea marine bacterium. Journal of the American Chemical Society, 111(19): Haefner B, Drugs from the deep: marine natural products as drug candidates. Drug Discovery Today, 8(12): He S, Wang H Q, Wu B et al, 2012a. Response surface methodology optimization of fermentation conditions for rapid and efficient accumulation of macrolactin a by marine Bacillus amyloliquefaciens ESB-2. Molecules, 18(1): He S, Wang H Q, Yan X J et al, 2012b. Preparative isolation and purification of macrolactin antibiotics from marine bacterium Bacillus amyloliquefaciens using high-speed counter-current chromatography in stepwise elution mode. Journal of Chromatography A, 1272: Ito Y, Recent advances in counter-current chromatography. Journal of Chromatography A, 538(1): 3 25 Jaruchoktaweechai C, Suwanborirux K, Tanasupawatt S et al, New macrolactins from a marine Bacillus sp. Sc026. Journal of Natural Products, 63(7): Kim H-H, Hwang S-Y, Kim W-G et al, Neuronal cell protection activity of macrolactin A produced by Actinomadura sp.. Journal of Microbiology and Biotechnology, 7(6): Kim H O, Lim J M, Joo J H et al, Optimization of submerged culture condition for the production of mycelial biomass and exopolysaccharides by Agrocybe cylindracea. Bioresource Technology, 96(10): Li H B, Chen F, Preparative isolation and purification of six diterpenoids from the Chinese medicinal plant Salvia miltiorrhiza by high-speed counter-current chromatography. Journal of Chromatography A, 925(1 2): Nishivama T, Matsuzaki K, Kovama H et al, In vitro activity of faropenem against beta-lactamase producing clinical isolates. The Japanese Journal of Antibiotics, 53(3): Owen R O, McCreath G E, Chase H A, A new approach to continuous counter-current protein chromatography: Direct purification of malate dehydrogenase from a Saccharomyces cerevisiae homogenate as a model system. Biotechnology and Bioengineering, 53(4): Tulp M, Bohlin L, Unconventional natural sources for future drug discovery. Drug Discovery Today, 9(10): Yang Q, Han W J, Zhang W J et al, 2009.Identification of a macrolactina antibiotic-producing marine Bacillus amyloliquefaciens JY-863 strain and optimization of its fermentation conditions. Pharmaceutical Biotechnology, 16(4): , 346

7 5 : (Bacillus amyloliquefacien) Macrolactin A 1077 OPTIMIZATION OF CARBON RESOURCE FOR MACROLACTIN A PRODUCTION FROM MARINE BACILLUS AMYLOLIQUEFACIEN HOU Dong-Yuan, WU Zu-Fang, ZHANG Xin (Ningbo University, Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Ningbo , China) Abstract To optimize the production of Macrolactin A from a marine Bacillus amyloliquefaciens ESB-2, we extract crude material with macroporous adsorptive resin and then purified in high-speed counter-current chromatography (HSCCC). The purity of separated component was determined and the influence of different carbon sources on Macrolactin A yield was detected, in high performance liquid chromatography (HPLC). The fermentation curve of marine Bacillus amyloliquefaciens was obtained under optimization condition. The results show that Macrolactin A could be purified quickly in HSCCC, and the purity of the product reached >95%. The carbon sources of glucose, sucrose, and maltose were conducive for the accumulation of Macrolactin A, and maltose was the best carbon source at concentration of 1%, under which the concentration of Macrolactin A could reach 18.5 mg/l and the production was more than doubled. Key words high-speed counter-current chromatography (HSCCC); Bacillus amyloliquefaciens; Macrolactin A; carbon resource optimization

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g 16 6 2018 11 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov. 2018 doi 10. 3969 /j. issn. 1672-3678. 2018. 06. 004 1 2 1 1 1. 330200 2. 330006 4 Box-Behnken 1 25 g /ml 121 92 min 2 6. 284%