World Journal of Microbiology and Biotechnology

Supplementary material for

Improving the yieldof (+)-terrein from the salt-tolerantAspergillus terreusPT06-2

ChengyingZhaoa,†, Lei Guoa,†, LipingWangb, GuoliangZhua, WeimingZhua,*

a Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China

bKey Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guiyang 550002, China

† These authorsmakeequalcontributiontothiswork.

* Corresponding author at: School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.

Tel./Fax: +86-532-8203-1268. E-mail address:.

Procedure of fungus fermentation and isolation of (+)-terrein in verification experiment.

The seed liquid ofA. terreus PT06-2 was prepared by cultivation for 3 days at 28 °C and 180 rpm in a 500-mL shake flask containing 150 mL liquid medium MI. The liquid with 3% inoculum size was seeded into a 500-mL shake flask containing 150 mL the optimal medium consisted of 13.1% NaCl, 3.6% starch, 2% sodium glutamate, 0.05% KCl, and adjustinginitial pH to 5 by 10% hydrochloric acid. After cultured for 18 days at 28 °C and 180 rpm, 350 mL ethyl acetate (EtOAc) was added to the fermentation broth and thenwas ultrasonic fragmentation for 1h. The EtOAc solution was separated and the water layer was extracted twice with 350 mLEtOAc. The EtOAc solutions were combined and concentrated under reduced pressure to give the crude extract. The crudeextract was resolved in MeOH at 55 °C and then cooled to room temperature. Colorlessneedles crystallized from the MeOH solution and were separated by suction filtration and dryness to give (+)-terrein (1281 mg) (8.54 g/L yield). The other four parallel experiments gave (+)-terrein with the yields of 8.21 g/L, 8.31 g/L, 8.09 g/L and 7.82 g/L, respectively. The average yield of(+)-terrein in five experiments is (8.20± 0.072) g/L. (+)-Terrein was identified by NMR spectra, specific rotation, CD and ECD calculation.

Physical data of (+)-terrein from A.terreusPT06-2

(+)-Terrein: colorless needle crystal;m.p. 125–126 oC; [α]D25+368 (c 0.1, acetone); UV (MeOH) λmax (log ε) 275.0 (4.27) nm; CD (c 0.1, MeOH) λmax (Δε) 211.5 (–2.20), 244.2 (+9.14), 271.4 (+7.25) and 346.5 (–1.62) nm;IR (KBr) νmax3416, 3380, 1695, 1637, 1118, 1085 cm-1;1H (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see TableS1; ESI-MS m/z 155.0 [M+H]+, 177.1 [M+Na]+.

Table S1. 1H (500 MHz) and 13C (125 MHz) NMR data of (+)-terrein (DMSO-d6, TMS, δ ppm)

Position / δC / δH (J in Hz)
1 / 203.6, qC
2 / 80.8, CH / 3.89, dd (2.7, 6.4)
3 / 76.4, CH / 4.50, dd (2.7, 7.4)
4 / 168.5, qC
5 / 124.7, CH / 6.02, s
6 / 125.5, CH / 6.37, dd (2.0, 15.9)
7 / 139.3, CH / 6.72, dq (6.9, 15.8)
8 / 19.1, CH3 / 1.88, dd (1.7, 6.9)
2-OH / 5.66, d (6.5)
3-OH / 5.78, d (7.4)

Fig. S1.The 1H-NMR spectrum of (+)-terrein in DMSO-d6

Fig. S2.The 13C-NMR spectrum of (+)-terrein in DMSO-d6

Fig. S3.The DEPT spectrum of (+)-terrein in DMSO-d6

Fig. S4.CD spectrum of (+)-terrein and the calculated ECD spectra of (+)- and (−)-terrein