Screening of Monacolin K-high-yielding Monascus strain by combinative mutation treatment

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1 264507~ Mycosystema Monascus purpureus 45s 1.0 Monacolin K Monascus purpureus ZT32 5 Monacolin K 219.9µg/mL 2 Monacolin K 8.33mg/g 3.3 Q939.5 A Screening of Monacolin K-high-yielding Monascus strain by combinative mutation treatment SUN Jia-Long 1,2,3,4 ZOU Xiao 4 LIU Ai-Ying 4* LIANG Zong-Qi 4 XIAO Tang-Fu 1 ( 1 State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese Academy of Sciences, Guiyang , China; 2 Graduate School of Chinese Academy of Sciences, Beijing , China; 3 Guizhou Institute of Environmental Science, Guiyang , China; 4 Institute of Fungus Resources, Guizhou University, Huaxi , China) Abstract: A strain of Monascus purpureus isolated from red yeast rice in Zhangzhou, Fujian Province, was treated with UV irradiation, and then the spore suspension was incubated in media containing LiCl. The exposure time under UV is 45s and the concentration of LiCl in the medium is 1.0.The mutant Monascus purpureus ZT32 was obtained and detected by ultraviolet spectrophotometry. The content of Monacolin K for the fifth subsequent generation of subcultures reached 219.9µg/mL, more two times than that for the original strain. In the solid-state fermentation of red yeast rice using the mutant, the production of Monacolin K attained 8.33mg/g, 3 times than that of the original strain. Key words: Monascus purprueus, UV irradiation, LiCl, liquid fermentation, solid-state fermentation Monascus 2001 * Corresponding author. ayliu6@yahoo.com.cn

2 (Heber et al ) 5 Monacolin Monacolin K Lovastatin HMG-CoA HMG-CoA HMG-CoA Endo 2000Monacolin K 1994Endo McDermott Monascus purpureus ZZMonascus rubber ZKMonascus purpureus GX Monascus sp. XM : g 1000mLpH min % 3% 3% 0.8% 0.8% 0.1% 1000mLpH K 5 50g 12h min mL /ml 9cm1999 1min 15s, 30s, 45s, 60s, 75s 90s 1h /ml 0.1mL d

3 4 Monacolin K /ml 0.1mL d Monacolin K Monacolin K d 30120r/min 13d Monacolin K d 5mL 0.05% Tween-80 50mL 250mL 30120r/min 2~4d 2% 50mL 250mL 30120r/min 13d Monacolin K Monacolin K Monacolin K 50mL 50mL ph h 5% Na 2 CO mL mL 20mL ph=3.0250r/min305h 5% Na 2 CO mL Monacolin K TLC Monacolin K µL 5mm :4:1 1h Rf

4 Monacolin K Monacolin K Sigma 2mg 20mL mL 10mL 10mL 238nm OD Monacolin K Monacolin K Monacolin K 4 Rf Monacolin K Monacolin K 238nm Monacolin K 1Y=0.0508X r= Y Monacolin K µg/mlx 238nm Monacolin K 2µg/mL14µg/mL Fig.1 The standard curve of Monacolin K. Monacolin K 1 ZZ Monacolin K ZZ

5 4 Monacolin K 511 ZZ 5d Monacolin K 5 Monacolin K 13d 13d Monacolin K Table 1 Comparison of Monacolin K production in various strains of Monascus Prepared strains of Monascus spp. Monacolin K µg/ml Content of Monacolin K (µg/ml Monascus purpureus GX 57.8 Monascus sp. XM 41.8 Monascus rubber ZK 52.8 Monascus purpureus ZZ 65.4 Fig.2 Growth curve of Monascus purpureus. 2.2 ZZ ZZ ZZ 2 45s 90% 7.6% 45s

6 Table 2 Selection of optimum exposure duration under UV for M. purpureus ZZ strain Duration treated UVs % Death rate% % Positive mutation rate% ZZ ZZ % 3.1% 1 Table 3 Selection of optimum concentration of LiCl for M. purpureus ZZ strain LiCl Concentration of LiCl % Death rate% % Positive mutation rate% s ZZ ZZ 45s % 4% Table 4 Selection optimum concentration of LiCl for M. purpureus ZZ strain mutation after 45s irradiation of UV LiCl Concentration of LiCl % Death rate% % Positive mutated rate%

7 4 Monacolin K ZZ ZZ 57 Monacolin K Table 5 Biomass and content of Monacolin K in the five mutated strains(zt) of M. purpureus in the first round mutation screening Strains g/50ml Biomassg/50mL Monacolin K µg/ml Content of Monacolin Kµg/mL ZZ ZT ZT ZT ZT ZT Monacolin K Table 6 Biomass and content of Monacolin K in the five mutated strains(zt) of M. purpureus in the second round mutation screening Strains g/50ml Biomassg/50mL Monacolin K µg/ml Content of Monacolin Kµg/mL ZZ ZT ZT ZT ZT ZT Monacolin K Table 7 Biomass and content of Monacolin K in the five mutated strainszt of M. purpureus in the third round mutation screening Strains g/50ml Biomassg/50mL Monacolin K µg/ml Content of Monacolin Kµg/mL ZZ ZT ZT ZT ZT ZT

8 ZZ ZZ ZT32ZT33 ZT35 5 Monacolin K 5 8 Monacolin K ZT32 Monacolin K Table 8 Biomass and content of Monacolin K in mutated strainszt of M. purpureus in the fifth generation of subcultures Strains g/50ml Biomassg/50mL Monacolin K µg/ml Content of Monacolin Kµg/mL ZZ ZT ZT ZT ZZ ZT32ZT33 ZT35 Monacolin K 9 Table 9 Solid-state fermentation test of M. purpureus ZZ mutated strainszt Strains Monacolin K mg/g Content of Monacolin Kmg/g ZZ 2.48 ZT ZT ZT (Barker1903) UV LiCl Monacolin K 2 Monacolin K 3 (Chen Hu2005) Monacolin K Monacolin K

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