264507~5162007 Mycosystema 1 550002 2 100039 3 550002 4 550000 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 1672-6472200704-0507-0516 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 550002, China; 2 Graduate School of Chinese Academy of Sciences, Beijing 100039, China; 3 Guizhou Institute of Environmental Science, Guiyang 550002, China; 4 Institute of Fungus Resources, Guizhou University, Huaxi 550000, 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. E-mail: ayliu6@yahoo.com.cn 2007-04-042007-06-21
508 26 (Heber et al.19992000) 5 Monacolin Monacolin K Lovastatin HMG-CoA HMG-CoA HMG-CoA Endo 2000Monacolin K 1994Endo 20002000 McDermott1999 1.1 1.1.1 Monascus purpureus ZZMonascus rubber ZKMonascus purpureus GX Monascus sp. XM 1.1.2 1.2 1.2.1 4:10 55 12 20g 1000mLpH 78 12130min 1.2.2 7% 3% 3% 0.8% 0.8% 0.1% 1000mLpH7819971999 1.2.3 K 5 50g 12h 121 30min1999 1.3 1.3.1 50mL 1.0 10 6 /ml 9cm1999 1min 15s, 30s, 45s, 60s, 75s 90s 1h1999 1999 1.3.2 1.0 10-4 1.0 10-5 1.0 10-6 /ml 0.1mL 0.6 0.8 1.0 1.2 3 30 10d
4 Monacolin K 509 1.3.3 10 1.0 10-4 1.0 10-5 1.0 10-6 /ml 0.1mL 0.6 0.8 1.0 3 30 10d 1.3.4 1999 200 20 Monacolin K 5 5 200 20 Monacolin K 5 1.4 1.4.1 5d 30120r/min 13d Monacolin K 20 1.4.2 5d 5mL 0.05% Tween-80 50mL 250mL 30120r/min 2~4d 2% 50mL 250mL 30120r/min 13d Monacolin K 3 1.5 Monacolin K 1.5.1 Monacolin K 50mL 50mL ph3.0 30 5h 5% Na 2 CO 3 60 20mL 2000 50 50mL 20mL ph=3.0250r/min305h 5% Na 2 CO 3 60 20mL 1998 1.5.2 Monacolin K TLC Monacolin K 1998 15µL 5mm :4:1 1h Rf
510 26 1.5.3 Monacolin K Monacolin K Sigma 2mg 20mL 00.20.40.60.81.01.2 1.4mL 10mL 10mL 238nm OD Monacolin K Monacolin K 2 2.1 4 Monacolin K 4 Rf 0.97 19984 Monacolin K Monacolin K 238nm Monacolin K 1Y=0.0508X0.00117r=0.999948 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 3 9 9 9 9 12
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 2.2.1 ZZ ZZ 2 45s 90% 7.6% 45s
512 26 Table 2 Selection of optimum exposure duration under UV for M. purpureus ZZ strain Duration treated UVs % Death rate% % Positive mutation rate% 15 62.5 3.8 30 80 4 45 90.1 7.6 60 92.2 5.3 75 97.3 0.1 90 99.4 0 2.2.2 ZZ ZZ 3 1 82.9% 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% 0.4 9.8 1.8 0.6 21.3 1.6 0.8 43.9 2.2 1 82.9 3.1 1.2 100 0 2.2.3 45s ZZ ZZ 45s 4 1 92% 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% 0.6 30 1.4 0.8 65 2.5 1 92 4
4 Monacolin K 513 2.2.4 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 2.48 65.4 ZT11 2.02 126.3 ZT12 2.12 110.5 ZT13 2.1 124.1 ZT14 2.26 117.6 ZT15 2.34 108.4 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 2.48 65.4 ZT21 2.09 125 ZT22 2.11 123 ZT23 2.21 118.3 ZT24 2.04 143.8 ZT25 2.38 98.6 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 2.48 65.4 ZT31 2.1 124.3 ZT32 1.89 223.4 ZT33 1.97 180.5 ZT34 2.13 121.6 ZT35 1.96 197.3
514 26 2.2.5 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 2.48 65.4 ZT32 1.78 219.9 ZT33 1.97 178.6 ZT35 1.88 201.7 2.2.6 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 ZT32 8.33 ZT33 6.77 ZT35 7.66 3 (Barker1903) UV LiCl Monacolin K 2 Monacolin K 3 (Chen Hu2005) Monacolin K Monacolin K
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