产西贝碱内生真菌的诱变及发酵条件的优化
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摘要
以本实验室保藏的、具有产西贝碱能力的平贝母内生真菌P7为实验菌株。
为解决P7菌株在长期保藏过程中出现的退化现象,采用宿主复壮法对其进行了复壮研究,即在培养过程中向培养基中加入平贝母药材,结果表明,当平贝母药材的加入量为3%时,对P7菌株的生长及西贝碱的积累均具有促进作用,且西贝碱产量可达到分离之初菌株西贝碱产量的95.2%,达到了一定的复壮效果,而当加入量超过6%以后,虽对P7菌株的生长仍具有促进作用,但西贝碱的积累却受到明显抑制。
为了得到西贝碱的高产菌株,采用传统的诱变方法对P7菌株先进行了单因素诱变,包括紫外、He-Ne激光和亚硝酸处理,在各自较优剂量范围内对其正突变率进行分析得出,对于P7菌株,紫外诱变的效果最好,亚硝酸次之,He-Ne激光较弱。并对各自的正突变菌株进一步筛选,分别得到不同诱变处理的优势菌株。再以它们作为二次出发菌株进行复合诱变处理,最终筛选得到一株高产菌株P-UH-3,其西贝碱产量为0.0881mg/L,菌丝体干重为2.80g/L,分别比原始出发菌株P7的西贝碱产量提高了89.9%,菌体干重提高了18.6%,诱变效果良好,而且P-UH-3菌株具有稳定的遗传特性。
为了进一步提高目的产物西贝碱的产量,对P-UH-3菌株的培养条件进行了优化研究。通过比较不同供试培养基中菌丝体生长和西贝碱积累情况,发现最适于菌丝体生长的固体培养基为PDA培养基,而最利于西贝碱积累的液体培养基为豆芽汁蔗糖培养基。并以豆芽汁培养基为基础对P-UH-3菌株的培养条件进行单因素和正交试验优化,结果表明,最利于西贝碱积累的发酵条件为：在豆芽汁培养基的基础上,加入葡萄糖10%,蛋白胨0.15%,培养基起始pH 7.0,种龄为36h,接种量为10%,最佳培养温度26℃,培养时间为7天。与初始培养条件相比,在优化后的发酵条件下,P-UH-3菌株西贝碱的产量为0.1258mg/L,增加了42.8%,而菌丝体干重的变化不明显。
The strain P7 is a sipeimine-producing endophytic fungus isolated from the bulbus of a medicinal plant Fritillaria ussuriensis.
To solve the degeneration problem of P7 during the course of preservation, the strain was rejuvenated by adding different concentration of Bulbus F. ussuriensis into culture medium. The results showed that, the biomass and sipeimine yield of the strain rejuvenated by adding 3% bulbus were effectively improved, while the bulbus concentration exceeded 6%, the biomass was also improved, but the sipeimine yield reduced.
In order to get mutant strain with higher sipeimine yield, different physical and chemical mutagens were used to mutagenize the initial strain P7, including UV, He-Ne laser and HNO2. Then comparing the positive mutation frequency within selected dosage, the results showed that the best single mutagen for P7 was UV, the second was HNO2, and He-Ne laser was the last. Then the dominant strains mutagenized by single mutagen as the second initial strains were further mutagenized, and a dominant strain P-UH-3 was selected, its sipeimine yield and biomass were 0.0881mg/L and 2.80g/L, respectively,89.9% and 18.6% higher than those of the initial strain P7. The yield was constant after successive culture of 10 generations.
In order to further improve the product yield, the culture conditions of the mutant strain P-UH-3 were then optimized. Based on the growth rate and the yield of sipeimine, the optimal solid culture medium was PDA and the liquid culture medium was bean sprouts medium. The suitable fermentation conditions for sipeimine accumulation of P-UH-3 were then further optimized by single-factor and the orthogonal experiment. The results showed that bean sprouts liquid medium as a basic culture medium, adjusted the glucose concentration to 10%, peptone 0.15%, pH 7.0, age of inoculum 36h, inoculation volume 10%, the temperature 26℃and culture period 7d. Comparing with the initial condition, the sipeimine yield was 0.1258mg/L, improved by 42.8%, but the change in biomass was not obvious.
为解决P7菌株在长期保藏过程中出现的退化现象,采用宿主复壮法对其进行了复壮研究,即在培养过程中向培养基中加入平贝母药材,结果表明,当平贝母药材的加入量为3%时,对P7菌株的生长及西贝碱的积累均具有促进作用,且西贝碱产量可达到分离之初菌株西贝碱产量的95.2%,达到了一定的复壮效果,而当加入量超过6%以后,虽对P7菌株的生长仍具有促进作用,但西贝碱的积累却受到明显抑制。
为了得到西贝碱的高产菌株,采用传统的诱变方法对P7菌株先进行了单因素诱变,包括紫外、He-Ne激光和亚硝酸处理,在各自较优剂量范围内对其正突变率进行分析得出,对于P7菌株,紫外诱变的效果最好,亚硝酸次之,He-Ne激光较弱。并对各自的正突变菌株进一步筛选,分别得到不同诱变处理的优势菌株。再以它们作为二次出发菌株进行复合诱变处理,最终筛选得到一株高产菌株P-UH-3,其西贝碱产量为0.0881mg/L,菌丝体干重为2.80g/L,分别比原始出发菌株P7的西贝碱产量提高了89.9%,菌体干重提高了18.6%,诱变效果良好,而且P-UH-3菌株具有稳定的遗传特性。
为了进一步提高目的产物西贝碱的产量,对P-UH-3菌株的培养条件进行了优化研究。通过比较不同供试培养基中菌丝体生长和西贝碱积累情况,发现最适于菌丝体生长的固体培养基为PDA培养基,而最利于西贝碱积累的液体培养基为豆芽汁蔗糖培养基。并以豆芽汁培养基为基础对P-UH-3菌株的培养条件进行单因素和正交试验优化,结果表明,最利于西贝碱积累的发酵条件为：在豆芽汁培养基的基础上,加入葡萄糖10%,蛋白胨0.15%,培养基起始pH 7.0,种龄为36h,接种量为10%,最佳培养温度26℃,培养时间为7天。与初始培养条件相比,在优化后的发酵条件下,P-UH-3菌株西贝碱的产量为0.1258mg/L,增加了42.8%,而菌丝体干重的变化不明显。
The strain P7 is a sipeimine-producing endophytic fungus isolated from the bulbus of a medicinal plant Fritillaria ussuriensis.
To solve the degeneration problem of P7 during the course of preservation, the strain was rejuvenated by adding different concentration of Bulbus F. ussuriensis into culture medium. The results showed that, the biomass and sipeimine yield of the strain rejuvenated by adding 3% bulbus were effectively improved, while the bulbus concentration exceeded 6%, the biomass was also improved, but the sipeimine yield reduced.
In order to get mutant strain with higher sipeimine yield, different physical and chemical mutagens were used to mutagenize the initial strain P7, including UV, He-Ne laser and HNO2. Then comparing the positive mutation frequency within selected dosage, the results showed that the best single mutagen for P7 was UV, the second was HNO2, and He-Ne laser was the last. Then the dominant strains mutagenized by single mutagen as the second initial strains were further mutagenized, and a dominant strain P-UH-3 was selected, its sipeimine yield and biomass were 0.0881mg/L and 2.80g/L, respectively,89.9% and 18.6% higher than those of the initial strain P7. The yield was constant after successive culture of 10 generations.
In order to further improve the product yield, the culture conditions of the mutant strain P-UH-3 were then optimized. Based on the growth rate and the yield of sipeimine, the optimal solid culture medium was PDA and the liquid culture medium was bean sprouts medium. The suitable fermentation conditions for sipeimine accumulation of P-UH-3 were then further optimized by single-factor and the orthogonal experiment. The results showed that bean sprouts liquid medium as a basic culture medium, adjusted the glucose concentration to 10%, peptone 0.15%, pH 7.0, age of inoculum 36h, inoculation volume 10%, the temperature 26℃and culture period 7d. Comparing with the initial condition, the sipeimine yield was 0.1258mg/L, improved by 42.8%, but the change in biomass was not obvious.
引文
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