建立基于多孔板发酵及酶标仪检测的高通量选育模型,提高诱变后硫酸新霉素高产菌株的筛选效率。 对影响酶标仪检测的主要影响因素1.0×10-4mol/L曲利本蓝的添加量(μL/mL)进行优化,并建立相应的分光光度法,对适用高通量筛选模型的微孔板进行选择,并对其发酵条件进行正交优化。结果表明,曲利本蓝的添加量为500 μL/mL,硫酸新霉素在1.284~10.272 U/mL,吸光度与样品浓度呈良好的线性关系(R2=0.998 8),平均回收率为98.468 4%,重复性RSD为3.270 0%;24孔板适用于高通量筛选模型,优化后发酵条件:转速、装液量和接种量分别为220 r/min、2 mL和8%,在此基础上,实际发酵效价达到(6 825±77) U/mL,较原发酵条件提高了27.40%。基于多孔板发酵及酶标仪检测建立高通量选育模型,能够达到诱变后快速筛选硫酸新霉素高产菌株的目的,也为后续高通量选育其他抗生素高产菌株奠定了基础。
In order to improve the efficiency of screening strains with high-yield of neomycin sulfate after mutagenesis, a high-throughput screening method based on well plates and microplate reader was developed. The adding amount of 1.0×10-4mol/L trypan blue was optimized, which was a main influencing factor of the method. The corresponding spectrophotometric method was also established. Well plates that were suitable for high-throughput screening were selected, and the fermentation conditions were optimized. The results showed that adding 500 μL/mL 1.0×10-4mol/L trypan blue, the absorbancy of neomycin sulfate showed a good linear relationship with concentrations in the range of 1.284-10.272 U/mL (R2=0.998 8). Besides, the average recovery rate was 98.47%, and the RSD was 3.27%. The 24-well plates were suitable for high-throughput screening, and further optimization increased the potency of fermentation by 27.40% to 6 825±77 U/mL. The optimal fermentation conditions were as follows: 220 r/min for speed, 2 mL medium volume, and 8% inoculum size. This study indicates that the high-throughput screening method based on well plates and microplate reader can rapidly screen strains with high-yield of neomycin sulfate after mutagenesis, which also laid the foundation for high-throughput screening and breeding of strains with high-yield of other antibiotics.
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