摘要
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^(-4)mol/L曲利本蓝的添加量(μL/m L)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500μL/m L,硫酸新霉素在1.284～10.272 U/m L,吸光度与样品浓度呈良好的线性关系(R^2=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 m L和8%,在此基础上,实际发酵效价达到(6 825±77)U/m L,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^-4 mol/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^-4 mol/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 ( R^2 =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.
作者
余飞
孙俊峰
刘鹏飞
刘艳
王洲
薛正莲
YU Fei;SUN Junfeng;LIU Pengfei;LIU Yan;WANG Zhou;XUE Zhenglian(Microorganism Fermentation Engineering and Technology Research Center of Anhui Province,Institute of Biologic & Chemical Engineering,Anhui Polytechnic University,Wuhu 241000,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2019年第8期162-167,177,共7页
Food and Fermentation Industries
关键词
弗氏链霉菌
硫酸新霉素
高通量
Streptomyces fradiae
neomycin
high-throughput screening
