L-天冬氨酸-α-脱羧酶高产菌株初筛方法的建立  

An Efficient Method for Screening High Yield Strains of L-Aspartate-α-Decarboxylase

作　　者：王姗姗[1] 余志良[1] 裘娟萍[1] 

机构地区：[1]浙江工业大学生物工程学院,浙江杭州310014

出　　处：《氨基酸和生物资源》2016年第2期58-63,共6页Amino Acids & Biotic Resources

摘　　要：通过试管法、平板法、Berthelot法和纸层析法对L-天冬氨酸-α-脱羧酶(PanD)高产菌株的初筛方法进行研究。分别检测转化体系中底物L-天冬氨酸减少引起的pH变化及产物CO2和β-丙氨酸的增加量,并用高效液相色谱法比较这四种方法的准确性。结果表明:CO2能溶于转化液,难以用倒置管收集完全;PanD系胞内酶,难以用平板点种法改变平板的pH;β-丙氨酸和L-天冬氨酸在Berthelot检测中吸收值均偏低,不适用于PanD高产菌的筛选;纸层析法可以直接检测β-丙氨酸和L-天冬氨酸,成本低,操作简单,具一定的精确度,可以较大规模地筛选PanD高产菌株。Test tube method,plate method,Berthelot method and filter paper chromatography were used and compared to develop a rapid method for screening high yield strains of L-aspartate-α-decarboxylase.The pH values,CO2 volume,L-aspartic acid andβ-alanine contents in transformation liquid were detected,and high performance liquid chromatography（HPLC）was used to assess the reliability and accuracy of these methods.Results showed that CO2 disolved in transformation liquid,it is hard to collect CO2 in Henderson＇s tube;PanD is an intracellular enzyme,and cannot cause detectable change of pH value in spot inoculation method;the absorption value ofβ-alanine and L-aspartic acid in Berthelot method is too low to be measured.Comparatively,filter paper chromatography can be used to directly and sensitively detectβ-alanine and L-aspartic acid content.Filter paper chromatography method is lower-cost,faster and more convenient compared with other methods.All these features allow this method for screening of recombinant strains with high PanD activity.

关 键 词：L-天冬氨酸-α-脱羧酶(PanD) Β-丙氨酸 L-天冬氨酸 高产菌株 筛选 

分 类 号：Q557.1[生物学—生物化学]