An immobilized cell membrane bioreaction system was developed to promote cell stability. The hollow fiber membrane bioreactor with immobilized Pseudomonas putida cells, operating in continual repeated batch operation ...An immobilized cell membrane bioreaction system was developed to promote cell stability. The hollow fiber membrane bioreactor with immobilized Pseudomonas putida cells, operating in continual repeated batch operation mode was used for producing D-p-hydroxyphenylglycine from DL-5-p-hydroxyphenyl hydantoin. The concentration of N-carbamyl-D-p-hydroxyphenylglycine and D-p-hydroxyphenylglycine in the efflux was analyzed by high performance liquid chromatography at different intervals.展开更多
D-p-hydroxyphenylglycine(D-HPG)is an important intermediate in the pharmaceutical industry,and it is commonly syn-thesized by cascading D-hydantoinase(DHase)and D-carbamoylase(DCase).In this study,the stability of DCa...D-p-hydroxyphenylglycine(D-HPG)is an important intermediate in the pharmaceutical industry,and it is commonly syn-thesized by cascading D-hydantoinase(DHase)and D-carbamoylase(DCase).In this study,the stability of DCase was identified as the main problem that limits its application.Therefore,the complexed structure of AkDCase(DCase from the Agrobacterium sp.strain KNK712)with the substrate N-carbamoyl-D-p-hydroxyphenylglycine(CpHPG)(with 2.52Åresolution)and catalytic mechanism were resolved.Based on the catalytic mechanism and electrostatic stabilization,salt bridge engineering was adopted to improve AkDCase thermostability.The best variant,AkDCase^(D30A),increased theTm by 2.91℃and half-life(t_(1/2))at 40 and 60℃by 18.43 h and 23.21 min,respectively.After AkDCase^(D30A) was assembled with GsDHase(DHase from Geobacillus stearothermophilus SD-1)in a single Escherichia coli cell,the recombinant strain could produce 29.53 g/L D-HPG within 12 h,with a 97%conversion and a 2.46 g/(L·h)space-time yield(STY).The titer of D-HPG increased by 40.55%compared to the E.coli cell harboring pETduet-1-AkDCase-GsDHase.The recombinant strain could be used for two cycles.Our research provides a basis for the industrial production of D-HPG.展开更多
文摘An immobilized cell membrane bioreaction system was developed to promote cell stability. The hollow fiber membrane bioreactor with immobilized Pseudomonas putida cells, operating in continual repeated batch operation mode was used for producing D-p-hydroxyphenylglycine from DL-5-p-hydroxyphenyl hydantoin. The concentration of N-carbamyl-D-p-hydroxyphenylglycine and D-p-hydroxyphenylglycine in the efflux was analyzed by high performance liquid chromatography at different intervals.
基金supported by the National Key R&D Program of China(Grant No.2021YFC2100100)the General Program of National Natural Science Foundation of China(22178146)+1 种基金the Program for Young Talents in Chinathe Fundamental Research Funds for the Central Universities(JUSRP622011).
文摘D-p-hydroxyphenylglycine(D-HPG)is an important intermediate in the pharmaceutical industry,and it is commonly syn-thesized by cascading D-hydantoinase(DHase)and D-carbamoylase(DCase).In this study,the stability of DCase was identified as the main problem that limits its application.Therefore,the complexed structure of AkDCase(DCase from the Agrobacterium sp.strain KNK712)with the substrate N-carbamoyl-D-p-hydroxyphenylglycine(CpHPG)(with 2.52Åresolution)and catalytic mechanism were resolved.Based on the catalytic mechanism and electrostatic stabilization,salt bridge engineering was adopted to improve AkDCase thermostability.The best variant,AkDCase^(D30A),increased theTm by 2.91℃and half-life(t_(1/2))at 40 and 60℃by 18.43 h and 23.21 min,respectively.After AkDCase^(D30A) was assembled with GsDHase(DHase from Geobacillus stearothermophilus SD-1)in a single Escherichia coli cell,the recombinant strain could produce 29.53 g/L D-HPG within 12 h,with a 97%conversion and a 2.46 g/(L·h)space-time yield(STY).The titer of D-HPG increased by 40.55%compared to the E.coli cell harboring pETduet-1-AkDCase-GsDHase.The recombinant strain could be used for two cycles.Our research provides a basis for the industrial production of D-HPG.
