(R)-2-Hydroxy-4-phenylbutyric acid ethyl ester((R)-HPBE)is an essential chiral intermediate in the synthesis of angiotensin-converting enzyme(ACE)inhibitors.Its production involves the highly selective asymmetric redu...(R)-2-Hydroxy-4-phenylbutyric acid ethyl ester((R)-HPBE)is an essential chiral intermediate in the synthesis of angiotensin-converting enzyme(ACE)inhibitors.Its production involves the highly selective asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate(OPBE),catalyzed by carbonyl reductase(CpCR),with efficient cofactor regeneration playing a crucial role.In this study,an in-situ coenzyme regeneration system was developed by coupling carbonyl reductase(CpCR)with glucose dehydrogenase(GDH),resulting in the construction of five recombinant strains capable of NADPH regeneration.Among these,the recombinant strain E.coli BL21-pETDuet-1-GDH-L-CpCR,where CpCR is fused to the C-terminus of GDH,demonstrated the highest catalytic activity.This strain exhibited an enzyme activity of 69.78 U/mg and achieved a conversion rate of 98.3%,with an enantiomeric excess(ee)of 99.9%during the conversion of 30 mM OPBE to(R)-HPBE.High-density fermentation further enhanced enzyme yield,achieving an enzyme activity of 1960 U/mL in the fermentation broth,which is 16.2 times higher than the volumetric activity obtained from shake flask fermentation.Additionally,the implementation of a substrate feeding strategy enabled continuous processing,allowing the strain to efficiently convert a final OPBE concentration of 920 mM,producing 912 mM of(R)-HPBE.These findings highlight the system’s improved catalytic efficiency,stability,and scalability,making it highly suitable for industrial-scale biocatalytic production.展开更多
Amorphophallus konjac is an important economic crop widely cultivated in Southeast Asia and Africa. However, A. konjac is seriously infected by soft rot pathogen. The endocellular acyl homoserine lactonase (AiiA) whic...Amorphophallus konjac is an important economic crop widely cultivated in Southeast Asia and Africa. However, A. konjac is seriously infected by soft rot pathogen. The endocellular acyl homoserine lactonase (AiiA) which is generated by Bacillus species has inhibitory effect on soft rot pathogen through disrupting the signal molecules (N-acylhomoserine lactones, AHL) of their Quorum Sensing system. The aim of our study is to obtain recombinant yeast which produces AiiA protein. The recombinant yeast Pichia pastoris GS115 was constructed to constitutive expression of the AiiA gene. The results of reverse transcript PCR analysis showed that the AiiA gene was expressed successfully in the yeast. Proteins extracted from YPDS showed the highest inhibition efficacy to E. carotovora compared with the other two mediums (YPD and LB) under tested conditions.展开更多
基金supported by the Zhejiang Provincial Science and Technology Plan Project(2024C03014)the National Nature Science Foundation of China(Project Nos.21978267,and 22078300)as well as the Natural Science Foundation of Heilongjiang Province(LH2022H107).
文摘(R)-2-Hydroxy-4-phenylbutyric acid ethyl ester((R)-HPBE)is an essential chiral intermediate in the synthesis of angiotensin-converting enzyme(ACE)inhibitors.Its production involves the highly selective asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate(OPBE),catalyzed by carbonyl reductase(CpCR),with efficient cofactor regeneration playing a crucial role.In this study,an in-situ coenzyme regeneration system was developed by coupling carbonyl reductase(CpCR)with glucose dehydrogenase(GDH),resulting in the construction of five recombinant strains capable of NADPH regeneration.Among these,the recombinant strain E.coli BL21-pETDuet-1-GDH-L-CpCR,where CpCR is fused to the C-terminus of GDH,demonstrated the highest catalytic activity.This strain exhibited an enzyme activity of 69.78 U/mg and achieved a conversion rate of 98.3%,with an enantiomeric excess(ee)of 99.9%during the conversion of 30 mM OPBE to(R)-HPBE.High-density fermentation further enhanced enzyme yield,achieving an enzyme activity of 1960 U/mL in the fermentation broth,which is 16.2 times higher than the volumetric activity obtained from shake flask fermentation.Additionally,the implementation of a substrate feeding strategy enabled continuous processing,allowing the strain to efficiently convert a final OPBE concentration of 920 mM,producing 912 mM of(R)-HPBE.These findings highlight the system’s improved catalytic efficiency,stability,and scalability,making it highly suitable for industrial-scale biocatalytic production.
文摘Amorphophallus konjac is an important economic crop widely cultivated in Southeast Asia and Africa. However, A. konjac is seriously infected by soft rot pathogen. The endocellular acyl homoserine lactonase (AiiA) which is generated by Bacillus species has inhibitory effect on soft rot pathogen through disrupting the signal molecules (N-acylhomoserine lactones, AHL) of their Quorum Sensing system. The aim of our study is to obtain recombinant yeast which produces AiiA protein. The recombinant yeast Pichia pastoris GS115 was constructed to constitutive expression of the AiiA gene. The results of reverse transcript PCR analysis showed that the AiiA gene was expressed successfully in the yeast. Proteins extracted from YPDS showed the highest inhibition efficacy to E. carotovora compared with the other two mediums (YPD and LB) under tested conditions.
