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High-level soluble and functional expression of Trigonopsis variabilis D-amino acid oxidase in Escherichia coli. 2014

Senwen Deng, and Erzheng Su, and Xiaoqiang Ma, and Shengli Yang, and Dongzhi Wei
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.

D-Amino acid oxidase is an important biocatalyst used in a variety of fields, and its economically justified level recombinant expression in Escherichia coli has not been established. To accomplish this, after a single Phe54Tyr substitution, fusion proteins of D-amino acid oxidase from Trigonopsis variabilis (TvDAO) with 6 × His-tags were constructed and expressed in E. coli. The effects of his-tags fusing position were revealed. Significant increase in holoenzyme percent and protein solubility made N-terminus tagged TvDAO (termed NHDAO) a suitable choice for TvDAO production. However, reduced cell growth and protein production rates were also observed for the NHDAO bearing strains. To optimize the performance of NHDAO production, changes of culture medium were tested. Finally, a production of 140 U/mL or 3.48 g active enzyme per liter which accounted for 41.4 % of the total protein, and a specific activity of 16.68 U/mg for the crude extract, were achieved in a 3.7 L fermenter in 28.5 h. This indicated a possibility for functional and economical TvDAO expression in E. coli to meet the industrial need.

UI MeSH Term Description Entries
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003605 D-Amino-Acid Oxidase dextro-Amino Acid Oxidase,D-Amino Acid Dehydrogenase,Acid Dehydrogenase, D-Amino,Acid Oxidase, dextro-Amino,D Amino Acid Dehydrogenase,D Amino Acid Oxidase,Dehydrogenase, D-Amino Acid,Oxidase, D-Amino-Acid,Oxidase, dextro-Amino Acid,dextro Amino Acid Oxidase
D004718 Saccharomycetales An order of fungi in the phylum Ascomycota that multiply by budding. They include the telomorphic ascomycetous yeasts which are found in a very wide range of habitats. Budding Yeast,Endomycetales,Endomycopsis,Yeast, Budding,Budding Yeasts,Endomycetale,Endomycopses,Saccharomycetale,Yeasts, Budding
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005656 Fungal Proteins Proteins found in any species of fungus. Fungal Gene Products,Fungal Gene Proteins,Fungal Peptides,Gene Products, Fungal,Yeast Proteins,Gene Proteins, Fungal,Peptides, Fungal,Proteins, Fungal
D015870 Gene Expression The phenotypic manifestation of a gene or genes by the processes of GENETIC TRANSCRIPTION and GENETIC TRANSLATION. Expression, Gene,Expressions, Gene,Gene Expressions

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