ABSTRACT
The genus Rhodococcus is of considerable interest in recent years, stemming from their diverse applications in biodegradation, bioremediation, biotransformation and biosurfactant. Using Nocardia/Rhodococcus-Escherichia coli shuttle plasmid pNV18.1 as the backbone vector, we tested the driven efficiency of promoters Ptac and PlacZ of E. coli and Pami-1/Pami-2 of R. ruber in host R. rhodochrous ATCC 33278 by overexpression of nitrile hydratase. Results showed that the specific activity of nitrile hydratase per dry cell weight in engineered Rhodococcus strains driven by Ptac, Pami-1, Pami-2 and PlacZ was 7.5, 6.3, 5.3 and 1.8 times of that in the wild, respectively. It indicated that these promoters could be well recognized by RNA polymerase of Rhodococcus. We further expressed the beta-galactosidase reporter gene (lacZ) in R. ruber driven by promoter PlacZ. Results indicated that lacZ was an appropriate reporter gene for genetic or metabolic engineering research of Rhodococcus.
Subject(s)
Escherichia coli , Genetics , Gene Expression Regulation, Bacterial , Genes, Reporter , Genetics , Lac Operon , Genetics , Promoter Regions, Genetic , Genetics , Rhodococcus , Genetics , beta-Galactosidase , GeneticsABSTRACT
D-amino acid oxidase (DAAO) is one of important industrial enzymes. To increase the solubility and activity of the TvDAAO from Trignoposis variabilis expressed in recombinant Escherichia coli (E. coli), a maltose binding protein (MBP) and Vitreoscilla hemoglobin (VHb) was introduced to fuse with N-terminal of the TvDAAO, respectively. Fusion protein of MBP-TvDAAO was constitutively expressed in JM105/pMKC-DAAO and inductively expressed in JM105/pMKL-DAAO. With respect to the control strain of BL21 (DE3)/pET-DAAO without MBP fusion, the constitutive fusion expression obtained 28% of soluble protein with 3.7 folds of solubility improvement. As for the inductive fusion expression, corresponding results changed to 17% and 1.8 folds, respectively. However, the DAAO activity significantly decreased in the MBP-fusing expression. Fusion protein of VHb-TvDAAO was constructed and inductively expressed in BL21 (DE3)/pET-VDAAO. Its DAAO activity highly reached 3.24 u/mL in flask culture, about 90% increase in contrast to the control without VHb.
Subject(s)
Bacterial Proteins , Genetics , Carrier Proteins , Genetics , D-Amino-Acid Oxidase , Genetics , Escherichia coli , Genetics , Metabolism , Maltose-Binding Proteins , Recombinant Fusion Proteins , Genetics , Truncated Hemoglobins , Genetics , Yeasts , GeneticsABSTRACT
To facilitate the expression of GL 7 ACA acylase gene in a recombinant E coli , a fragment of the gene, in which the signal peptide was deleted by PCR method, was inserted into a prokaryotic expression vector, pET 28a By colony PCR method screening, a recombinant plasmid pET ACY was obtained and then transformed into the expression host BL21 (DE3) The influences of induction conditions such as IPTG concentration, the time of induction and the induction temperature on the expression of the recombinant protein were investigated Under optimal condition, the enzyme activity could reach 266 U/L Finally, the recombinant GL 7 ACA acylase can be easily isolated to a purity of about 80% by a simple anion ion exchange chromatography with enzyme activity recovery of 50%