Difference between revisions of "Part:BBa K1972004"
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The DszB desulfurase catalyzes the rate-limiting step of the 4S-pathway and the Y63F amino acid substitution was previously reported to enhance its activity and stability. Therefore, the Y63F amino acid substitution of the DszB desulfurase was performed. | The DszB desulfurase catalyzes the rate-limiting step of the 4S-pathway and the Y63F amino acid substitution was previously reported to enhance its activity and stability. Therefore, the Y63F amino acid substitution of the DszB desulfurase was performed. | ||
| − | [[File:T--SCUT-China A--u24.png|900px||center|]] | + | [[File:T--SCUT-China A--u24.png|900px||center|Figure 1. Bio-circuit of BBa_K1972013 after our third optimization]] |
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Furthermore, the desulfurization experiment showed that the activities of enzyme C and A were still stronger than B, which caused undesirable accumulation of intermediate products (DBTO2/HBPS), seriously affecting the activity of enzyme B. The promoters of dsz genes were further adjusted. Gene dszB, dszC and dszA were controlled by the tac promoter, which was strong enough and endogenous for E.coli. At the same time, dszD was under the weak lac promoter as an independent operon, making the expression of enzyme D relatively weak, with the purpose of indirectly attenuating the effect of the enzyme A and C (as shown in Figure 1). | Furthermore, the desulfurization experiment showed that the activities of enzyme C and A were still stronger than B, which caused undesirable accumulation of intermediate products (DBTO2/HBPS), seriously affecting the activity of enzyme B. The promoters of dsz genes were further adjusted. Gene dszB, dszC and dszA were controlled by the tac promoter, which was strong enough and endogenous for E.coli. At the same time, dszD was under the weak lac promoter as an independent operon, making the expression of enzyme D relatively weak, with the purpose of indirectly attenuating the effect of the enzyme A and C (as shown in Figure 1). | ||
Revision as of 15:08, 25 October 2016
dszA (a monooxygenase gene ), E.coli-optimized
DszA is a monoxygenase which can convert DBTsulfone (DBTO2) to 2-(2-hydroxybiphenyl) sulfinate (HBPS) during the 4S pathway, in which dibenzothiophene (DBT) undergoes three successive oxidation steps and one a hydrolytic step leading to the formation of 2-hydroxybiphenyl (2HBP). And DszA require reducing equivalents (FMNH2) supplied by a flavinreductase (DszD) while working.
This single monooxygenase gene was synthesized whith E.coli codon optimized by Generay and the sequence was found in NCBI (GenBank Accession number L37363.1)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 583
Illegal AgeI site found at 687
Illegal AgeI site found at 970 - 1000COMPATIBLE WITH RFC[1000]
The DszB desulfurase catalyzes the rate-limiting step of the 4S-pathway and the Y63F amino acid substitution was previously reported to enhance its activity and stability. Therefore, the Y63F amino acid substitution of the DszB desulfurase was performed.
Furthermore, the desulfurization experiment showed that the activities of enzyme C and A were still stronger than B, which caused undesirable accumulation of intermediate products (DBTO2/HBPS), seriously affecting the activity of enzyme B. The promoters of dsz genes were further adjusted. Gene dszB, dszC and dszA were controlled by the tac promoter, which was strong enough and endogenous for E.coli. At the same time, dszD was under the weak lac promoter as an independent operon, making the expression of enzyme D relatively weak, with the purpose of indirectly attenuating the effect of the enzyme A and C (as shown in Figure 1).
Figure 2. The desulfurization results of Recombinant strain BL21-dszBACD (optimized) tested by HPLC
The desulfurization efficiency of the recombinant strain BL21-dszBACD (optimized) is greatly improved.