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| Cloning and expression optimization of acetyl-CoA carboxylase acb gene from oil-producing walnut endophyte |
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| KeyWord:walnut endophyte oil-producing acetyl-CoA carboxylase cloning expression optimization |
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| Abstract: |
| To explore the biological activity of the bacterial heterogeneous acetyl-CoA carboxylase β subunit and its influence on the acetyl-CoA carboxylase activity, the acetyl-CoA carboxylase carboxyltransferase subunit beta(β-CT) gene(acb gene) was amplified by PCR technology using the genomic DNA of a high oil-producing walnut endophyte B. subtilis HB1310 as template, the expression vector pET-28a-acb was constructed and expressed in E. coli BL21, and the induced expression conditions of this acb gene were further optimized. The results showed that the acetyl-CoA carboxylase acb gene was expressed in E. coli BL21 with a molecular weight of 25-35 kDa. Moreover, the optimal induced expression conditions were determined as follows: isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration 1.0 mmol/L, induction time 6 h, and induction initial pH 8.0. Under the optimal conditions, the acetyl-CoA carboxylase activity of the engineering bacteria reached (4.11±0.03) U/mL, which was 39.7% higher than that of the wild bacteria. In conclusion, acetyl-CoA carboxylase β-CT is an acetyl-CoA carboxylase subunit with good biological activity. |
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