Autor: |
许思远,张永贵,赵沛,曹娟娟,张琴XU Siyuan,ZHANG Yonggui,ZHAO Pei,CAO Juanjuan,ZHANG Qin |
Jazyk: |
English<br />Chinese |
Rok vydání: |
2023 |
Předmět: |
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Zdroj: |
Zhongguo youzhi, Vol 48, Iss 4, Pp 99-104 (2023) |
Druh dokumentu: |
article |
ISSN: |
1003-7969 |
DOI: |
10.19902/j.cnki.zgyz.1003-7969.220108 |
Popis: |
为了探究细菌异质性乙酰辅酶A羧化酶β亚基生物活性及其对乙酰辅酶A羧化酶酶活影响,以高产油核桃内生细菌B. subtilis HB1310基因组DNA为模板,利用PCR技术扩增其乙酰辅酶A羧化酶羧基转移酶β亚基(β-CT)基因(acb基因),构建pET-28a-acb载体并在E. coli BL21中表达,进一步对乙酰辅酶A羧化酶acb基因的诱导表达条件进行了优化。结果表明:乙酰辅酶A羧化酶acb基因在E. coli BL21中实现了表达,分子质量在25~35 kDa之间;重组蛋白最佳诱导表达条件为诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)浓度1.0 mmol/L、诱导时间6 h、诱导初始pH 8.0,在此条件下工程菌的乙酰辅酶A羧化酶活性可达(4.11±0.03) U/mL,较野生菌提高39.7%。综上,乙酰辅酶A羧化酶β-CT为具有较好生物活性的乙酰辅酶A羧化酶亚基。 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. |
Databáze: |
Directory of Open Access Journals |
Externí odkaz: |
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