植物乳杆菌P-8羟基脂肪酸脱氢酶的克隆表达及活性鉴定
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摘要
以植物乳杆菌P-8的基因组DNA为模板扩增出其羟基脂肪酸脱氢酶基因,对其进行生物信息学分析,结果表明该基因共编码286个氨基酸,蛋白质理论分子质量约为32 k Da,理论等电点为5. 15,不含跨膜区,是1个亲水性的细胞质蛋白。Ser、Thr、Tyr磷酸化位点个数分别为3、3、7。蛋白质的二级结构主要构成方式为α螺旋、无规则卷曲和β折叠。将此基因连接到p ET-28a(+)质粒载体中构建重组质粒p ET28a-DH,并转化入大肠杆菌E. coli Transetta (DE3)中获得重组大肠杆菌。重组菌在16℃条件下,经0. 1 mmol/L IPTG诱导16 h有可溶性表达,经Western Blot验证确定为重组羟基脂肪酸脱氢酶。通过镍柱亲和层析,得到纯化的重组酶。重组酶经初步活性检测,结果显示重组酶可以转化10-羟基-顺-12-十八碳烯酸为10-氧代-顺-12-十八碳烯酸,本研究为进一步探明植物乳杆菌P-8转化共轭亚油酸的机制奠定了基础。
The hydroxy fatty acid short chain dehydrogenase gene(CLA-DH) was amplified by PCR using the genomic DNA of Lactobacillus plantarum P-8 as a template. Bioinformatics analysis showed that this gene encoded 286 amino acids with a theoretical molecular mass of about 32 kDa,and a theoretical isoelectric point of 5. 15. It was a hydrophilic cytoplasmic protein with no trans-membrane region. Its phosphorylation sites of serine,threonine,and tyrosine were 3,3,and 7,respectively. The secondary structure of the protein mainly composed of α-helices,random coils,and β-sheets. The recombinant plasmid pET28 a-DH was constructed by inserting CLA-DH into p ET-28 a(+),and then transformed into E. coli Transetta to obtain a compounded E. coli for expression. The recombinant bacteria had soluble expression at 16 ℃ after 16 h induction with 0. 1 mmol/L IPTG. The results from Western Blot confirmed that the expression was recombinant CLA-DH. The purified recombinant CLA-DH was obtained by nickel column affinity chromatography. The preliminary activity assay of the recombinant CLA-DH showed that 10-hydroxy-cis-12-octadecenoic acid could be converted into 10-oxo-cis-12-octadecenoic acid,which laid a foundation for further analysis of the mechanism of transforming conjugated linoleic acid by L. plantarum P-8.
The hydroxy fatty acid short chain dehydrogenase gene(CLA-DH) was amplified by PCR using the genomic DNA of Lactobacillus plantarum P-8 as a template. Bioinformatics analysis showed that this gene encoded 286 amino acids with a theoretical molecular mass of about 32 kDa,and a theoretical isoelectric point of 5. 15. It was a hydrophilic cytoplasmic protein with no trans-membrane region. Its phosphorylation sites of serine,threonine,and tyrosine were 3,3,and 7,respectively. The secondary structure of the protein mainly composed of α-helices,random coils,and β-sheets. The recombinant plasmid pET28 a-DH was constructed by inserting CLA-DH into p ET-28 a(+),and then transformed into E. coli Transetta to obtain a compounded E. coli for expression. The recombinant bacteria had soluble expression at 16 ℃ after 16 h induction with 0. 1 mmol/L IPTG. The results from Western Blot confirmed that the expression was recombinant CLA-DH. The purified recombinant CLA-DH was obtained by nickel column affinity chromatography. The preliminary activity assay of the recombinant CLA-DH showed that 10-hydroxy-cis-12-octadecenoic acid could be converted into 10-oxo-cis-12-octadecenoic acid,which laid a foundation for further analysis of the mechanism of transforming conjugated linoleic acid by L. plantarum P-8.
引文
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[10] KIM Y,HIRAI S,GOTO T,et al. Potent PPARαactivator derived from tomato juice,13-oxo-9,11-Octadecadienoic acid,decreases plasma and hepatic triglyceride in obese diabetic mice[J]. Plos One,2012,7(2):e31 317.
[11]杜美婷,陈慧泽,赵国芬.植物乳杆菌P8产共轭亚油酸条件的优化[J].生物学杂志,2012,29(3):92-95.
[2] JONES E L,SHINGFIELD K J,KOHEN C,et al. Chemical,physical,and sensory properties of dairy products enriched with conjugatedlinoleic acid[J]. Journal of Dairy Science,2005,88(8):2 923-2 937.
[3] CRUMB D J. Conjugated linoleic acid(CLA)-An overiew[J]. Internatiomal Journal of Applied Research in Natural Products,2011,4(3):12-18
[4] COOK M E,PARIZA M W. Dietetic foods containing conjugated linoleic acids:WO,US 5760082 A[P]. 1998.
[5]刘晓华,曹郁生,陈燕.微生物生产共轭亚油酸的研究[J].食品与发酵工业,2003,29(9):69-72.
[6] LIN T Y,HUANG T H,CHEN G T S J. Conjugated linoleic acid production by immobilized cells of lactobacillus delbruckii ssp bulgaricus and lactobacillus acidophilus[J].Food Chem,2005,92(1):23-28
[7]杨波.乳酸菌生物转化共轭亚油酸的研究[D].无锡:江南大学,2014.
[8] TAKEUCHI M,KISHINO S,PARK S B,et al. Characterization of hydroxy fatty acid dehydrogenase involved in polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum,AKU 1009a[J]. Journal of Molecular Catalysis B Enzymatic,2015,117:7-12.
[9] KISHINO S,PARK S B,TAKEUCHI M,et al. Novel multi-component enzyme machinery in lactic acid bacteria catalyzing CC double bond migration useful for conjugated fatty acid synthesis[J]. Biochemical&Biophysical Research Communications,2011,416(1-2):193.
[10] KIM Y,HIRAI S,GOTO T,et al. Potent PPARαactivator derived from tomato juice,13-oxo-9,11-Octadecadienoic acid,decreases plasma and hepatic triglyceride in obese diabetic mice[J]. Plos One,2012,7(2):e31 317.
[11]杜美婷,陈慧泽,赵国芬.植物乳杆菌P8产共轭亚油酸条件的优化[J].生物学杂志,2012,29(3):92-95.