Cloning, purification, and refolding of human paraoxonase-3 expressed in Escherichia coli and its characterization

Human paraoxonase (hPON3) is a high density lipoprotein-related glycoprotein with multi-enzymatic properties and antioxidant activity which is proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. In this study, hPON3 gene was amplified from Human Fetal Liver Marathon-Ready cDNA and expressed in Escherichia coli. A majority of the expressed protein existed as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 and refolded in vitro. The refolded rhPON3 was purified by DEAE-Sepharose Fast Flow and its purity was up to 90%. The Km and Vmax values of refolded rhPON3, in respect to phenylacetate hydrolysis were 7.47 +/- 2.14 mM and 66 +/- 17 U/min/mg (n = 3). The Km and Vmax values of refolded rhPON3, in respect to dihydrocoumarin hydrolysis were 0.83 +/- 0.21 mM and 621 +/- 66 U/min/mg (n = 3). The refolded rhPON3 exhibited similar antioxidant activity to that of rhPON3 purified from the soluble fraction of cell lysate and could effectively protect LDL from Cu2+ induced oxidation.     

Carbohydrate analysis of Mortierella alpina by colorimetry and HPLC–ELSD to reveal accumulation differences of sugar and lipid

Biotechnology Letters - To establish reliable methods for the extraction and quantification of the total carbohydrate and intracellular saccharides from Mortierella alpina and study the changes...     

Comparison of Biochemical Activities between High and Low Lipid-Producing Strains of Mucor circinelloides: An Explanation for the High Oleaginicity of Strain WJ11

The oleaginous fungus, Mucor circinelloides, is one of few fungi that produce high amounts of γ-linolenic acid (GLA); however, it usually only produces <25% lipid. Nevertheless, a new strain (WJ11) isolated in this laboratory can produce lipid up to 36% (w/w) cell dry weight (CDW). We have investigated the potential mechanism of high lipid accumulation in M. circinelloides WJ11 by comparative biochemical analysis with a low lipid-producing strain, M. circinelloides CBS 277.49, which accumulates less than 15% (w/w) lipid. M. circinelloides WJ11 produced more cell mass than that of strain CBS 277.49, although with slower glucose consumption. In the lipid accumulation phase, activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in strain WJ11 were greater than in CBS 277.49 by 46% and 17%, respectively, and therefore may provide more NADPH for fatty acid biosynthesis. The activities of NAD⁺:isocitrate dehydrogenase and NADP⁺:isocitrate dehydrogenase, however, were 43% and 54%, respectively, lower in WJ11 than in CBS 277.49 and may retard the tricarboxylic acid cycle and thereby provide more substrate for ATP:citrate lyase (ACL) to produce acetyl-CoA. Also, the activities of ACL and fatty acid synthase in the high lipid-producing strain, WJ11, were 25% and 56%, respectively, greater than in strain CBS 277.49. These enzymes may therefore cooperatively regulate the fatty acid biosynthesis in these two strains.     

Two-stage pH control combined with oxygen-enriched air strategies for the highly efficient production of EPA by Mortierella alpina CCFM698 with fed-batch fermentation

Bioprocess and Biosystems Engineering - Dissolved oxygen and pH are critical factors influencing cell growth and metabolism. In our previous work, we constructed the recombinant strain Mortierella...     

一种新型IIa类细菌素的克隆表达和活性鉴定

NB-C1为一种潜在的IIa类细菌素基因,为实现其在大肠杆菌中的高效可溶表达,首先构建了NB-C1蛋白与绿色荧光蛋白 (GFP) 的融合表达载体pIVEX 2.4d-GFP-NB-C1,然后将构建的表达载体转化大肠杆菌BL21(DE3) pLysS,经诱导表达后,重组蛋白GFP-NB-C1以可溶的形式存在于细胞内。经Ni-NTA亲和层析柱分离纯化后,重组融合蛋白的纯度大于95％,产量达36.1 mg/L。抑菌试验表明,纯化后的重组蛋白对单核细胞增生李斯特氏菌具有明显的抑制作用。     

Increased fatty acid unsaturation and production of arachidonic acid by homologous over-expression of the mitochondrial malic enzyme in Mortierella alpina

Malic enzyme (ME) catalyses the oxidative decarboxylation of l-malate to pyruvate and provides NADPH for intracellular metabolism, such as fatty acid synthesis. Here, the mitochondrial ME (mME) gene from Mortierella alpina was homologously over-expressed. Compared with controls, fungal arachidonic acid (ARA; 20:4 n?6) content increased by 60 % without affecting the total fatty acid content. Our results suggest that enhancing mME activity may be an effective mean to increase industrial production of ARA in M. alpina.     

A hexaploid triticale 4D (4B) substitution line confers superior stripe rust resistance

Triticale lines tend to become less resistant to stripe rust and other fungal diseases over time and exhibit relatively limited genetic diversity. Therefore, it is important that new triticale varieties with superior agronomic traits are continually produced to enrich the available genetic pool. In this study, a new hexaploid triticale line (K14-827-1), which was derived from the progenies of a wheat–rye–Psathyrostachys huashanica trigeneric hybrid, was identified and analyzed using genomic and fluorescence in situ hybridizations, seed protein profiling, and molecular markers. Meiotic pairing studies suggested that the mean chromosomal configuration of K14-827-1 was 2n = 42 = 0.24 I + 18.23 II (ring) + 2.65 II (rod). The in situ hybridization karyotyping results indicated that K14-827-1 was a 4D (4B) substitution line, consisting of complete R and A genomes and chromosomes 4D, 1B–3B, and 5B–7B. Simple sequence repeat analysis of K14-827-1 confirmed that wheat chromosome 4B had been substituted by chromosome 4D. The seed protein profiling results uncovered polymorphic 75K γ-secalin and low-molecular-weight glutenin subunits between K14 - 827-1 and its recurrent triticale parent (Zhongsi828). Furthermore, the K14-827-1 plants were highly resistant to the stripe rust pathogen (Puccinia striiformis f. sp. tritici) prevalent in China, including race V26/Gui22, than Zhongsi828 plants at the seedling and adult stages. This new hexaploid triticale line may be useful for diversifying triticale germplasms and breeding new varieties with improved forage grass traits.     

利用非经典分泌蛋白质实现脂肪酶A的分泌表达

【目的】以枯草芽孢杆菌脂肪酶A(Lipase A)为报告蛋白,尝试利用4种非经典分泌蛋白质及其前50个氨基酸作为分泌信号以实现其分泌表达。【方法】我们扩增了脂肪酶A的编码基因和非经典分泌蛋白质的编码序列,构建了8种针对脂肪酶A的分泌表达载体,并转化至枯草芽孢杆菌WB800菌株,通过测定重组菌株的酶活、利用蛋白质电泳和免疫印迹等技术检测脂肪酶A的分泌情况【结果】以Pdh A的氨基酸序列和Sod A、Eno的前50氨基酸序列作为分泌信号的重组菌株较好的实现了脂肪酶A的分泌表达。【结论】部分非经分泌蛋白质的编码基因或其前50个氨基酸序列能够引导脂肪酶A分泌至细胞外。