RESUMO
With the genomic DNA of strain EJS-3 as the template, we amplified the gene of fibrinolytic enzyme from Paenibacillus polymyxa (PPFE-I) by PCR. We purified the PCR product and ligated it into pMD19-T. After DNA sequencing, we cloned the PPFE-I gene into expression vector pET-DsbA and transformed it into Escherichia coli BL21(DE3). Upon induction of IPTG, we found that the activity of recombinant fibrinolytic enzyme fused with DsbA expressed in Escherichia coli was 228 IU/mL. SDS-PAGE analysis showed that the recombinant enzyme was soluble and accounted for about 18.4% of total cell protein. Western blotting demonstrated that the recombinant protein was DsbA-PPFE-I. We purified the recombinant enzyme by Ni affinity chromatography, thrombin digestion and sephadex G-100 gel-filtration, and identified the molecular weight of purified product to be 66.3 kDa with MALDI-TOF mass spectrometry. The purified enzyme exhibited distinct fibrinolytic activity on fibrin plate.
Assuntos
Escherichia coli/metabolismo , Fibrinolíticos/metabolismo , Paenibacillus/química , Paenibacillus/enzimologia , Proteínas Recombinantes de Fusão/biossíntese , Antifibrinolíticos/farmacologia , Clonagem Molecular , Escherichia coli/genética , Vetores Genéticos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
OBJECTIVE: To produce Proteus vulgaris lipase (PVL) in large quantities, we cloned and expressed the lipase gene in Escherichia coli. METHODS: We cloned PVL gene by PCR method and then inserted the open reading frame of PVL gene into pET-DsbA and pMBP-P vectors. PVL gene was expressed in E. coli with the introduction of isopropyl beta-D-1-thiogalactopyranoside (IPTG). We also studied the optimal culture conditions, including the concentrations of glucose, IPTG and ampicillin, induction temperature, and pH value of the medium. The characteristics of recombinant lipase were examined after affinitive purification by His-chelating affinity chromatography. RESULTS: The open reading frame of PVL gene consisted of 864 base pairs, encoding a protein of 287 amino acids. The sequence was deposited to GenBank with the accession number FJ643627. The gene was expressed in E. coli and active lipase was obtained from E. coli BL21 cells by the induction of IPTG, and the lipase production reached 192. 2 U/mL in BL21 [pET-PVL] after culture for 15 h at 15 degrees C. The maximum production was obtained by culturing BL21 cells in LB medium (pH 8.5) with 15 mg/mL glucose and 200 mg/L ampicillin, as well as adding 100 mg/L IPTG at OD600 of 1.2. A single protein band of 31 kDa was displayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after affinitive purification. The properties of lipase expressed in E. coli were similar to the native one, which could hydrolyze all three esters of triglyceride. CONCLUSION: We have succeeded in over-expressing the lipase gene from P. vulgaris in E. coli, and this research has laid a foundation for improvement and industrial application of this lipase.
Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular , Expressão Gênica , Lipase/genética , Proteus vulgaris/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Lipase/química , Lipase/metabolismo , Dados de Sequência MolecularRESUMO
A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of 605 amino acids. The gene was fused to the pPICZalphaA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-alpha factor signal peptide under the control of the AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around 94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum temperature and pH of the purified enzyme were 40 degrees C and 6.0, respectively. Over 88% of maximum activity was maintained below 40 degrees C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver-Burk plotting revealed that rGOD exhibited a K (m) value of 16.95 mM and a K (cat) value of 484.26 s(-1).
Assuntos
Aspergillus niger/enzimologia , Glucose Oxidase/metabolismo , Pichia/metabolismo , Sequência de Aminoácidos , Biocatálise , Clonagem Molecular , Expressão Gênica , Glucose Oxidase/genética , Glucose Oxidase/isolamento & purificação , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Pichia/química , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Lipases are important biocatalysts that are widely used in food processing and bio-diesel production. However, organic solvents could inactivate some lipases during applications. Therefore, the efficient cloning and expression of the organic solvent-tolerant lipase is important to its application. In this work, we first found out an organic solvent-tolerant lipase from Staphylococcus saprophyticus M36 and amplified the 741 bp Lipase gene lip3 (GenBank Accession No. FJ979867), by PCR, which encoded a 31.6 kD polypeptide of 247 amino acid residues. But the lipase shared 83% identity with tentative lip3 gene of Staphylococcus saprophyticus (GenBank Accession No. AP008934). We connected the gene with expression vector pET-DsbA, transformed it into Escherichia coli BL21 (DE3), and obtained the recombinant pET-DsbA-lip3. With the induction by 0.4 mmol/L of isopropyl beta-D-thiogalactopyranoside at pH 8.0, OD600 1.0, 25 degrees C for 12 h, the lipase activity reached up to 25.8 U/mL. The lipase expressed was stable in the presence of methanol, n-hexane, and isooctane, n-heptane.
