Dissertation > Excellent graduate degree dissertation topics show

Cloning and Expression of Organic Solvent Tolerant Lipase Gene from Staphylococcus Saprophyticus M36

Author: TangYanLi
Tutor: LuZhaoXin
School: Nanjing Agricultural College
Course: Of Food Science
Keywords: Staphylococcus saprophyticus lipase gene cloning and expression Escherichia coli Bacillus subtilis site-specific mutagenesis
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 5
Quote: 0
Read: Download Dissertation


Biodiesel is a form of biomass energy with the advantages of environmental friendly, regeneration, and low energy consumption. It can resolve the crisis yielded by the depletion of oil and environmental contamination, so biodiesel become more attractive recently. The synthesize technology of biodiesel by bioconversion method have the advantages of moderation condition, simple procedure, low energy consumption and environmental friendly compared with the traditional catalyze method. However, it is difficult for the development and industrialization of biodiesel produced by bioconversion method due to the high cost of lipase, toxicity effect of organic solvent to lipase and the special catalysis of lipase. This thesis mainly studied the cloning of the organic solvent-tolerant lipase gene lip3 from Staphylococcus saprophyticus M36, and the expression of the lipase in Escherichia coli and Bacillus subtilis. It also studies the site-specific mutagenesis of lip5 gene to get organic solvent-tolerant lipase. Main study results are as following:1. Genomic DNA was extracted form Staphylococcus saprophyticus M36. Then specific primers were designed according to the tentative lipase III sequence and the lip3 with the full length of 741bp was amplified. According to the conserved region sequence of lipase V, the specific primers were designed. With the specific primer and degenerate oligonucleotides, the lip5 of 804bp was amplified and then sequenced by Site finding-PCR. GenBank Accession Numbers of lip3 and lip5 are FJ979867 and FJ979868 respectively.2. Lip3 was fused with the expression vector pET-DsbA, and transformed into Escherichia coli BL21 (DE3), and finally obtained the recombinant pET-DsbA-lip3. It was certified that lip3 was expressed correctly through SDS-PAGE electrophoresis; active stain and chromogenic agar plate. With the induction by 0.4 mmol/L of Isopropyl beta-D-thiogalactopyranoside at pH 8.0, OD600 1.0,25℃for 12 h, the recombinase activity reached up to 25.8 U/mL. The fusion protein ligand of BL21/pET-DsbA-lip3 was removed by Nickel column, and the electrophoretically pure lip3 protein was obstained. The optimal pH of the recombinase is 8.0 and the optimal temperature is 25℃, and it was stable in the presence of methanol, n-hexane, and isooctane, n-heptane. The activity of BL21/pET-lip3 recombinase activity was raised by Mg2+ andβ-mercaptoethanol.3. Lip3 was highly expressed in B. subtilis DB104 using pHT43 as the vector. The optimal pH of pHT43-lip3 is 8.0 and the optimal temperature is 50℃. Nearly 50% of the activity was maintained in the presence of ethanol, isopropanol and acetone after 1 h. The recombinase activity was raised by isoamylol, Na+, K+ and P-mercaptoethanol.4. We constructed the mutant gene lip5D93S, via over-lap extension PCR. Organic solvent-tolerant lipase was produced by expression of the mutant in B. subtilis. It was proved that Asp in the active centre of lip5 was replaced by Ser, which resulted in inactive lip5 recombinase. Lip5D93s was ligated with the expression vector pHT43, and transformed into B. subtilis DB104. The properties of recombinase pHT43-lip5D93S were studied. The optimal pH is 8.0 and the optimal temperature is 40℃. And 50% of activity was retained in the presence of isopropanol, n-hexane, n-heptane and acetone after 1 h.

Related Dissertations

  1. Penicillium Expansum TS414 Lipase in Pichia Pastori: Expression, Purification and Enantioselective Esterification of (R, S)-naproxen,Q814
  2. Studies on Fermentation Optimization, Purification and Enzyme Characteristics of Lipase from Aspergillus Oryzae FS-1,TQ925.6
  3. Cloning and Expression of Growth Hormone Gene in Cobia (Rachycentron Canadus),Q786
  4. Directed Evolution of Lipase from Penicillium Expansum FS1884 by Error Prone PCR,Q78
  5. Isolation Entophytes and Study on the Fermentation of Bacillus Subtilis tiD7 from Rehmannia,TQ461
  6. Optimization of Fermentation Conditions, Purification, Cloning and Expression of a Cold-active Lipase from Pseudomonas Sp.RT-1,TQ925
  7. Isolation and Characterization of Carbendazim-Degrading Strains, Cloning and Expression of the mheI Gene,X172
  8. Expression of VP2 Gene of Infectious Bursal Disease Virus and Preparation of Monoclonal Antibodies Against the Recombinant VP2 Protein,S852.65
  9. Isolation and Identification of Avian Pathogenic Escherichia Coli from Chicken and Characterizations of Its Virulence-Associated Genes,S852.61
  10. Isolation, Identification and Degrading Characteristic of a Cyhalofop-Butyl-Degrading Bacteria, and Cloning, Expression of a Cyhalofop-Butyl-Hydrolyzing Gene chbH,X172
  11. Improvement in Electroporation Transformation Efficiency of Gene for Bacillus Subtilis with Trehalose,Q78
  12. Screening of Bacterial Strains with High α-Galactosidase Activity and Its Gene Cloning, Expression, Purification and Characterization,TQ925
  13. Screening and Isolation of One γ-PGA Producing Bacterium and Bilogical Effect of Bio-Organic Fertilizer,TQ922.1
  14. Lipase-catalyzed Transesterification of Lard to Produce L-ascorbyl Fatty Acid Esters,TS221
  15. Antifungal Mechanism of Fengycin Against Rhizopus Stolonifer,TS201.3
  16. Extraction of the Lipase from Pseudomonas Cepacia S31 and Its Applications in Food,TS201.25
  17. Cucumber Growth Promotion and Wilt Disease Suppression of Bacillus Subtilis SQR9 and Their Mechanisms,S436.421
  18. Antagonistic Action and Bio-Control Effect of Bacillus Subtilis Strain Fmbj Upon the Postharvest Pathogen Rhizopus Stolonifer,S476
  19. Molecular Biological Characteristics of Multiple Porcine Fc Gamma RⅡB Sub-isoforms Generated by Alternative Splicing,S828
  20. Alleviating Effects of Bacillus Subtilis on the Cucumber under Salt Stress in Substrate Culture,S642.2

CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
© 2012 www.DissertationTopic.Net  Mobile