Dissertation Project Presentation Bhavik Thakar

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This is the presentation of my Final Year Dissertation project. The project along with this presentation earned me 84% in the final year and 10/10 GPA. Though it is a brief version of my Thesis, I’m sure it conveys with clarity.

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Dissertation Project Presentation Bhavik Thakar

  1. 1. “STUDIES ON EXPRESSION AND CHARACTERIZATION OF ARECOMBINANT AMYLASE FROM MICROBISPORA SP.” Presented by: BHAVIK THAKAR M. Tech (Integrated) in Biotechnology Under the Guidance of: Dr. Neelu Nawani Associate Professor, Department of Industrial Biotechnology,Dr. D. Y. Patil Biotechnology and Bioinformatics Institute
  2. 2. INTRODUCTION Microbispora Microbispora Actinomycetales Thermophillic Mesophillic Streptosporangiaceae• Significance: Produces industrially important Metabolites• Microbispora sp. V2: An isolate obtained from thermal springs which could grow at 50oC and was chosen for the studies on thermostability of its enzymes. 2
  3. 3. INTRODUCTION• Microbispora sp. V2 produces many hydrolytic enzymes, amongst which is a unique oligosaccharide forming amylase which hydrolyses starch into maltopentaoside.• The amylase gene was cloned in DH5α strain of E.coli using shotgun cloning and transformed using pUC18 plasmid vector.• The recombinant clones were screened through Blue-White Screening technique.• The recombinant strain 31 expressing starch hydrolytic activity was selected and grown on Luria agar containing Ampicillin. 3
  4. 4. METHODS AND RESULTS Expression of Starch degrading activity IPTG (Isopropyl Thiogalactoside) was spread on Luria Agar plates containing Ampicillin and Starch After absorption of IPTG in the medium, a single isolated colony from the culture plate of recombinant E.coli strain 31 was spot inoculated After incubating it for 24 hours at 370C, the plate was flooded with Iodine solution After sometime, the plate was observed under bright light 4
  5. 5. METHODS & RESULTS  A Zone of Clearance was formedSection of the plate showing Zone White colonies visible on Blue - Whiteof Clearance screening 5
  6. 6. METHODS & RESULTS Transformation of DH5α with pUC18 without gene insert The E.coli cells were made competent by calcium chloride method These competent cells were transformed with pUC18 vector without amylase gene insert 6
  7. 7. METHODS & RESULTS Isolation of Plasmid DNA A single isolated colony of the transformed E.coli cells was suspended in 50µl of 10mM EDTA (pH 8.0) To each tube, 50µl of NSS (NaOH, Sucrose, SDS) solution was added and vortexed These tubes were then incubated at 700C in a water bath for 5 minutes and then allowed to reach room temperature To each tube, 1.5µl of 4M KCL solution was added, vortexed and incubated on ice for 5 minutes 7
  8. 8. METHODS & RESULTS On centrifugation, the supernatant obtained was used for plasmid DNA analysis Plasmid DNA Analysis • Agarose gel electrophoresis was used to analyse the pUC18 plasmid DNA for its successful transformation in E.coli DH5α • This was done by running parallel Electrophoresis of transformed E.coli DH5α containing pUC18 plasmid without amylase gene insert along with cloned E.coli strain 31(containing pUC18 plasmid with amylase gene insert) and 1Kb ladder 8
  9. 9. METHODS & RESULTS The migration efficiency changed on transformation due to increase in Molecular weight of the genome. LANE A: Recombinant strain 31 LANE B: pUC18 plasmid DNA LANE C: 1Kb LadderPlasmid Profile of pUC18 DNA 9
  10. 10. METHODS & RESULTS IPTG Induction for production of recombinant Amylase A single isolated colony from overnight plates was inoculated in fresh Luria broth + Ampicillin and incubated until the OD at A600 reached 0.6 – 0.8 1% of this broth was inoculated in fresh broth medium and again kept for incubation until A600 reached 0.6-0.8 The culture was then induced with IPTG and incubated for 12 hours Localization of recombinant Amylase enzyme Extracellular fraction Cytoplasmic/Periplasmic (Secreted) fraction 10
  11. 11. METHODS & RESULTS Extraction of recombinant amylase enzyme Post induction, the extracellular fraction of the culture was harvested by centrifugation (Extracts) Pellet obtained after centrifugation was suspended in CelLytic-BTM buffer and incubated for 30 minutes The suspension was centrifuged and the supernatant was collected This supernatant consists of the cytoplasmic/periplasmic fraction of amylase enzyme (Lysates) 11
  12. 12. METHODS & RESULTS Amylase is present in the Lysate sample of recombinant strain 31 on induction with IPTG The amylase so produced is an α-amylase Graph of Enzyme activity of amylase on starch azure 7 Enzyme activity (Units/ml) 6.182 6 5 4 3 2.256 2 1.277 1.522 1.384 1.195 1 0 Rec. 31 Rec. 31 DH5α [IPTG] DH5α Tr. DH5α Tr. DH5α [IPTG] [Uninduced] [Uninduced] [IPTG] [Uninduced] Samples & their type of Induction 13
  13. 13. METHODS & RESULTS  Significance of using IPTG: • Overexpression of recombinant α-amylase enzyme • Enters into the cell in a concentration dependent manner • Cannot be cleaved by β-galactosidase Recombinant strain 31 showed negligible β - cyclodextrinase activity 14
  14. 14. METHODS & RESULTS pH Optimum α-Amylase enzyme activity (Units/ml) Graph of Optimum pH for amylase activity 0.03 0.025 0.024 0.02 0.018 0.018 0.018 0.016 0.015 0.014 0.01 0.007 0.005 0.005 0 2 3 4 5 6 7 8 9 10 11 pH  The α-amylase optimally degrades starch at pH 7.0 17
  15. 15. METHODS & RESULTS Temperature Optimum Graph of Optimum Temperature for amylase activity 0.02 α-Amylase enzyme activity 0.018 0.018 0.016 (Units/ml) 0.014 0.012 0.01 0.008 0.006 0.005 0.005 0.004 0.003 0.002 0.001 0 20 30 40 50 60 70 80 pH The α-amylase optimally degrades starch at 300C Temperature 18
  16. 16. METHODS & RESULTS Protein Analysis on SDS-PAGE M 2 3 4 5 6 7 8 9 10 11 12 13 M Lane M – Protein Molecular Weight Marker Lane 3 – Sonicated lysed DH5α Lane 6 – IPTG induced strain 31 Lysate Lane 9 – IPTG induced DH5α Lysate Lane 11 – Uninduced strain 31 Lysate Lane 12 – Uninduced DH5α Lysate  Sonication disrupts the enzyme and hence lysis buffer was better for cell lysis 19
  17. 17. CONCLUSION Amylase is overproduced on induction with IPTG It is produced in the Periplasm The recombinant amylase is an α-amylase It shows activity upto 6.182 Units/ml It has very less or no β-cyclodextrinase activity It digests starch with Specific activity of 10.185 Units/mg It optimally degrades starch at pH 7.0 and temperature 300C 20
  18. 18. BIBLIOGRAPHYo Bernfeld, P. (1951). “Enzymes of starch degradation and synthesis”. Advances in enzymology 12, 379-481o Benjamin N. Mijts and Bharat K. C. Patel (2002). “Cloning, sequencing and expression of an aamylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme”. Microbiology, 148, 2343–2349.o Sambrook, J., E. F. Fritsch, and T. Maniatis (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.o Noriyuki Doukyu, Watara Yamagishi, Hirokazu Kuwahara, and Hiroyasu Ogino (2008). “A Maltooligosachharide-Forming Amylase Gene from Brachybacterium sp. Strain LB25: Cloning and Expression in Escherichia coli”. Biosci.Biotechnol., 72 (9), 2444-2447, 2008.o Pierre Cornelis, Colette Digneffe and Karine Willemot (1982), "Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli", Molecular And General Genetics MGG, Volume 186, Number 4, 507-511o Vigal T, Gil JA, Daza A, García-González MD, Martín JF. (1991), "Cloning, characterization and expression of an alpha-amylase gene from Streptomyces griseus IMRU3570", Mol Gen Genet; 225(2):278-88.o M.W. Fogarty (1983),”Microbial Amylases. In: Microbial Enzymes and Biotechnology”, W.M. Fogarty (Ed.), Applied Science Publishers Ltd., London, UK pp. 1–92.

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