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Title: Study of Purification and Characterization of Versatile Peroxidase from Wild White Rot Fungi Collected from Himachal Pradesh
Researcher: Thakur Neha
Guide(s): Tripathi Astha
Keywords: Biotechnology and Applied Microbiology
Life Sciences
University: Shoolini University of Biotechnology and Management Sciences
Completed Date: 2019
Abstract: newlineABSTRACT newlineThe collection of 253 wild mushroom fruiting bodies was done from different ecological regions of Himachal Pradesh, India in 2014. Out of 253 fruiting bodies only 40 pure cultures were obtained and tested for qualitative test of enzymes Laccase and Manganese peroxidase (MnP). Out of 40 mycelial cultures only 6 showed positive results for both enzymes. On the basis of macroscopic features the 6 mushrooms showed similarity with Morchella sp. (1/14), Trametes sp.1 (6/14), Ramaria sp. (74/14), Helvella sp. (102/14), Amanita sp. (105/14), Trametes sp.2 (144/14). Quantitative screening of ligninolytic enzymes was done with the 6 selected mushrooms and both species of Trametes showed good activity for versatile peroxidase enzyme. newlineThe optimal environmental conditions of Trametes species was then determined by establishing variability in growth response on solid and liquid media over a range of different growth parameters. Both Trametes spp. showed best mycelial growth and biomass production at 30°C and at pH 7. Synthetic medium with different concentrations of glucose and ammonium tartrate was used for determining the ratio of carbon and nitrogen and both varieties showed good mycelial growth and biomass production with the ratio 20:2 and 20:1; while in Trametes sp.1 mycelial consistency was thick with the ratio 20:1. newlineAfter optimization, confirmation experiment for the presence of versatile peroxidase was conducted for both Trametes spp, - Trametes sp.2 showed highest production of versatile peroxidase and was further selected for purification and molecular identification. Pure mycelial culture of Trametes sp.2 was used in PCR gene assay using ITS region of rRNA as target for amplification which showed 99-100% homology with published NCBI sequences and identified as Trametes versicolor. The Purification of versatile peroxidase from Trametes versicolor was done by using 3L of modified Kirk medium. The protein extracted from ammonium sulphate was precipitated at 75%. The precipitated protein was dialyzed at 20mM Sodium tartrate buffer and protein was adsorbed on Q-sepharose fast flow column elution towards different concentrations. Trametes versicolor showed 42KDa band in 200mM sodium acetate buffer in anion exchange chromatography after 12% SDS-PAGE and stained with silver staining. The enzymatic activity was determined by substrate named Reactive black 5. The characterization was done with FTIR and MALDI-TOF techniques. In FTIR the Trametes versicolor showed various newlinex newlinepolysaccharides with different functional groups. In MALDI-TOF, the peptide mapping analysis showed the amino acid sequences of 17 peptide fragments and protein coverage of 66% for Trametes versicolor. It can be concluded from the present studies that Trametes versicolor possess good potential for exploiting the properties of versatile peroxidase enzyme which has been further optimized and characterized at molecular level by using FTIR, MALDI-TOF technique. So the further studies should focus on developing an efficient heterologous expression system for this enzyme which would generate great commercial industrial value and on other side holds a great potential in eco-friendly processes like Bioremediation and Biorefienery. newlineKeywords: Extracellular ligninolytic enzymes: Laccase, Manganese peroxidase, Lignin peroxidase, Aryl alcohol oxidase, Versatile Peroxidase. Mass spectrometry, Fourier transforms infrared spectroscopy, Nutrient Rich Medium, Nutrient Poor Medium.
Pagination: 107
Appears in Departments:Faculty Of Biotechnology

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01front page.pdfAttached File16.18 kBAdobe PDFView/Open
02 certificates.pdf726.95 kBAdobe PDFView/Open
06 table of contents.pdf45.33 kBAdobe PDFView/Open
07 acknowledgement.pdf10.17 kBAdobe PDFView/Open
08 list of abbreviations.pdf242.82 kBAdobe PDFView/Open
09 list of tables.pdf15.81 kBAdobe PDFView/Open
10 list of figure.pdf96.63 kBAdobe PDFView/Open
11 abstract.pdf12.71 kBAdobe PDFView/Open
12 chapter 1.pdf343.23 kBAdobe PDFView/Open
13 chapter 2.pdf496.48 kBAdobe PDFView/Open
14 chapter 3.pdf350.45 kBAdobe PDFView/Open
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16 chapter 5.pdf209.98 kBAdobe PDFView/Open
17 chapter 6.pdf7.72 kBAdobe PDFView/Open
18 chapter 7.pdf296.1 kBAdobe PDFView/Open
19 chapter 8.pdf106.55 kBAdobe PDFView/Open
20 cahpter 9.pdf157.52 kBAdobe PDFView/Open
21 paper 1.pdf309.89 kBAdobe PDFView/Open
22 paper 2.pdf555.2 kBAdobe PDFView/Open
23 paper 3.pdf578.23 kBAdobe PDFView/Open
80_recommendation.pdf24.91 kBAdobe PDFView/Open

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