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Subject: Biotechnology × Author: Parul × End date: 2024 × Start date: 2020 ×
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    ThesisItemOpen Access
    Isolation and purification of proteins from Tinospora cordifolia against Pseudomonas aeruginosa
    (College of Horticulture and Forestry Dr YSP UHF, Neri, Hamirpur(H.P.), 2023-05-14) Parul; Sharma, Sneh
    The present investigation on the “Isolation and purification of proteins from Tinospora cordifoliaagainst Pseudomonas aeruginosa.” were carried out in the Department of Biotechnology, College of Horticulture and Forestry (Dr. Y.S. Parmar University of Horticulture and Forestry), Neri Hamirpur (HP) during 2021-2022. Tinospora cordifolia is a deciduous shrub. It is a well-recognized for its medicinal properties in Indian Ayurveda system. It is one of the most versatile shrub commonly known as “giloy”. The plant gained attention due to its various biological activities such as anti-diabetic, anti-microbial, anti-allergic, anti-oxidant and anti-cancer activities. The plant parts contains various chemical constituents such as alkaloids, steroids, glycosides and polysaccharides. The study was undertaken for isolation and purification of antibacterial protein of Tinospora cordifolia collected from Hamirpur district (Neri), Himachal Pradesh. Analysis was done by stem ethanolic, methanolic, extract of samples taken from Neri village. It was found that methanol act as better solvent for extraction of proteins. Ammonium sulphate precipitation of methaolic crude extract showed that 60-70% precipitation was found best for precipitation of proteins. Further, 60-70% precipitates was subject for dialysis followed by ion exchange chromatography. The fractions obtained after ion exchange chromatography was assayed by antibacterial activity against test organism. The active fractions were pooled together and carried out for SDS-PAGE. The total protein content present in methanolic crude extract was 2.056 mg/ml. The ammonium sulphate precipitation showed 10.833±0.928 mm zone of inhibition and after purification the antibacterial activity increased to 15±0.289 mm. The partial characterization of proteins revealed that the antibacterial proteins was stable at optimal pH 8, heat stable at 40 ℃ temperature and stable at low salt concentration. For future aspects, the stem extract and protein can be further exploited for its other properties such as its sequencing.
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    ThesisItemOpen Access
    “Studies on Protoplast fusion of Trichoderma”
    (College of Horticulture and Forestry Neri, Hamirpur (H.P.), 2021-12-04) Parul; Dhiman, Karuna
    In the present investigation protoplast fusion between Trichoderma Harzianum MTCC 936 and Trichoderma virens MTCC 1376 was done to develop a method to genetically recombine the important characteristics of two parental strain i.e. mutated strain of Trichoderma virens having high growth enhancement feature and Trichoderma harzianum strain having good biocontrol capability. The main purpose of this study was to produce a useful strain having properties from both fungi so to use single strain instead if using fungi individually or in combination. Viable protoplast from mycelium of both fungal strain were released using 100 mg/ml lysing enzyme and 0.2 mg/ml β-glucuronidase in 1.2 M sorbitol as osmotic stabilizer. Interspecific fusion was carried out using 40% Peg as fusogen and fusants were regenerated on PRMM media. 10 fusants were selected on the basis of morphological markers i.e. spore colouration, colony edge, ring formation and mycelia growth out of which 5 fast growing fusants (F1, F4, F6, F7 and F10) on 2% colloidal chitin medium were selected medium were selected for further studies. All the fusants showed morphological variations. Protein pattern of fusant and parental strain was compared using SDS PAGE and it was depicted that some fusants was hybrid of parental strain having bands from both the parent whereas other fusants contain different banding pattern than the parental strains. Molecular characterization of parental strain and selected fusants was investigated by RAPD markers and seven primer showed selective amplification in parental strain and fusants. Cluster analysis was carried out which depicted that F1 and F4 fusants were similar to each other at 0.80 coefficient value. Cell wall degrading activity was compared and it was observed that some fusants showed 4.4 fold increase in chitinase and 3 fold increase in β-1,3-glucanase activity as compared to parental strains. Antifungal activity of fusants and parental strains were tested against Fusarium oxyporum. Most fusants showed antagonistic activity against Fusarium oxyporum. Fusants F1 showed highest percent inhibition (70.18) against Fusarium oxyporum.