Purification of patatin-like protein from skim latex of hevea brasiliensis and evaluation of its antimicrobial activity /
Under the higher income of natural rubber production, Malaysia is still repressed with the striving cost for its waste management and the mounting environmental problems due to its polluting skim latex, a byproduct of the high ammoniated concentrate latex processing. This skim latex is not easily re...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
Gombak, Selangor :
Kulliyyah of Engineering, International Islamic University Malaysia,
2010
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| Subjects: | |
| Online Access: | http://studentrepo.iium.edu.my/handle/123456789/5155 |
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| Summary: | Under the higher income of natural rubber production, Malaysia is still repressed with the striving cost for its waste management and the mounting environmental problems due to its polluting skim latex, a byproduct of the high ammoniated concentrate latex processing. This skim latex is not easily recycled into new rubber products, so finding new uses for this rubber waste is a major concern. Cytosolic serum (C-serum) extracted from fresh Hevea brasiliensis latex has been identified to contain Hev b7 also known as patatin-like-protein. This protein is homologous to patatin found in potato tubers that exhibits lipid acyl hydrolase activity which may play a role in the defense mechanism against plant parasites. Therefore, this study is carried out to develop a purification strategy for patatin-like-protein from the byproduct of latex processing that is skim latex. Three sequential steps of column chromatography technique was conducted to purify the target protein namely an ion exchange chromatography on DEAE Sepharose™, gel filtration chromatography on Sephacryl™, and hydrophobic interaction chromatography on Phenyl Sepharose™. A statistical study using a full-factorial design of experiment by Statistica® software was introduced to the ion exchange chromatography in order to investigate the relationship between the pH and concentration of Tris-HCl protein buffer towards the recovery of total protein content, total lipid acyl hydrolase (LAH) activity, and protein purity thus, came up to an optimized condition of protein buffer. The success of this three steps column chromatography purification technique was assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate the protein purity, Bradford's protein assay to quantify the protein yield, enzymatic assay of LAH activity to measure total activity of active protein, and immunoassays through Dot and Western blot to confirm it as a patatin-like-protein. An antimicrobial susceptibility testing method against several pathogenic bacteria was included to investigate its potential as a natural antimicrobial agent. Based on statistical and chromatogram outputs, patatin-like-protein from skim latex is optimally purified at 17 mM Tris-HCl buffer, pH 8.7 on ion exchange column chromatography and 2M of NaCl on hydrophobic interaction column chromatography. The purified protein showed a single band of an estimated molecular weight of 45 kDa on SDS-PAGE and was confirmed to be patatin-like-protein by a strong signal obtained on Dot and Western blots, immunodetected against rabbit polyclonal anti-Hev b7 from C-serum. As much as 3L skim latex serum was successfully collected from 5L crude skim latex and a total purified protein of 26 mg with 1.5 μmole/min/mg of LAH specific activity was obtained from this crude. Positive detections of antimicrobial activity were observed on Salmonella paratyphi IMR S1401 and Pseudomonas aeruginosa IMR P137 which point out the similar characteristic of the purified patatin from skim latex and the one from potato. The study was significant as an initial attempt to purify patatin-like-protein from skim latex and thus established an alternative way of utilizing the natural rubber waste. |
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| Item Description: | Abstract in English and Arabic. "A dissertation submitted in partial fulfilment of the requirements for the degree of Master of Science (Biotechnology Engineering)."--On t.p. |
| Physical Description: | xx, 187 leaves : ill. ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 155-162). |