Design, synthesis, and cloning of industrial lipase
Issued Date
2024
Copyright Date
1993
Resource Type
Language
eng
File Type
application/pdf
No. of Pages/File Size
xiv, 217 leaves : ill.
Access Rights
open access
Rights
ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (M.Sc. (Biochemistry))--Mahidol University, 1993
Suggested Citation
Bifani, Juan Pablo, 1964- Design, synthesis, and cloning of industrial lipase. Thesis (M.Sc. (Biochemistry))--Mahidol University, 1993. Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/100918
Title
Design, synthesis, and cloning of industrial lipase
Author(s)
Advisor(s)
Abstract
Two different approaches have been pursued in the search for a lipase with potential industrial applications. One approach concentrated on the design, synthesis, assembly, and cloning of an industrial lipase, while the second involved an extensive search, identification, and purification of a lipase-secreting thermophile and its lipase protein from endogenous sources in Thailand. A synthetic lipase gene (SLG) pJPUCLipI was designed and synthesized based on the reported amino acid sequence of Pseudomonas fragi IFO 3458 and IFO 12049. The construct pJPUCLipI has 32 regularly-spaced unique restriction sites allowing for specific and rapid cassette mutagenesis. The entire gene has been transformed and split into two vectors, but not joined together into a single insert. The first clone pFLip3/4 is 311 nucleotides long, including the conserved region Ser(83) and 24 unique restriction sites. The fidelity of the insert was tested by restriction analysis, sequencing, and colony hybridization. The second clone pFLip5/6 consists of 108 nucleotides and 8 restriction sites. It was generated by polymerase chain reaction (PCR) and cloned as a blunt end insert. The wild-type lipase model from P. fragi 3458 and another highly-homologous gene from P. fragi IFO 12049 were amplified by PCR, cloned, and expressed in E. coli. A simple protocol for the screening, identification, and isolation of lipase-secreting thermophiles was developed. Thirteen thermophilic bacteria displaying esterase activity with potential lipolytic characteristics were isolated from hot-water springs from Chiangmai, Thailand. The growth of one of the isolates, strain TL71, was enhanced and suppressed through the regulation of culture conditions, growth medium, pH, triglycerides, free fatty acids, and most notably, polysaccharides. Through temperature regulation of cell growth, it was determined that the polysaccharide concentration required for maximum lipase secretion was finite. Cell arrest resulting from either the addition of potassium cyanide or low temperature, both of which counter-balanced the enhancement effect of polysaccharides, lent support to the "detachment theory". Partially-purified lipase concentrate from strain TL71 was subjected to thermostability determinations. Lipase TL71.-2c proved to be highly thermostable as compared with other reports, thus displaying a very promising potential for industrial applications. A simple protocol involving extraction, purification, hydrolysis, ligation, and cloning of genomic DNA was used to obtain three positive clones of the lipase gene of thermophile TL71. Screenings of the desired clones were performed by consecutive assays with tributyrin agar plates, p-nitrophenylpalmitate assay, and a combination of olive oil-rhodamine, olive oil-calcium chloride or olive oil-saturated copper techniques.
Description
Biochemistry (Mahidol University 1993)
Degree Name
Master of Science
Degree Level
Master's degree
Degree Department
Faculty of Science
Degree Discipline
Biochemistry
Degree Grantor(s)
Mahidol University