Chapter title |
Stabilization of Enzymes by Using Thermophiles
|
---|---|
Chapter number | 21 |
Book title |
Microbial Steroids
|
Published in |
Methods in molecular biology, July 2017
|
DOI | 10.1007/978-1-4939-7183-1_21 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7182-4, 978-1-4939-7183-1
|
Authors |
Ana Luisa Ribeiro, Mercedes Sánchez, Aurelio Hidalgo, José Berenguer, Ribeiro, Ana Luisa, Sánchez, Mercedes, Hidalgo, Aurelio, Berenguer, José |
Abstract |
Manufactured steroid compounds have many applications in the pharmaceutical industry. Due to the chemical complexity and chirality of steroids, there is an increasing demand for enzyme-based bioconversion processes to replace those based on chemical synthesis. In this context, thermostability of the involved enzymes is a highly desirable property as both the increased half-life of the enzyme and the enhanced solubility of substrates and products will improve the yield of the reactions. Metagenomic libraries from thermal environments are potential sources of thermostable enzymes of prokaryotic origin, but the number of expected hits could be quite low for enzymes handling substrates such as steroids, rarely found in prokaryotes. An alternative to metagenome screening is the selection of thermostable variants of well-known steroid-processing enzymes. Here we review and detail a protocol for such selection, where error-prone PCR (epPCR) is used to introduce random mutations into a gene to create a variants library for further selection of thermostable variants in the thermophile Thermus thermophilus. The method involves the use of folding interference vectors where the proper folding of the enzyme of interest at high temperature is linked to the folding of a reporter encoding a selectable property such as thermostable resistance to kanamycin, leading to a life-or-death selection of variants of reinforced folding independently of the activity of the enzyme. |
X Demographics
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 1 | 100% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 1 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 19 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Researcher | 4 | 21% |
Student > Doctoral Student | 2 | 11% |
Student > Bachelor | 2 | 11% |
Student > Master | 2 | 11% |
Student > Ph. D. Student | 1 | 5% |
Other | 1 | 5% |
Unknown | 7 | 37% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 5 | 26% |
Agricultural and Biological Sciences | 4 | 21% |
Immunology and Microbiology | 1 | 5% |
Chemistry | 1 | 5% |
Unknown | 8 | 42% |