Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): A Versatile Tool for Generating Targeted Libraries

Herman, Asael; Tawfik, Dan S.

doi:10.1002/9783527634026.ch19

Cited by 7 publications

(10 citation statements)

References 24 publications

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“…Therefore, we substituted it to tryptophan in order to generate π interactions between these two residues. A combinatorial library based on these eight residues was generated and screened (Herman and Tawfik 2007).…”

Section: Resultsmentioning

confidence: 99%

“…Eight oligonucleotides were designed and obtained from Sigma-Aldrich (Rehovot, Israel) (see Table S1 in supplementary material). DNA library of lip T6His with randomization of the eight specific mutations was created by using the ISOR protocol (Incorporating Synthetic Oligonucleotides via Gene Reassembly (Herman and Tawfik 2007)) with modifications as previously described (Dror et al 2014). After the random hybridization of the oligonucleotides, the assembly product was amplified using Bnested^PCR reaction.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus

Dror

Kanteev

Kagan

et al. 2015

Appl Microbiol Biotechnol

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Enzymatic production of biodiesel by transesterification of triglycerides and alcohol, catalyzed by lipases, offers an environmentally friendly and efficient alternative to the chemically catalyzed process while using low-grade feedstocks. Methanol is utilized frequently as the alcohol in the reaction due to its reactivity and low cost. However, one of the major drawbacks of the enzymatic system is the presence of high methanol concentrations which leads to methanol-induced unfolding and inactivation of the biocatalyst. Therefore, a methanol-stable lipase is of great interest for the biodiesel industry. In this study, protein engineering was applied to substitute charged surface residues with hydrophobic ones to enhance the stability in methanol of a lipase from Geobacillus stearothermophilus T6. We identified a methanol-stable variant, R374W, and combined it with a variant found previously, H86Y/A269T. The triple mutant, H86Y/A269T/R374W, had a half-life value at 70 % methanol of 324 min which reflects an 87-fold enhanced stability compared to the wild type together with elevated thermostability in buffer and in 50 % methanol. This variant also exhibited an improved biodiesel yield from waste chicken oil compared to commercial Lipolase 100L® and Novozyme® CALB. Crystal structures of the wild type and the methanol-stable variants provided insights regarding structure-stability correlations. The most prominent features were the extensive formation of new hydrogen bonds between surface residues directly or mediated by structural water molecules and the stabilization of Zn and Ca binding sites. Mutation sites were also characterized by lower B-factor values calculated from the X-ray structures indicating improved rigidity.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus

Dror

Kanteev

Kagan

et al. 2015

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…Additionally, limited knowledge of the sequence-structure-function relationship may necessitate semirational and random mutagenesis approaches. Methods including error-prone PCR, site-saturation mutagenesis (Labrou, 2010), DNA shuffling (Stemmer, 1994), and incorporating synthetic oligonucleotides via recombination (ISOR) (Herman & Tawfik, 2007) have all been used to create diversity in a random or semirandom manner.…”

Section: Rational Semirational and Random Librariesmentioning

confidence: 99%

Bioprospecting in the Genomic Age

Hicks¹,

Prather²

2014

Advances in Applied Microbiology

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“…Once decisions have been made about which mutations to retain, which to incorporate, and which to discard have been made, subsequent libraries can be constructed through any number of combinatorial library generation techniques, including fully [87] or semisynthetic DNA shuffling [16,88,89]. An advantage of the machine learning-guided approach to evolution is that combinatorial libraries devoted to searching high gradient directions of the sequence-function landscape can be constructed and screened in parallel with ongoing diversity generation efforts.…”

Section: Machine Learning-guided Strategiesmentioning

confidence: 99%

Principles of Enzyme Optimization for the Rapid Creation of Industrial Biocatalysts

Fox

Giver

2010

Enzyme Technologies

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Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): A Versatile Tool for Generating Targeted Libraries

Cited by 7 publications

References 24 publications

Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus

Structural insights into methanol-stable variants of lipase T6 from Geobacillus stearothermophilus

Bioprospecting in the Genomic Age

Principles of Enzyme Optimization for the Rapid Creation of Industrial Biocatalysts

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