scholarly journals Protein engineering of fungal xylanase

2007 ◽  
Author(s):  
◽  
Dawn Elizabeth Stephens
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
Chlorine Dioxide ◽  
Dna Recombination ◽  
Thermomyces Lanuginosus ◽  
Dna Shuffling ◽  
Genetic Level ◽  
The Creation ◽  
Pre Treatment ◽  
Paper Pulps

Protein engineering technologies, such as directed evolution and DNA recombination, are often used to modify enzymes on a genetic level for the creation of useful industrial catalysts. Pre-treatment of paper pulps with xylanases have been shown to decrease the amounts of toxic chlorine dioxide used to bleach pulp. This study was undertaken to improve the thermal and alkaline stabilities of the xylanase from the fungus Thermomyces lanuginosus using ep-PCR and DNA shuffling.

scholarly journals Affinity maturation: highlights in the application of in vitro strategies for the directed evolution of antibodies

2021 ◽  
Author(s):  
Denice T.Y. Chan ◽  
Maria A.T. Groves
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
Sequence Space ◽  
Therapeutic Potential ◽  
Dna Recombination ◽  
Affinity Maturation ◽  
Therapeutic Antibodies ◽  
Effective Strategies ◽  
Key Features

Affinity maturation is a key technique in protein engineering which is used to improve affinity and binding interactions in vitro, a process often required to fulfil the therapeutic potential of antibodies. There are many available display technologies and maturation methods developed over the years, which have been instrumental in the production of therapeutic antibodies. However, due to the inherent limitations in display capacity of these technologies, accommodation of expansive and complex library builds is still a challenge. In this article, we discuss our recent efforts in the affinity maturation of a difficult antibody lineage using an unbiased approach, which sought to explore a larger sequence space through the application of DNA recombination and shuffling techniques across the entire antibody region and selections using ribosome display. We also highlight the key features of several display technologies and diversification methods, and discuss the strategies devised by different groups in response to different challenges. Particular attention is drawn to examples which are aimed at the expansion of sequence, structural or experimental diversity through different means and approaches. Here, we provide our perspectives on these methodologies and the considerations involved in the design of effective strategies for the directed evolution of antibodies.

scholarly journals Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

2021 ◽  
Vol 22 (3) ◽  
pp. 1157
Author(s):  
Pablo Aza ◽  
Felipe de Salas ◽  
Gonzalo Molpeceres ◽  
David Rodríguez-Escribano ◽  
Iñigo de la Fuente ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
Signal Peptide ◽  
Laccase Activity ◽  
Laccase Production ◽  
Wide Range ◽  
Mating Factor ◽  
Conserved Amino Acids ◽  
Basidiomycete Fungi

Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of S. cerevisiae cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.

scholarly journals Application of DNA Shuffling as a Tool for Hydrolase Activity Improvement of Pseudomonas Strain

2020 ◽  
Vol 11 (1) ◽  
pp. 7735-7745
Keyword(s):  
Protein Engineering ◽  
Protoplast Fusion ◽  
Concentration Gradient ◽  
First Generation ◽  
Human Life ◽  
Genome Shuffling ◽  
Dna Shuffling ◽  
High Concentration ◽  
Mutant Strains ◽  
High Concentrations

Biotechnology is considered one of the most influential technologies in various areas of human life, including health, economics, and the environment. Protein engineering is one of the major biotechnology tools in the field of modification and advancement of biocatalysts capabilities. Among the most effective protein engineering methods, in particular, to improve the industrial strain capabilities, is the shuffling genome method. This study aimed to follow knowledge and biocatalysts engineering techniques based on DNA shuffling methods. In the first step, two procedures were followed (DES method and compatibility according to the concentration gradient of Diazinon) to obtain mutant strains. Acquired mutant strains from both methods were resistant to high concentrations of poison up to 3000 mg/L. The activity of these strains also demonstrated their elevated activity compared to parent samples. The highest activity was related to four strains IR1.G1, IR1.D8, IR1.D4, and IR1.D5, which were 0.234 U/ml, 0.1 U/ml, 0.098 U/ml, and 0.066 U/ml, respectively. The improved strain was obtained via the concentration gradient of the diazinon method (IRL1.G1 strain) in comparison with IRL1.D8 strain (owning highest activity through DES method) possesses excessive activity in 3000 mg/L concentration of Diazinon. The evaluated results of first-generation genome shuffling of strains (the first round of protoplast fusion) also indicated that those shuffled strains with the ability to grow in the vicinity of the toxin (3000 mg/L concentration of Diazinon) showed better activity than obtained mutated strains by both methods (concentration gradient of the toxin and the DES method). In the final stage, the best results were related to IRL1.F2, IRL1.F3, and IRL1.F1 shuffled strains with 0.541 mg/L, 0.523 mg/L, and 0.509 mg/L, respectively. The highest activity belonged to the IRL1.F2 genome shuffled strain (first round of protoplast fusion). This strain could grow in a high concentration of toxin, and also, the activity was increased 30, 3.6, and 2.3 times in comparison with the parent strain (IRL1), IRL.D8 mutant, and IRL1.G1, respectively.

Continuous directed evolution for strain and protein engineering

2018 ◽  
Vol 53 ◽  
pp. 158-163 ◽  
Author(s):  
Simon d’Oelsnitz ◽  
Andrew Ellington
Keyword(s):  
Protein Engineering ◽  
Directed Evolution

Nucleotide Exchange and Excision Technology DNA Shuffling and Directed Evolution

2010 ◽  
pp. 333-344 ◽  
Author(s):  
Janina Speck ◽  
Sabine C. Stebel ◽  
Katja M. Arndt ◽  
Kristian M. Müller
Keyword(s):  
Directed Evolution ◽  
Dna Shuffling ◽  
Nucleotide Exchange

scholarly journals Lipophilic Extractives from Eucalyptus globulus Pulp during Kraft Cooking Followed by TCF and ECF Bleaching

Holzforschung ◽  
2001 ◽  
Vol 55 (3) ◽  
pp. 260-264 ◽  
Author(s):  
A. Gutiérrez ◽  
J. Romero ◽  
J.C. del Río
Keyword(s):  
Gas Chromatography ◽  
Chlorine Dioxide ◽  
Eucalyptus Globulus ◽  
Gas Chromatography Mass Spectrometry ◽  
Chemical Analyses ◽  
Wood Extractives ◽  
Elemental Chlorine ◽  
Kraft Cooking ◽  
Ecf Bleaching ◽  
Paper Pulps

SummaryThe chemical composition of lipophilic extractives in paper pulps fromEucalyptus globuluswood during kraft cooking followed by TCF (“totally chlorine free”) and ECF (“elemental chlorine free”) bleaching sequences has been determined by gas chromatography and gas chromatography-mass spectrometry. The chemical analyses revealed that the composition of the lipophilic extractives in pulp after kraft cooking and TCF bleaching with hydrogen peroxide was similar to that ofE. globuluswood extractives, sitosterol and sitosterol esters being the predominant compounds. In contrast, the presence of these compounds was almost negligible in pulp after ECF bleaching with chlorine dioxide and only the saturated sterol stigmastanol, in both free and esterified forms, survived the bleaching.

Secondary effluent reclamation: combination of pre-treatment and disinfection technologies

2008 ◽  
Vol 57 (12) ◽  
pp. 1963-1968 ◽  
Author(s):  
L. Alcalde ◽  
M. Folch ◽  
J. C. Tapias ◽  
F. Martínez ◽  
S. Enguídanos ◽  
...  
Keyword(s):  
Ultraviolet Light ◽  
Chlorine Dioxide ◽  
Peracetic Acid ◽  
Additional Treatment ◽  
Secondary Effluent ◽  
E Coli ◽  
Disinfection Methods ◽  
Physico Chemical ◽  
Treatment Train ◽  
Pre Treatment

A study was carried out to evaluate the efficiency of secondary effluent additional treatment, using a combination of pre-treatments (ring filter, physico-chemical and infiltration-percolation) followed by disinfection methods (chlorine dioxide, peracetic acid and ultraviolet light). Three different indicator microorganisms were determined: E. coli, total coliforms and somatic bacteriophages. The results show better efficiency of physico-chemical and infiltration-percolation processes. Bacteriophages were eliminated to a lesser extent than bacterial indicators in all the treatment systems. Chlorine dioxide and peracetic acid seems to be more efficient in disinfection than ultraviolet light when a ring filter is the pre-treatment used. For the same doses and contact times, the efficiency of the disinfection methods is higher when the pre-treatment used is the physico-chemical or the infiltration-percolation system. The final effluent quality from the physico-chemical treatment train and the infiltration-percolation treatment train, followed by the disinfectants, achieves an E. coli content that allows the reuse in most of the uses described in the Spanish legislation for wastewater reuse.

scholarly journals Shuffling the Neutral Drift of Unspecific Peroxygenase inSaccharomyces cerevisiae

2018 ◽  
Vol 84 (15) ◽  
Author(s):  
Javier Martin-Diaz ◽  
Carmen Paret ◽  
Eva García-Ruiz ◽  
Patricia Molina-Espeja ◽  
Miguel Alcalde
Keyword(s):  
Genetic Drift ◽  
Dna Recombination ◽  
Dna Shuffling ◽  
Content Type ◽  
Neutral Drift ◽  
Oxygenase Activity ◽  
Improved Stability ◽  
Neutral Mutations ◽  
Neutral Genetic Drift

ABSTRACTUnspecific peroxygenase (UPO) is a highly promiscuous biocatalyst, and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO inSaccharomyces cerevisiae, so that neutral mutations were simply added and recombinedin vivo. With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded active UPO variants were generated. After only eight rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with changes in peroxidative and peroxygenative activities, kinetic thermostability, and enhanced tolerance to organic solvents. With an average of 4.6 substitutions introduced per clone, neutral mutations covered approximately 10% of the protein sequence. Accordingly, this study opens new avenues for UPO design by bringing together neutral genetic drift and DNA recombinationin vivo.IMPORTANCEFungal peroxygenases resemble the peroxide shunt pathway of cytochrome P450 monoxygenases, performing selective oxyfunctionalizations of unactivated C-H bonds in a broad range of organic compounds. In this study, we combined neutral genetic drift andin vivoDNA shuffling to generate highly functional peroxygenase mutant libraries. The panel of neutrally evolved peroxygenases showed different activity profiles for peroxygenative substrates and improved stability with respect to temperature and the presence of organic cosolvents, making the enzymes valuable blueprints for emerging evolution campaigns. This association of DNA recombination and neutral drift is paving the way for future work in peroxygenase engineering and, from a more general perspective, to any other enzyme system heterologously expressed inS. cerevisiae.

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