keyword
MENU ▼
Read by QxMD icon Read
search

"Saturation mutagenesis"

keyword
https://www.readbyqxmd.com/read/28284313/one-step-combined-focused-eppcr-and-saturation-mutagenesis-for-thermostability-evolution-of-a-new-cold-active-xylanase
#1
Juan Pablo Acevedo, Manfred T Reetz, Juan A Asenjo, Loreto P Parra
Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry...
May 2017: Enzyme and Microbial Technology
https://www.readbyqxmd.com/read/28255534/the-structure-of-toho1-%C3%AE-lactamase-in-complex-with-penicillin-reveals-the-role-of-tyr105-in-substrate-recognition
#2
Patricia S Langan, Venu Gopal Vandavasi, Kevin L Weiss, Jonathan B Cooper, Stephan L Ginell, Leighton Coates
The role of the conserved residue Tyr105 in class A β-lactamases has been the subject of investigation using both structural studies and saturation mutagenesis. Both have shown that while it does not need to be strictly conserved for activity, it is important for substrate recognition. With this in mind we determined the crystal structure of Toho1 β-lactamase at 15 K to 1.10 Å resolution in complex with penicillin. As expected a ring-opened penicillin molecule bound to Ser70 the catalytic nucleophile, can clearly be seen in electron density in the active site...
December 2016: FEBS Open Bio
https://www.readbyqxmd.com/read/28251763/a-13-week-research-based-biochemistry-laboratory-curriculum
#3
Scott T Lefurgy, Emily C Mundorff
Here, we present a 13-week research-based biochemistry laboratory curriculum designed to provide the students with the experience of engaging in original research while introducing foundational biochemistry laboratory techniques. The laboratory experience has been developed around the directed evolution of an enzyme chosen by the instructor, with mutations designed by the students. Ideal enzymes for this curriculum are able to be structurally modeled, solubly expressed, and monitored for activity by UV/Vis spectroscopy, and an example curriculum for haloalkane dehalogenase is given...
March 2, 2017: Biochemistry and Molecular Biology Education
https://www.readbyqxmd.com/read/28218758/variant-aware-saturating-mutagenesis-using-multiple-cas9-nucleases-identifies-regulatory-elements-at-trait-associated-loci
#4
Matthew C Canver, Samuel Lessard, Luca Pinello, Yuxuan Wu, Yann Ilboudo, Emily N Stern, Austen J Needleman, Frédéric Galactéros, Carlo Brugnara, Abdullah Kutlar, Colin McKenzie, Marvin Reid, Diane D Chen, Partha Pratim Das, Mitchel A Cole, Jing Zeng, Ryo Kurita, Yukio Nakamura, Guo-Cheng Yuan, Guillaume Lettre, Daniel E Bauer, Stuart H Orkin
Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants...
February 20, 2017: Nature Genetics
https://www.readbyqxmd.com/read/28218303/a-combination-of-mutational-and-computational-scanning-guides-the-design-of-an-artificial-ligand-binding-controlled-lipase
#5
Marco Kaschner, Oliver Schillinger, Timo Fettweiss, Christina Nutschel, Frank Krause, Alexander Fulton, Birgit Strodel, Andreas Stadler, Karl-Erich Jaeger, Ulrich Krauss
Allostery, i.e. the control of enzyme activity by a small molecule at a location distant from the enzyme's active site, represents a mechanism essential for sustaining life. The rational design of allostery is a non-trivial task but can be achieved by fusion of a sensory domain, which responds to environmental stimuli with a change in its structure. Hereby, the site of domain fusion is difficult to predict. We here explore the possibility to rationally engineer allostery into the naturally not allosterically regulated Bacillus subtilis lipase A, by fusion of the citrate-binding sensor-domain of the CitA sensory-kinase of Klebsiella pneumoniae...
February 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28170244/structure-activity-relationship-and-molecular-mechanics-reveal-the-importance-of-ring-entropy-in-the-biosynthesis-and-activity-of-a-natural-product
#6
Hai L Tran, Katrina W Lexa, Olivier Julien, Travis S Young, Christopher T Walsh, Matthew P Jacobson, James A Wells
Macrocycles are appealing drug candidates due to their high affinity, specificity, and favorable pharmacological properties. In this study, we explored the effects of chemical modifications to a natural product macrocycle upon its activity, 3D geometry, and conformational entropy. We chose thiocillin as a model system, a thiopeptide in the ribosomally encoded family of natural products that exhibits potent antimicrobial effects against Gram-positive bacteria. Since thiocillin is derived from a genetically encoded peptide scaffold, site-directed mutagenesis allows for rapid generation of analogues...
February 22, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28165493/a-noise-trimming-and-positional-significance-of-transposon-insertion-system-to-identify-essential-genes-in-yersinia-pestis
#7
Zheng Rong Yang, Helen L Bullifent, Karen Moore, Konrad Paszkiewicz, Richard J Saint, Stephanie J Southern, Olivia L Champion, Nicola J Senior, Mitali Sarkar-Tyson, Petra C F Oyston, Timothy P Atkins, Richard W Titball
Massively parallel sequencing technology coupled with saturation mutagenesis has provided new and global insights into gene functions and roles. At a simplistic level, the frequency of mutations within genes can indicate the degree of essentiality. However, this approach neglects to take account of the positional significance of mutations - the function of a gene is less likely to be disrupted by a mutation close to the distal ends. Therefore, a systematic bioinformatics approach to improve the reliability of essential gene identification is desirable...
February 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28163026/engineering-of-cyp106a2-for-steroid-9%C3%AE-and-6%C3%AE-hydroxylation
#8
Julia Nikolaus, Kim Thoa Nguyen, Cornelia Virus, Jan L Riehm, Michael Hutter, Rita Bernhardt
CYP 106A2 from Bacillus megaterium ATCC 13368 has been described as a 15β-hydroxylase showing also minor 11α-, 9α- and 6β-hydroxylase activity for progesterone conversion. Previously, mutant proteins with a changed selectivity towards 11α-OH-progesterone have already been produced. The challenge of this work was to create mutant proteins with a higher regioselectivity towards hydroxylation at positions 9 and 6 of the steroid molecule. 9α-hydroxyprogesterone exhibits pharmaceutical importance, because it is a useful intermediate in the production of physiologically active substances which possess progestational activity...
April 2017: Steroids
https://www.readbyqxmd.com/read/28134951/converting-pasteurella-multocida%C3%AE-2-3-sialyltransferase-1-pmst1-to-a-regioselective-%C3%AE-2-6-sialyltransferase-by-saturation-mutagenesis-and-regioselective-screening
#9
John B McArthur, Hai Yu, Jie Zeng, Xi Chen
A microtiter plate-based screening assay capable of determining the activity and regioselectivity of sialyltransferases was developed. This assay was used to screen two single-site saturation libraries of Pasteurella multocidaα2-3-sialyltransferase 1 (PmST1) for α2-6-sialyltransferase activity and total sialyltransferase activity. PmST1 double mutant P34H/M144L was found to be the most effective α2-6-sialyltransferase and displayed 50% reduced donor hydrolysis and 50-fold reduced sialidase activity compared to the wild-type PmST1...
February 21, 2017: Organic & Biomolecular Chemistry
https://www.readbyqxmd.com/read/28109425/beyond-the-natural-proteome-nondegenerate-saturation-mutagenesis-methodologies-and-advantages
#10
M M Ferreira Amaral, L Frigotto, A V Hine
Beyond the natural proteome, high-throughput mutagenesis offers the protein engineer an opportunity to "tweak" the wild-type activity of a protein to create a recombinant protein with required attributes. Of the various approaches available, saturation mutagenesis is one of the core techniques employed by protein engineers, and in recent times, nondegenerate saturation mutagenesis is emerging as the approach of choice. This review compares the current methodologies available for conducting nondegenerate saturation mutagenesis with traditional, degenerate saturation and briefly outlines the options available for screening the resulting libraries, to discover a novel protein with the required activity and/or specificity...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28096490/comprehensive-essentiality-analysis-of-the-mycobacterium-tuberculosis-genome-via-saturating-transposon-mutagenesis
#11
Michael A DeJesus, Elias R Gerrick, Weizhen Xu, Sae Woong Park, Jarukit E Long, Cara C Boutte, Eric J Rubin, Dirk Schnappinger, Sabine Ehrt, Sarah M Fortune, Christopher M Sassetti, Thomas R Ioerger
For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed...
January 17, 2017: MBio
https://www.readbyqxmd.com/read/28092444/engineering-of-the-bacillus-circulans-%C3%AE-galactosidase-product-specificity
#12
Huifang Yin, Tjaard Pijning, Xiangfeng Meng, Lubbert Dijkhuizen, Sander S van Leeuwen
Microbial β-galactosidase enzymes are widely used as biocatalysts in industry to produce prebiotic galactooligosaccharides (GOS) from lactose. GOS mixtures are used as beneficial additives in infant formula to mimic the prebiotic effects of human milk oligosaccharides (hMOS). The structural variety in GOS mixtures is significantly lower than in hMOS. Since this structural complexity is considered as the basis for the multiple biological functions of hMOS, it is important to broaden the variety of GOS structures...
February 7, 2017: Biochemistry
https://www.readbyqxmd.com/read/28074907/insights-into-an-evolutionary-strategy-leading-to-antibiotic-resistance
#13
Chun-Feng D Hou, Jian-Wei Liu, Charles Collyer, Nataša Mitić, Marcelo Monteiro Pedroso, Gerhard Schenk, David L Ollis
Metallo-β-lactamases (MBLs) with activity towards a broad-spectrum of β-lactam antibiotics have become a major threat to public health, not least due to their ability to rapidly adapt their substrate preference. In this study, the capability of the MBL AIM-1 to evade antibiotic pressure by introducing specific mutations was probed by two alternative methods, i.e. site-saturation mutagenesis (SSM) of active site residues and in vitro evolution. Both approaches demonstrated that a single mutation in AIM-1 can greatly enhance a pathogen's resistance towards broad spectrum antibiotics without significantly compromising the catalytic efficiency of the enzyme...
January 11, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28074221/exploring-the-thermostable-properties-of-halohydrin-dehalogenase-from-agrobacterium-radiobacter-ad1-by-a-combinatorial-directed-evolution-strategy
#14
Zhiyun Wu, Wenfeng Deng, Yapei Tong, Qian Liao, Dongmin Xin, Huashun Yu, Juan Feng, Lixia Tang
As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained...
January 10, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28073929/improving-heterologous-production-of-phenylpropanoids-in-saccharomyces-cerevisiae-by-tackling-an-unwanted-side-reaction-of-tsc13-an-endogenous-double-bond-reductase
#15
Beata Joanna Lehka, Michael Eichenberger, Walden Emil Bjørn-Yoshimoto, Katherina Garcia Vanegas, Nicolaas Buijs, Niels Bjerg Jensen, Jane Dannow Dyekjær, Håvard Jenssen, Ernesto Simon, Michael Naesby
Phenylpropanoids, such as flavonoids and stilbenoids, are of great commercial interest, and their production in S. cerevisiae is a very promising strategy. However, to achieve commercially viable production, each step of the process must be optimized. We looked at carbon loss, known to occur in the heterologous flavonoid pathway in yeast, and identified an endogenous enzyme, the enoyl reductase Tsc13, which turned out to be responsible for the accumulation of phloretic acid via reduction of p-coumaroyl-CoA...
January 10, 2017: FEMS Yeast Research
https://www.readbyqxmd.com/read/28064476/chemoselective-coupling-preserves-the-substrate-integrity-of-surface-immobilized-oligonucleotides-for-emulsion-pcr-based-gene-library-construction
#16
Marie L Malone, Valerie J Cavett, Brian M Paegel
Combinatorial bead libraries figure prominently in next-generation sequencing and are also important tools for in vitro evolution. The most common methodology for generating such bead libraries, emulsion PCR (emPCR), enzymatically extends bead-immobilized oligonucleotide PCR primers in emulsion droplets containing a single progenitor library member. Primers are almost always immobilized on beads via noncovalent biotin-streptavidin binding. Here, we describe covalent bead functionalization with primers (∼10(6) primers/2...
January 9, 2017: ACS Combinatorial Science
https://www.readbyqxmd.com/read/28050632/amino-acid-substitutions-in-random-mutagenesis-libraries-lessons-from-analyzing-3000-mutations
#17
Jing Zhao, Victorine Josiane Frauenkron-Machedjou, Tsvetan Kardashliev, Anna Joëlle Ruff, Leilei Zhu, Marco Bocola, Ulrich Schwaneberg
The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis...
January 3, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28049717/a-novel-hect-ubiquitin-ligase-regulating-chemotaxis-and-development-in-dictyostelium-discoideum
#18
Barbara Pergolizzi, Enrico Bracco, Salvatore Bozzaro
Cyclic AMP binding to G protein-coupled receptors orchestrates chemotaxis and development in Dictyostelium. By activating the RasC-TORC2-AKT/PKB module, cAMP regulates cell polarization during chemotaxis. TORC2 also mediates GPCR-dependent stimulation of adenylyl cyclase A (ACA), enhancing cAMP relay and developmental gene expression. Thus, mutants defective in the TORC2 Pia/Rictor subunit are impaired in chemotaxis and development. Near-saturation mutagenesis of a Pia/Rictor mutant by random gene disruption led to selection of two suppressor mutants, in which spontaneous chemotaxis and development were restored...
January 3, 2017: Journal of Cell Science
https://www.readbyqxmd.com/read/28044225/simple-mssm-a-simple-and-efficient-method-for-simultaneous-multi-site-saturation-mutagenesis
#19
Feng Cheng, Jian-Miao Xu, Chao Xiang, Zhi-Qiang Liu, Li-Qing Zhao, Yu-Guo Zheng
OBJECTIVE: To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. RESULTS: A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons...
January 2, 2017: Biotechnology Letters
https://www.readbyqxmd.com/read/28044007/modification-of-the-peroxygenative-peroxidative-activity-ratio-in-the-unspecific-peroxygenase-from-agrocybe-aegerita-by-structure-guided-evolution
#20
Diana M Mate, Miguel A Palomino, Patricia Molina-Espeja, Javier Martin-Diaz, Miguel Alcalde
Unspecific peroxygenase (UPO) is a heme-thiolate peroxidase capable of performing with high-selectivity C-H oxyfunctionalizations of great interest in organic synthesis through its peroxygenative activity. However, the convergence of such activity with an unwanted peroxidative activity encumbers practical applications. In this study, we have modified the peroxygenative:peroxidative activity ratio (P:p ratio) of UPO from Agrocybe aegerita by structure-guided evolution. Several flexible loops (Glu1-Pro35, Gly103-Asp131, Ser226-Gly243, Gln254-Thr276 and Ty293-Arg327) were selected on the basis on their B-factors and ΔΔG values...
January 1, 2017: Protein Engineering, Design & Selection: PEDS
keyword
keyword
78213
1
2
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read
×

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"