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"Saturation mutagenesis"

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https://www.readbyqxmd.com/read/28638047/semi-rational-engineering-of-a-thermostable-aldo-keto-reductase-from-thermotoga-maritima-for-synthesis-of-enantiopure-ethyl-2-hydroxy-4-phenylbutyrate-ehpb
#1
Zhiguo Wang, Shuo Zhou, Shuangling Zhang, Sa Zhang, Fangmeng Zhu, Xiaolu Jin, Zhenming Chen, Xiaoling Xu
A novel aldo-keto reductase Tm1743 characterized from Thermotoga maritima was explored as an effective biocatalyst in chiral alcohol production. Natural Tm1743 catalyzes asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) at high efficiency, but the production of, ethyl (S)-2-hydroxy-4-phenylbutyrate ((S)-EHPB), which is less desirable, is preferred with an enantiomeric excess (ee) value of 76.5%. Thus, altering the enantioselectivity of Tm1743 to obtain the more valuable product (R)-EHPB for angiotensin drug synthesis is highly desired...
June 21, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28637270/in-planta-recapitulation-of-isoprene-synthases-evolution-from-ocimene-synthases
#2
Mingai Li, Jia Xu, Alberto Algarra Alarcon, Silvia Carlin, Enrico Barbaro, Luca Cappellin, Violeta Velikova, Urska Vrhovsek, Francesco Loreto, Claudio Varotto
Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated.We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases...
June 16, 2017: Molecular Biology and Evolution
https://www.readbyqxmd.com/read/28601895/site-saturation-mutagenesis-library-construction-and-screening-for-specific-broad-spectrum-single-domain-antibodies-against-multiple-cry1-toxins
#3
Lingxia Jiao, Yuan Liu, Xiao Zhang, Beibei Liu, Cunzheng Zhang, Xianjin Liu
Potential ecological environmental and food safety risks of various Cry toxins of Bacillus thuringiensis (Bt) in transgenic food have received gradually increasing attention, which urged to establish an efficient and broad-spectrum detection technology for Cry toxins. Based on the single-domain antibody (sdAb) A8 against Bt Cry1Ab toxin screened from the humanized domain antibody library, the key amino acids of sdAb (A8) binding five kinds of Cry1 toxins were predicted using homology modeling and molecular docking technology, and the results showed that 105th asparagine, 106th arginine, 107th valine, and 114th arginine, respectively, located in heavy-chain complementarity-determining region 3 were common key amino acid sites...
June 11, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28571627/directed-evolution-of-thermomyces-lanuginosus-lipase-to-enhance-methanol-tolerance-for-efficient-production-of-biodiesel-from-waste-grease
#4
Kaiyuan Tian, Kee Tai, Bryan Jian Wei Chua, Zhi Li
Engineering a methanol tolerant lipase is of great importance in biodiesel production. Here, the first semi-rational method for directed enzyme evolution to enhance methanol tolerance by targeting high B-factor residues for iterative saturation mutagenesis (ISM) is reported. The best double mutant, TLL-S105C/D27R, retained 71% of its original activity after incubation in methanol, showing 30% greater methanol tolerance than TLL. TLL-S105C/D27R also displayed 27% higher activity over TLL. Structure modelling suggested that the increased stability of TLL-S105C/D27R was caused by the formation of a new hydrogen bond which stabilized the protein structure...
May 19, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28530095/rational-design-of-bacillus-coagulans-nl01-l-arabinose-isomerase-and-use-of-its-f279i-variant-in-d-tagatose-production
#5
Zhaojuan Zheng, Wending Mei, Meijuan Xia, Qin He, Jia Ouyang
d-Tagatose is a prospective functional sweetener that can be produced by l-arabinose isomerase (AI) from d-galactose. To improve the activity of AI toward d-galactose, the AI of Bacillus coagulans was rationally designed on the basis of molecular modeling and docking. After alanine scanning and site-saturation mutagenesis, variant F279I that exhibited improved activity toward d-galactose was obtained. The optimal temperature and pH of F279I were determined to be 50 °C and 8.0, respectively. This variant possessed 1...
May 30, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28526714/constans-imparts-dna-sequence-specificity-to-the-histone-fold-nf-yb-nf-yc-dimer
#6
Nerina Gnesutta, Roderick W Kumimoto, Swadhin Swain, Matteo Chiara, Chamindika Siriwardana, David Stephen Horner, Ben F Holt, Roberto Mantovani
NF-Y is a heterotrimeric transcription factor that binds CCAAT elements. The NF-Y trimer is composed of a Histone Fold Domain (HFD) dimer (NF-YB/NF-YC) and NF-YA, which confers DNA sequence-specificity. NF-YA shares a conserved domain with the CCT (CONSTANS, CONSTANS-LIKE, TOC1) proteins. We show that CONSTANS (CO/BBX1), a master flowering regulator, forms a trimer with Arabidopsis NF-YB2/NF-YC3 to efficiently bind the CORE element of the FT promoter. We term this complex NF-CO. Using saturation mutagenesis EMSAs and RNA-Seq profiling of co, nf-yb, and nf-yc mutants, we identify CCACA elements as the core NF-CO binding site...
May 19, 2017: Plant Cell
https://www.readbyqxmd.com/read/28508385/improving-the-temperature-characteristics-and-catalytic-efficiency-of-a-mesophilic-xylanase-from-aspergillus-oryzae-aoxyn11a-by-iterative-mutagenesis-based-on-in-silico-design
#7
Xue-Qing Li, Qin Wu, Die Hu, Rui Wang, Yan Liu, Min-Chen Wu, Jian-Fang Li
To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly(21) with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11A(G21I), was selected from the mutagenesis library, E...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28504265/in-vitro-evolution-of-an-influenza-broadly-neutralizing-antibody-is-modulated-by-hemagglutinin-receptor-specificity
#8
Nicholas C Wu, Geramie Grande, Hannah L Turner, Andrew B Ward, Jia Xie, Richard A Lerner, Ian A Wilson
The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. However, the factors that influence the evolution of high-affinity bnAbs remain elusive. We therefore explore the functional sequence space of bnAb C05, which targets the receptor-binding site (RBS) of influenza haemagglutinin (HA) via a long CDR H3. We combine saturation mutagenesis with yeast display to enrich for C05 variants of CDR H3 that bind to H1 and H3 HAs...
May 15, 2017: Nature Communications
https://www.readbyqxmd.com/read/28469274/crispr-cpf1-assisted-genome-editing-of-corynebacterium-glutamicum
#9
Yu Jiang, Fenghui Qian, Junjie Yang, Yingmiao Liu, Feng Dong, Chongmao Xu, Bingbing Sun, Biao Chen, Xiaoshu Xu, Yan Li, Renxiao Wang, Sheng Yang
Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86-100%...
May 4, 2017: Nature Communications
https://www.readbyqxmd.com/read/28464395/specificity-effects-of-amino-acid-substitutions-in-promiscuous-hydrolases-context-dependence-of-catalytic-residue-contributions-to-local-fitness-landscapes-in-nearby-sequence-space
#10
Christopher D Bayer, Bert van Loo, Florian Hollfelder
Catalytic promiscuity can facilitate evolution of enzyme functions-a multifunctional catalyst may act as a springboard for efficient functional adaptation. We test the effect of single mutations on multiple activities in two groups of promiscuous AP superfamily members to probe this hypothesis. We quantify the effect of site-saturating mutagenesis of an analogous, nucleophile-flanking residue in two superfamily members: an arylsulfatase (AS) and a phosphonate monoester hydrolase (PMH). Statistical analysis suggests that no one physicochemical characteristic alone explains the mutational effects...
June 1, 2017: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/28464223/casting-eppcr-ceppcr-a-simple-random-mutagenesis-method-to-generate-high-quality-mutant-libraries
#11
Jianhua Yang, Anna J Ruff, Marcus Arlt, Ulrich Schwaneberg
During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g., high temperature, extreme pH, ionic liquids, or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is random mutagenesis by error-prone polymerase chain reaction (epPCR). However, recent reports show that epPCR often fails to produce >70% of beneficial positions/amino acid exchanges which improve enzyme properties such as organic solvent or ionic liquid resistance...
May 2, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28425285/directed-evolution-of-carbonyl-reductase-from-rhodosporidium-toruloides-and-its-application-in-stereoselective-synthesis-of-tert-butyl-3r-5s-6-chloro-3-5-dihydroxyhexanoate
#12
Zhi-Qiang Liu, Lin Wu, Xiao-Jian Zhang, Ya-Ping Xue, Yu-Guo Zheng
tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl reductase RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH). For the activity of RtSCR9 to be improved, random mutagenesis and site-saturation mutagenesis were performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys, and mut-Phe156Gln). These mutants exhibited 1.94-, 3...
May 10, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28422960/predicting-the-impact-of-lynch-syndrome-causing-missense-mutations-from-structural-calculations
#13
Sofie V Nielsen, Amelie Stein, Alexander B Dinitzen, Elena Papaleo, Michael H Tatham, Esben G Poulsen, Maher M Kassem, Lene J Rasmussen, Kresten Lindorff-Larsen, Rasmus Hartmann-Petersen
Accurate methods to assess the pathogenicity of mutations are needed to fully leverage the possibilities of genome sequencing in diagnosis. Current data-driven and bioinformatics approaches are, however, limited by the large number of new variations found in each newly sequenced genome, and often do not provide direct mechanistic insight. Here we demonstrate, for the first time, that saturation mutagenesis, biophysical modeling and co-variation analysis, performed in silico, can predict the abundance, metabolic stability, and function of proteins inside living cells...
April 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28387245/design-and-application-of-a-lactulose-biosensor
#14
Jieyuan Wu, Peixia Jiang, Wei Chen, Dandan Xiong, Linglan Huang, Junying Jia, Yuanyuan Chen, Jian-Ming Jin, Shuang-Yan Tang
In this study the repressor of Escherichia coli lac operon, LacI, has been engineered for altered effector specificity. A LacI saturation mutagenesis library was subjected to Fluorescence Activated Cell Sorting (FACS) dual screening. Mutant LacI-L5 was selected and it is specifically induced by lactulose but not by other disaccharides tested (lactose, epilactose, maltose, sucrose, cellobiose and melibiose). LacI-L5 has been successfully used to construct a whole-cell lactulose biosensor which was then applied in directed evolution of cellobiose 2-epimerase (C2E) for elevated lactulose production...
April 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28381551/production-of-homogeneous-glycoprotein-with-multisite-modifications-by-an-engineered-n-glycosyltransferase-mutant
#15
Qitao Song, Zhigang Wu, Yueyuan Fan, Woran Song, Peiru Zhang, Li Wang, Faxing Wang, Yangyang Xu, Peng G Wang, Jiansong Cheng
Naturally occurring N-glycoproteins exhibit glycoform heterogeneity with respect to N-glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). However, access to well-defined glycoproteins is always important for both basic research and therapeutic purposes. As a result, there has been a substantial effort to identify and understand the catalytic properties of N-glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study we found that ApNGT, a newly discovered cytoplasmic N-glycosyltransferase from Actinobacillus pleuropneumoniae, has strict selectivity toward the residues around the Asn of N-glycosylation sequon by screening a small library of synthetic peptides...
May 26, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28284313/one-step-combined-focused-eppcr-and-saturation-mutagenesis-for-thermostability-evolution-of-a-new-cold-active-xylanase
#16
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
#17
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
#18
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
#19
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...
April 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
#20
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
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