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"Saturation mutagenesis"

John B McArthur, Hai Yu, Nova Tasnima, Christie M Lee, Andrew J Fisher, Xi Chen
The lack of α2-6-linkage specific sialidases limits the structural and functional studies of sialic acid-containing molecules. Photobacterium damselae α2-6-sialyltransferase (Pd2,6ST) was shown previously to have α2-6-specific, but weak, sialidase activity. Here we develop a high-throughput blue-white colony screening method to identify Pd2,6ST mutants with improved α2-6-sialidase activity from mutant libraries generated by sequential saturation mutagenesis. A triple mutant (Pd2,6ST S232L/T356S/W361F) has been identified with 101-fold improved activity, high α2-6-sialyl linkage selectivity, good activity in cleaving two common sialic acid forms N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc)...
March 15, 2018: ACS Chemical Biology
Alessandra Piccirilli, Paola Sandra Mercuri, Moreno Galleni, Massimiliano Aschi, André Matagne, Gianfranco Amicosante, Mariagrazia Perilli
GES-type β-lactamases are a group of enzymes that have evolved their hydrolytic activity towards carbapenems. In this study the role of residue 174 inside the Ω-loop of GES-1 and GES-5 was investigated. GES-1P174E and GES-5P174E mutants, selected by site-saturation mutagenesis, were purified and kinetically characterized. Comparing with GES-1 and GES-5 wild-type enzymes, GES-1P174E and GES-5P174E mutants exhibited lower kcat and kcat /Km values for cephalosporins and penicillins. Concerning carbapenems, the two mutants shared higher kcat values but lower Km values respect to those calculated for GES-1 and GES-5...
March 5, 2018: Antimicrobial Agents and Chemotherapy
Kritika Gupta, Raghavan Varadarajan
Where convenient phenotypic readouts are available, saturation mutagenesis coupled to deep sequencing provides a rapid and facile method to infer sequence determinants of protein structure, stability and function. We provide brief descriptions and currently available options for the various steps involved, and mention limitations of current implementations. We also highlight recent applications such as estimating relative stabilities and affinities of protein variants, mapping epitopes, protein model discrimination and prediction of mutant phenotypes...
March 2, 2018: Current Opinion in Structural Biology
Fucheng Zhu, Tianyue Jiang, Bin Wu, Bingfang He
Metalloprotease PT121Y114S , an effective catalyst for Z-aspartame synthesis under the substrate (Z-Asp:l-Phe-OMe) molar ratio of 1:5, was obtained previously. Herein, a computational strategy combining molecular dynamics simulation of the enzyme-substrate complex with binding free energy (ΔG) calculations was established to guide the further engineering of PT121Y114S . One His224 residue proximal to the PT121Y114S active site was selected on the basis of the difference in ΔG decomposition of PT121Y114S toward l-Phe-NH2 and l-Phe-OMe...
July 1, 2018: Food Chemistry
Shubhangi Kaushik, Sérgio M Marques, Prashant Khirsariya, Kamil Paruch, Lenka Libichova, Jan Brezovsky, Zbynek Prokop, Radka Chaloupkova, Jiri Damborsky
The traditional way of rationally engineering enzymes to change their biocatalytic properties utilizes the modifications of their active sites. Another emerging approach is the engineering of structural features involved in the exchange of ligands between buried active sites and the surrounding solvent. However, surprisingly little is known about the effects of mutations that alter the access tunnels on the enzymes' catalytic properties, and how these tunnels should be redesigned to allow fast passage of cognate substrates and products...
February 25, 2018: FEBS Journal
Chao Wang, Shuang Chen, Hong-Bin Zhang, Yao Li, Xue-Qin Hu
Dextran produced by dextransucrase hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown. Previous studies have investigated the relationships between structure and linkage specificity of the dextransucrase DSR from Leuconostoc mesenteroides by the site-directed mutagenesis of the catalytic pocket. The glycosidic linkage of dextran produced by mutant enzymes changed slightly by 3% to 20%. The mutagenesis dextransucrases were constructed by inserting an amino acid into a catalytic pocket to investigate the product specificities of dextransucrase thoroughly...
February 2, 2018: International Journal of Biological Macromolecules
Xiaoyan Ning, Yanli Zhang, Tiantian Yuan, Qingbin Li, Jian Tian, Weishi Guan, Bo Liu, Wei Zhang, Xinxin Xu, Yuhong Zhang
Glucose oxidase (GOD, EC. specifically catalyzes the reaction of β-d-glucose to gluconic acid and hydrogen peroxide in the presence of oxygen, which has become widely used in the food industry, gluconic acid production and the feed industry. However, the poor thermostability of the current commercial GOD is a key limiting factor preventing its widespread application. In the present study, amino acids closely related to the thermostability of glucose oxidase from Penicillium notatum were predicted with a computer-aided molecular simulation analysis, and mutant libraries were established following a saturation mutagenesis strategy...
January 31, 2018: International Journal of Molecular Sciences
Ge Qu, Jing Zhao, Ping Zheng, Jibin Sun, Zhoutong Sun
Screening is the bottleneck of directed evolution. In order to address this problem, a series of novel semi-rational designed strategies have been developed based on combinatorial active-site saturation test and iterative saturation mutagenesis, including single code saturation mutagenesis, double code saturation mutagenesis and triple code saturation mutagenesis. By creation of "small and smart" high qualified mutant libraries and combinatorial mutagenesis of specific sites, these new strategies have been successfully applied in multiparameter optimization, e...
January 25, 2018: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Zhi Zou, Diana M Mate, Kristin Rübsam, Felix Jakob, Ulrich Schwaneberg
Sortase-catalyzed ligations have emerged as powerful tools for the site-specific ligation of peptides and proteins in material science and biocatalysis. In this work, a directed sortase evolution strategy (SortEvolve) has been developed as a general high-throughput screening (HTS) platform to improve activity of sortase A (Application 1) and to perform directed laccase evolution through a semi-purification process in 96-well microtiter plate (MTP) (Application 2). A semi-purification process in polypropylene MTP (PP-MTP) is achieved through the anchor peptide LCI which acts as adhesion promoter...
January 24, 2018: ACS Combinatorial Science
Ge Qu, Richard Lonsdale, Peiyuan Yao, Guangyue Li, Beibei Liu, Manfred T Reetz, Zhoutong Sun
Directed evolution of stereo- or regioselective enzymes as catalysts in asymmetric transformations is of particular interest in organic synthesis. Upon evolving these biocatalysts, screening is the bottleneck. To beat the numbers problem most effectively, methods and strategies for building "small but smart" mutant libraries have been developed. Herein, we compared two different strategies regarding the application of triple-code saturation mutagenesis (TCSM) at multiresidue sites of the Thermoanaerobacter brockii alcohol dehydrogenase by using distinct reduced amino-acid alphabets...
January 4, 2018: Chembiochem: a European Journal of Chemical Biology
Almog Bregman-Cohen, Batel Deri, Shiran Maimon, Yael Pazy, Ayelet Fishman
2-Hydroxybiphenyl 3-monooxygenase (HbpA) is a flavin-containing NADH-dependent aromatic hydroxylase that oxidizes a broad range of 2-substituted phenols. In order to modulate its activity and selectivity, several residues in the active site pocket were investigated by saturation mutagenesis. Variant M321A demonstrated altered regioselectivity by oxidizing for the first time 3-hydroxybiphenyl, thus enabling the production of a new antioxidant, 3,4-dihydroxybiphenyl, with similar ferric reducing capacity as the well-studied piceatannol...
January 3, 2018: Chembiochem: a European Journal of Chemical Biology
John S Chorba, Adri M Galvan, Kevan M Shokat
Proprotein convertase subtilisin/kexin type 9 (PCSK9) down-regulates the low-density lipoprotein (LDL) receptor, elevating LDL cholesterol and accelerating atherosclerotic heart disease, making it a promising cardiovascular drug target. To achieve its maximal effect on the LDL receptor, PCSK9 requires autoproteolysis. After cleavage, PCSK9 retains its prodomain in the active site as a self-inhibitor. Unlike other proprotein convertases, however, this retention is permanent, inhibiting any further protease activity for the remainder of its life cycle...
February 9, 2018: Journal of Biological Chemistry
Shengdong Ke, Vincent Anquetil, Jorge Rojas Zamalloa, Alisha Maity, Anthony Yang, Mauricio A Arias, Sergey Kalachikov, James J Russo, Jingyue Ju, Lawrence A Chasin
To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively...
December 14, 2017: Genome Research
Du-San Baek, Jeong-Ho Kim, Ye-Jin Kim, Yong-Sung Kim
Neuropilin-1 (NRP1), which functions as a coreceptor for vascular endothelial growth factor (VEGF) and is implicated in vascular permeability and tumorigenesis, has been targeted by peptides that specifically bind to the VEGF-binding region on NRP1. Like natural VEGF ligands, all known peptides with NRP1-binding activity bind only through a carboxy (C)-terminal R/K-x-x-R/K sequence motif (x stands for any amino acids); this strict requirement is called the C-end rule (CendR). Here, we report immunoglobulin Fc-fused NRP1-specific peptides deviating from CendR...
February 5, 2018: Molecular Pharmaceutics
Aitao Li, Carlos G Acevedo-Rocha, Zhoutong Sun, Tony Cox, Jia Lucy Xu, Manfred T Reetz
Saturation mutagenesis (SM) constitutes a widely used technique in the directed evolution of selective enzymes as catalysts in organic chemistry and in the manipulation of metabolic paths and genomes, but the quality of the libraries is far from optimal due to the inherent amino acid bias. Herein, it is shown how this fundamental problem can be solved by applying high-fidelity solid-phase chemical gene synthesis on silicon chips followed by efficient gene assembly. Limonene epoxide hydrolase was chosen as the catalyst in the model desymmetrization of cyclohexene oxide with the stereoselective formation of (R,R)- and (S,S)-cyclohexane-1,2-diol...
February 2, 2018: Chembiochem: a European Journal of Chemical Biology
Shu-Ping Zou, Yu-Guo Zheng, Qun Wu, Zhi-Cai Wang, Ya-Ping Xue, Zhi-Qiang Liu
Enantioselective hydrolysis of epoxides by epoxide hydrolase (EH) is one of the most attractive approaches for the synthesis of chiral epoxides. So far, attempts to develop an efficient epoxide hydrolase -mediated biotransformation have been limited by either the low activity or insufficient enantioselectivity of epoxide hydrolase. In this study, iterative saturation mutagenesis (ISM) of epoxide hydrolase from Agrobacterium radiobacter AD1 (ArEH) was performed for efficient production of (R)-epichlorohydrin...
November 18, 2017: Applied Microbiology and Biotechnology
Gur Pines, James D Winkler, Assaf Pines, Ryan T Gill
The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations...
November 14, 2017: MBio
Guangyue Li, Maximilian J L J Fürst, Hamid Reza Mansouri, Anna K Ressmann, Adriana Ilie, Florian Rudroff, Marko D Mihovilovic, Marco W Fraaije, Manfred T Reetz
Baeyer-Villiger monooxygenases (BVMOs) and evolved mutants have been shown to be excellent biocatalysts in many stereoselective Baeyer-Villiger transformations, but industrial applications are rare which is partly due to the insufficient thermostability of BVMOs under operating conditions. In the present study, the substrate scope of the recently discovered thermally stable BVMO, TmCHMO from Thermocrispum municipale, was studied. This revealed that the wild-type (WT) enzyme catalyzes the oxidation of a variety of structurally different ketones with notable activity and enantioselectivity, including the desymmetrization of 4-methylcyclohexanone (99% ee, S)...
November 29, 2017: Organic & Biomolecular Chemistry
Eunok Jung, Beom Gi Park, Hee-Wang Yoo, Joonwon Kim, Kwon-Young Choi, Byung-Gee Kim
CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed saturation mutagenesis was attempted using a semi-rational approach that combined structure-based computational analysis and subsequent saturation mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover number (TTN) and catalytic efficiency (k cat/K M) toward palmitic acid compared to wild-type, respectively...
January 2018: Applied Microbiology and Biotechnology
Yanbing Zhu, Chaochao Qiao, Hebin Li, Lijun Li, Anfeng Xiao, Hui Ni, Zedong Jiang
This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A total of 10 single-site mutants were chosen using the PoPMuSiC program, and two mutants of K253N and P314T showed enhanced thermal stability. By saturation mutagenesis and thermostability analysis, K253H and P314T were the best mutants at the two sites. Combinational mutations of K253H, P314T and H260L were subsequently introduced, and the best mutant of K253H/H260L was selected. Thermal inactivation analysis showed the half-life (t1/2) value at 55°C for K253H/H260L was 7...
November 4, 2017: International Journal of Biological Macromolecules
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