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iterative saturation mutagenesis

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
Chengtuo Niu, Linjiang Zhu, Xin Xu, Qi Li
Higher thermostability is required for 1,3-1,4-β-glucanase to maintain high activity under harsh conditions in the brewing and animal feed industries. In this study, a comprehensive and comparative analysis of thermostability in bacterial β-glucanases was conducted through a method named spatial compartmentalization of mutational hotspots (SCMH), which combined alignment of homologous protein sequences, spatial compartmentalization, and molecular dynamic (MD) simulation. The overall/local flexibility of six homologous β-glucanases was calculated by MD simulation and linearly fitted with enzyme optimal enzymatic temperatures...
February 2017: Applied Microbiology and Biotechnology
Yan Duan, Lina Ba, Jianwei Gao, Xianxing Gao, Dunming Zhu, René M de Jong, Daniel Mink, Iwona Kaluzna, Zhanglin Lin
ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket...
October 2016: Applied Microbiology and Biotechnology
Kai Wu, Hualei Wang, Lifeng Chen, Haiyang Fan, Zhiqiang Zhao, Dongzhi Wei
Enantiopure styrene oxide (SO) and its derivatives are important building blocks for chiral synthesis. In this study, we developed an attractive "1-pot, 2-step" chemoenzymatic approach for producing enantiopure SO with 100 % theoretical yield. This approach involved asymmetric reduction of α-chloroacetophenone by an alcohol dehydrogenase (ADH; step 1), followed by base-induced ring closure (epoxidation) of enantiopure 2-chloro-1-phenylethanol produced by the ADH (step 2). By-product formation during epoxidation was suppressed to <1 % by adding methyl tert-butyl ether (MTBE) as the second phase...
October 2016: Applied Microbiology and Biotechnology
Xiao-Jing Luo, Jian Zhao, Chun-Xiu Li, Yun-Peng Bai, Manfred T Reetz, Hui-Lei Yu, Jian-He Xu
Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling...
November 2016: Biotechnology and Bioengineering
Meng Wang, Huimin Zhao
The advantages of computational design and directed evolution are complementary, and only through combined and iterative use of both approaches, a daunting task such as protein-ligand interaction design, can be achieved efficiently. Here, we describe a systematic strategy to combine structure-guided computational design, iterative site saturation mutagenesis, and yeast two-hybrid system (Y2H)-based phenotypic screening to engineer novel and orthogonal interactions between synthetic ligands and human estrogen receptor α (hERα) for the development of novel gene switches...
2016: Methods in Molecular Biology
Zhoutong Sun, Ylva Wikmark, Jan-E Bäckvall, Manfred T Reetz
Directed evolution of stereo- and regioselective enzymes constitutes a prolific source of catalysts for asymmetric transformations in organic chemistry. In this endeavor (iterative) saturation mutagenesis at sites lining the binding pocket of enzymes has emerged as the method of choice, but uncertainties regarding the question of how to group many residues into randomization sites and how to choose optimal upward pathways persist. Two new approaches promise to beat the numbers problem effectively. One utilizes a single amino acid as building block for the randomization of a 10-residue site, the other also employs only one but possibly different amino acid at each position of a 9-residue site...
April 4, 2016: Chemistry: a European Journal
Yun Hee Choi, Jong Hoon Kim, Bum Seok Park, Byung-Gee Kim
α1,3-Fucosyltransferase (α1,3-FucT) is essential for the biosynthesis of biologically active α1,3-fucosyloligosacchairdes (3-FOs) from human milk oligosaccharides (HMO), particularly 3-fucosyllactose (3-FL) trisaccharide. α1,3-FucT from Helicobacter pylori 26695 (FutA) accepts lactose and LacNAc as glycan acceptors and has a very low level of expression in Escherichia coli, and it shows a low catalytic activity for lactose in the large-scale synthesis of 3-FL. To overcome the poor solubility of FutA, codon optimization, and systematic truncation of the protein at the C-terminus with only one heptad repeat remaining (Δ52 FutA) were conducted to yield 150-200 mg/L of soluble protein of FutA and resulting in more than an 18-fold increase in the 3-FL yield...
August 2016: Biotechnology and Bioengineering
Yanru Liu, Liqing Qiu, Jianzhong Huang, Bingchun Zhao, Zuozhen Wang, Xiaolan Zhu, Yuanyuan Gao, Zhengyu Shu
OBJECTIVE: We improved the thermostability of LipA from Burkholderia cecapia ZYB002 by protein engineering technology to expand the application of lipase LipA. METHOD: On the basis of B-factor value of lipase LipA, series of potential mutation hotspots were selected for iterative saturation mutagenesis and the corresponding small mutation gene libraries were then constructed to screen the hyperthermal variants. RESULTS: From the above mutation libraries, we obtained a series of mutants whose enzyme half-life at 55 degrees C increased by 1...
June 4, 2015: Wei Sheng Wu Xue Bao, Acta Microbiologica Sinica
Xin Yin, Jian-Fang Li, Chun-Juan Wang, Die Hu, Qin Wu, Ying Gu, Min-Chen Wu
Feruloyl or ferulic acid esterase (Fae, EC catalyzes the hydrolysis of ester bonds between polysaccharides and phenolic acid compounds in xylan side chain. In this study, the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii was increased by iterative saturation mutagenesis (ISM). Two amino acids, Ser33 and Asn92, were selected for saturation mutagenesis according to the B-factors analyzed by B-FITTER software and ΔΔG values predicted by PoPMuSiC algorithm. After screening the saturation mutagenesis libraries constructed in Pichia pastoris, 15 promising variants were obtained...
December 2015: Applied Microbiology and Biotechnology
Shosuke Yoshida, Junichi Enoki, Robert Kourist, Kenji Miyamoto
A bacterial arylmalonate decarboxylase (AMDase) catalyzes asymmetric decarboxylation of unnatural arylmalonates to produce optically pure (R)-arylcarboxylates without the addition of cofactors. Previously, we designed an AMDase variant G74C/C188S that displays totally inverted enantioselectivity. However, the variant showed a 20,000-fold reduction in activity compared with the wild-type AMDase. Further studies have demonstrated that iterative saturation mutagenesis targeting the active site residues in a hydrophobic pocket of G74C/C188S leads to considerable improvement in activity where all positive variants harbor only hydrophobic substitutions...
2015: Bioscience, Biotechnology, and Biochemistry
Carole L Yauk, Iain B Lambert, M E Bette Meek, George R Douglas, Francesco Marchetti
The Organisation for Economic Cooperation and Development's (OECD) Adverse Outcome Pathway (AOP) programme aims to develop a knowledgebase of all known pathways of toxicity that lead to adverse effects in humans and ecosystems. A Users' Handbook was recently released to provide supplementary guidance on AOP development. This article describes one AOP-alkylation of DNA in male premeiotic germ cells leading to heritable mutations. This outcome is an important regulatory endpoint. The AOP describes the biological plausibility and empirical evidence supporting that compounds capable of alkylating DNA cause germ cell mutations and subsequent mutations in the offspring of exposed males...
December 2015: Environmental and Molecular Mutagenesis
Chao Guo, Yanpu Chen, Yu Zheng, Wei Zhang, Yunwen Tao, Juan Feng, Lixia Tang
Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and β-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained...
April 2015: Applied and Environmental Microbiology
Loreto P Parra, Juan P Acevedo, Manfred T Reetz
Phenylacetone monooxygenase (PAMO) is an exceptionally robust Baeyer-Villiger monooxygenase, which makes it ideal for potential industrial applications. However, its substrate scope is limited, unreactive cyclohexanone being a prominent example. Such a limitation is unfortunate, because this particular transformation in an ecologically viable manner would be highly desirable, the lactone and the respective lactam being of considerable interest as monomers in polymer science. We have applied directed evolution in search of an active mutant for this valuable C-C activating reaction...
July 2015: Biotechnology and Bioengineering
Letian Song, Adrian Tsang, Michel Sylvestre
Xylanases are used in many industrial processes including pulp bleaching, baking, detergent, and the hydrolysis of plant cell wall in biofuels production. In this work we have evolved a single domain GH10 xylanase, Xyn10A_ASPNG, from Aspergillus niger to improve its thermostability. We introduced a rational approach involving as the first step a computational analysis to guide the design of a mutagenesis library in targeted regions which identified thermal important residues that were subsequently randomly mutagenized through rounds of iterative saturation mutagenesis (ISM)...
June 2015: Biotechnology and Bioengineering
Susan K Boehlein, Janine R Shaw, Jon D Stewart, Bradford Sullivan, L Curtis Hannah
Iterative saturation mutagenesis (ISM) has been used to improve the thermostability of maize endosperm ADP-glucose pyrophosphorylase (AGPase), a highly-regulated, rate-limiting and temperature-sensitive enzyme essential for starch biosynthesis. The thermo-sensitivity of heterotetrameric AGPase has been linked to grain loss in cereals and improving this property might therefore have direct impacts on grain yield. Nine amino acids were selected for site-saturation mutagenesis on the basis of elevated B-factors in the crystal structure of the closest available homolog (a small subunit homotetramer of potato AGPase)...
February 15, 2015: Archives of Biochemistry and Biophysics
Xiong Wang, Kai Zheng, Huayu Zheng, Hongli Nie, Zujun Yang, Lixia Tang
Iterative saturation mutagenesis (ISM) has been shown to be a powerful method for directed evolution. In this study, the approach was modified (termed M-ISM) by combining the single-site saturation mutagenesis method with a DC-Analyzer-facilitated combinatorial strategy, aiming to evolve novel biocatalysts efficiently in the case where multiple sites are targeted simultaneously. Initially, all target sites were explored individually by constructing single-site saturation mutagenesis libraries. Next, the top two to four variants in each library were selected and combined using the DC-Analyzer-facilitated combinatorial strategy...
December 20, 2014: Journal of Biotechnology
Zhi-Gang Zhang, Richard Lonsdale, Joaquin Sanchis, Manfred T Reetz
Structure-based directed evolution utilizing iterative saturation mutagenesis (ISM) has been applied to phenyl acetone monooxygenase (PAMO), a thermally robust Baeyer-Villiger monooxygenase, in the quest to access a mutant which displays reversed enantioselectivity in the asymmetric sulfoxidation of prochiral thioethers. Whereas WT PAMO leads to 90% ee in the sulfoxidation of p-methylbenzyl methyl thioether with preference for the (S)-sulfoxide, the evolved mutant I67Q/P440F/A442N/L443I is 95% (R)-selective in the reaction of this and other thioethers...
December 10, 2014: Journal of the American Chemical Society
Kiran S Gajula, Peter J Huwe, Charlie Y Mo, Daniel J Crawford, James T Stivers, Ravi Radhakrishnan, Rahul M Kohli
Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9-11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement...
September 2014: Nucleic Acids Research
Alexander Dennig, Jan Marienhagen, Anna Joëlle Ruff, Ulrich Schwaneberg
Multi-site-saturation mutagenesis allows altering of "localizable" properties such as activity and selectivity and enables the discovery of cooperative amino acid substitutions which are unlikely to be discovered by saturating single codons individually or iteratively. The herein presented method "OmniChange" does not require any DNA modifying enzyme (e.g., endonucleases or ligases), and diverse mutant libraries with up to five simultaneously saturated positions are generated in a robust and technically simple manner in four steps...
2014: Methods in Molecular Biology
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