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semi rational mutagenesis

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
Tea Pavkov-Keller, Kerstin Steiner, Mario Faber, Martin Tengg, Helmut Schwab, Mandana Gruber-Khadjawi, Karl Gruber
Friedel-Crafts alkylation of aromatic systems is a classic reaction in organic chemistry, for which regiospecific mono-alkylation, however, is generally difficult to achieve. In nature, methyltransferases catalyze the addition of methyl groups to a wide range of biomolecules thereby modulating the physico-chemical properties of these compounds. Specifically, S-adenosyl-L-methionine dependent C-methyltransferases possess a high potential to serve as biocatalysts in environmentally benign organic syntheses. Here, we report on the high resolution crystal structure of CouO, a C-methyltransferase from Streptomyces rishiriensis involved in the biosynthesis of the antibiotic coumermycin A1...
2017: PloS One
Jian-Miao Xu, Feng Cheng, Fang-Tian Fu, Hai-Feng Hu, Yu-Guo Zheng
L-2-aminobutyric acid (L-ABA) as a precursor for the anticonvulsant and the antituberculotic is a key intermediate in the chemical and pharmaceutical industries. Recently, leucine dehydrogenase (LeuDH) with NAD(+) regeneration was developed for L-ABA production on a large scale. Previously, the L-ABA yield was improved by optimizing conversion conditions, including cofactor regeneration and enzyme immobilization but not protein engineering on LeuDH due to lacking an applicable high-throughput screening (HTS) method...
December 20, 2016: Applied Biochemistry and Biotechnology
Jorick Franceus, Tom Verhaeghe, Tom Desmet
Statistical analysis of a protein multiple sequence alignment can reveal groups of positions that undergo interdependent mutations throughout evolution. At these so-called correlated positions, only certain combinations of amino acids appear to be viable for maintaining proper folding, stability, catalytic activity or specificity. Therefore, it is often speculated that they could be interesting guides for semi-rational protein engineering purposes. Because they are a fingerprint from protein evolution, their analysis may provide valuable insight into a protein's structure or function and furthermore, they may also be suitable target positions for mutagenesis...
August 11, 2016: Journal of Industrial Microbiology & Biotechnology
Matthew Deaner, Hal S Alper
Control of gene expression is crucial to optimize metabolic pathways and synthetic gene networks. Promoters and terminators are stretches of DNA upstream and downstream (respectively) of genes that control both the rate at which the gene is transcribed and the rate at which mRNA is degraded. As a result, both of these elements control net protein expression from a synthetic construct. Thus, it is highly important to discover and engineer promoters and terminators with desired characteristics. This chapter highlights various approaches taken to catalogue these important synthetic elements...
June 9, 2016: Advances in Biochemical Engineering/biotechnology
Sandra Notonier, Łukasz Gricman, Jürgen Pleiss, Bernhard Hauer
The regioselective terminal hydroxylation of alkanes and fatty acids is of great interest in a variety of industrial applications, such as in cosmetics, in fine chemicals, and in the fragrance industry. The chemically challenging activation and oxidation of non-activated C-H bonds can be achieved with cytochrome P450 enzymes. -CPRBM3 is an artificial fusion construct consisting of the heme domain from Marinobacter aquaeolei and the reductase domain of CYP102A1 from Bacillus megaterium. It has the ability to hydroxylate medium- and long-chain fatty acids selectively at their terminal positions...
August 17, 2016: Chembiochem: a European Journal of Chemical Biology
Xiaoyue Wang, Guanglu Wang, Xinli Li, Jing Fu, Tao Chen, Zhiwen Wang, Xueming Zhao
Adenylosuccinate synthetase (EC. encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242...
August 10, 2016: Journal of 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
Tom Verhaeghe, Karel De Winter, Magali Berland, Rob De Vreese, Matthias D'hooghe, Bernard Offmann, Tom Desmet
Despite the growing importance of prebiotics in nutrition and gastroenterology, their structural variety is currently still very limited. The lack of straightforward procedures to gain new products in sufficient amounts often hampers application testing and further development. Although the enzyme sucrose phosphorylase can be used to produce the rare disaccharide kojibiose (α-1,2-glucobiose) from the bulk sugars sucrose and glucose, the target compound is only a side product that is difficult to isolate. Accordingly, for this biocatalyst to become economically attractive, the formation of other glucobioses should be avoided and therefore we applied semi-rational mutagenesis and low-throughput screening, which resulted in a double mutant (L341I_Q345S) with a selectivity of 95% for kojibiose...
March 4, 2016: Chemical Communications: Chem Comm
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
Hongli Pu, Bo Lü, Dongxu Zhao, Chun Li
To improve bond selectivity of recombinant β-glucuronidase in Escherichia coli (PGUS-E), based on the PGUS-E structure guidance, three key points R329, T369 and N467 were identified to be responsible for the bond selectivity of PGUS-E, and further saturation mutagenesis was conducted. Two positive mutants R329K and T369V were obtained by a combined selection technique of thin-layer chromatography and high performance liquid chromatography. Compared to PGUS-E, the bond selectivity of mutants R329K and T369V increased by 26...
July 2015: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Shao Thing Teoh, Sastia Putri, Yukio Mukai, Takeshi Bamba, Eiichiro Fukusaki
BACKGROUND: Traditional approaches to phenotype improvement include rational selection of genes for modification, and probability-driven processes such as laboratory evolution or random mutagenesis. A promising middle-ground approach is semi-rational engineering, where genetic modification targets are inferred from system-wide comparison of strains. Here, we have applied a metabolomics-based, semi-rational strategy of phenotype improvement to 1-butanol tolerance in Saccharomyces cerevisiae...
2015: Biotechnology for Biofuels
Grace Spatafora, John Corbett, Louis Cornacchione, William Daly, Diego Galan, Michael Wysota, Patrick Tivnan, Justin Collins, Dillon Nye, Talya Levitz, Wendy A Breyer, Arthur Glasfeld
UNLABELLED: Streptococcus mutans is the causative agent of dental caries, a significant concern for human health, and therefore an attractive target for therapeutics development. Previous work in our laboratory has identified a homodimeric, manganese-dependent repressor protein, SloR, as an important regulator of cariogenesis and has used site-directed mutagenesis to map functions to specific regions of the protein. Here we extend those studies to better understand the structural interaction between SloR and its operator and its effector metal ions...
November 2015: Journal of Bacteriology
Zhenming Chen, Yuanhui Ma, Mengyan He, Hongyang Ren, Shuo Zhou, Dunyue Lai, Zhiguo Wang, Linshu Jiang
Semi-rational directed evolution was applied to the D5 variant of monoamine oxidase from Aspergillus niger (MAO-N-D5) with the aim of deriving the more desirable (R)-mexiletine through the kinetic resolution of mexiletine enantiomers. Although MAO-N-D5 shows no activity towards rac-mexiletine, theoretical molecular docking studies revealed the potential binding conformations of both mexiletine enantiomers and MAO-N-D5. The key factors affecting the catalytic activity and specificity were identified. Based on the docking results, six residues in the binding pocket and along the binding pathway were selected as key sites for saturation mutagenesis of MAO-N-D5...
August 2015: Applied Biochemistry and Biotechnology
Serwanja Jamil, Meng-Han Liu, Yong-Mei Liu, Rui-Zhi Han, Guo-Chao Xu, Ye Ni
Due to its systemic arginine degradation, arginine deiminase (ADI) has attracted attentions as an anti-tumor drug. Its low activity at physiological conditions among other limitations has necessitated its engineering for improved properties. The present study describes the hydrophobic mutagenesis and semi-rational engineering of ADI from Pseudomonas plecoglossicida (PpADI). Using an improved ADI variant M13 (D38H/A128T/E296K/H404R/I410L) as parent, site saturation mutagenesis at position 162 resulted in an over 20 % increase in protein solubility...
July 2015: Applied Biochemistry and Biotechnology
Dorte M Larsen, Christian Nyffenegger, Maria M Swiniarska, Anders Thygesen, Mikael L Strube, Anne S Meyer, Jørn D Mikkelsen
Enzymatic conversion of pectinaceous biomasses such as potato and sugar beet pulp at high temperatures is advantageous as it gives rise to lower substrate viscosity, easier mixing, and increased substrate solubility and lowers the risk of contamination. Such high-temperature processing requires development of thermostable enzymes. Talaromyces stipitatus was found to secrete endo-1,4-β-galactanase when grown on sugar beet pectin as sole carbon source. The mature protein contained 353 AA and the MW was estimated to 36...
May 2015: Applied Microbiology and Biotechnology
David Teze, Franck Daligault, Vincent Ferrières, Yves-Henri Sanejouand, Charles Tellier
A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions. In order to use them as catalysts for oligosaccharide synthesis, the balance between these two competing reactions has to be shifted toward transglycosylation. We previously designed a semi-rational approach to convert the Thermus thermophilus β-glycosidases into transglycosidases by mutating highly conserved residues located around the -1 subsite. In an attempt to verify that this strategy could be a generic approach to turn glycosidases into transglycosidases, Geobacillus stearothermophilus α-galactosidase (AgaB) was selected in order to obtain α-transgalactosidases...
April 2015: Glycobiology
Mark J Calcott, David F Ackerley
Non-ribosomal peptide synthetases (NRPS) are large modular enzymes that govern the synthesis of numerous biotechnologically relevant products. Their mode of action is frequently compared to an assembly line, in which each module acts in a semi-autonomous but coordinated manner to add a specific monomer to a growing peptide chain, unfettered by ribosomal constraints. The modular nature of these systems offers tantalising prospects for synthetic biology, wherein the assembly line is re-engineered at a genetic level to generate a specific or combinatorial modified product...
December 2014: Biotechnology Letters
Sabrina Hoebenreich, Felipe E Zilly, Carlos G Acevedo-Rocha, Matías Zilly, Manfred T Reetz
Efficient and economic methods in directed evolution at the protein, metabolic, and genome level are needed for biocatalyst development and the success of synthetic biology. In contrast to random strategies, semirational approaches such as saturation mutagenesis explore the sequence space in a focused manner. Although several combinatorial libraries based on saturation mutagenesis have been reported using solid-phase gene synthesis, direct comparison with traditional PCR-based methods is currently lacking. In this work, we compare combinatorial protein libraries created in-house via PCR versus those generated by commercial solid-phase gene synthesis...
March 20, 2015: ACS Synthetic Biology
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