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https://www.readbyqxmd.com/read/29124283/semi-rational-engineering-of-cyp153a35-to-enhance-%C3%AF-hydroxylation-activity-toward-palmitic-acid
#1
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...
November 9, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29080350/identification-of-key-residues-modulating-the-stereoselectivity-of-nitrile-hydratase-towards-rac-mandelonitrile-by-semi-rational-engineering
#2
Zhongyi Cheng, Lukasz Peplowski, Wenjing Cui, Yuanyuan Xia, Zhongmei Liu, Jialei Zhang, Michihiko Kobayashi, Zhemin Zhou
Optically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high-value chiral chemicals is often time-, money- and resource-intensive; traditional semi-rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot-spot residues. In this study, through ligand-protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity towards rac-mandelonitrile (MAN)...
October 28, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/29057958/evolving-stability-and-ph-dependent-activity-of-the-high-redox-potential-botrytis-aclada-laccase-for-enzymatic-fuel-cells
#3
Stefan Scheiblbrandner, Erik Breslmayr, Florian Csarman, Regina Paukner, Johannes Führer, Peter L Herzog, Sergey V Shleev, Evgeny M Osipov, Tamara V Tikhonova, Vladimir O Popov, Dietmar Haltrich, Roland Ludwig, Roman Kittl
Fungal high redox potential laccases are proposed as cathodic biocatalysts in implantable enzymatic fuel cells to generate high cell voltages. Their application is limited mainly through their acidic pH optimum and chloride inhibition. This work investigates evolutionary and engineering strategies to increase the pH optimum of a chloride-tolerant, high redox potential laccase from the ascomycete Botrytis aclada. The laccase was subjected to two rounds of directed evolution and the clones screened for increased stability and activity at pH 6...
October 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28832623/variants-of-sequence-family-b-thermococcus-kodakaraensis-dna-polymerase-with-increased-mismatch-extension-selectivity
#4
Claudia Huber, Andreas Marx
Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error...
2017: PloS One
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
#5
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/28583351/protein-engineering-of-cyp105s-for-their-industrial-uses
#6
REVIEW
Kaori Yasuda, Hiroshi Sugimoto, Keiko Hayashi, Teisuke Takita, Kiyoshi Yasukawa, Miho Ohta, Masaki Kamakura, Shinichi Ikushiro, Yoshitsugu Shiro, Toshiyuki Sakaki
Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D3 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation...
June 2, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28571627/directed-evolution-of-thermomyces-lanuginosus-lipase-to-enhance-methanol-tolerance-for-efficient-production-of-biodiesel-from-waste-grease
#7
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/28284313/one-step-combined-focused-eppcr-and-saturation-mutagenesis-for-thermostability-evolution-of-a-new-cold-active-xylanase
#8
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/28152088/crystal-structure-and-catalytic-mechanism-of-couo-a-versatile-c-methyltransferase-from-streptomyces-rishiriensis
#9
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
https://www.readbyqxmd.com/read/28000046/semi-rational-engineering-of-leucine-dehydrogenase-for-l-2-aminobutyric-acid-production
#10
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...
July 2017: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/27514664/correlated-positions-in-protein-evolution-and-engineering
#11
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...
May 2017: Journal of Industrial Microbiology & Biotechnology
https://www.readbyqxmd.com/read/27277391/promoter-and-terminator-discovery-and-engineering
#12
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...
2018: Advances in Biochemical Engineering/biotechnology
https://www.readbyqxmd.com/read/27251775/semirational-protein-engineering-of-cyp153am-aq-cprbm3-for-efficient-terminal-hydroxylation-of-short-to-long-chain-fatty-acids
#13
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. CYP153AM.aq. -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
https://www.readbyqxmd.com/read/27234879/directed-evolution-of-adenylosuccinate-synthetase-from-bacillus-subtilis-and-its-application-in-metabolic-engineering
#14
Xiaoyue Wang, Guanglu Wang, Xinli Li, Jing Fu, Tao Chen, Zhiwen Wang, Xueming Zhao
Adenylosuccinate synthetase (EC. 6.3.4.4) 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
https://www.readbyqxmd.com/read/27234138/semi-rational-engineering-of-cytochrome-cyp153a-from-marinobacter-aquaeolei-for-improved-%C3%AF-hydroxylation-activity-towards-oleic-acid
#15
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
https://www.readbyqxmd.com/read/27225474/practical-two-step-synthesis-of-enantiopure-styrene-oxide-through-an-optimized-chemoenzymatic-approach
#16
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
https://www.readbyqxmd.com/read/26858011/converting-bulk-sugars-into-prebiotics-semi-rational-design-of-a-transglucosylase-with-controlled-selectivity
#17
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
https://www.readbyqxmd.com/read/26804479/solubilization-and-iterative-saturation-mutagenesis-of-%C3%AE-1-3-fucosyltransferase-from-helicobacter-pylori-to-enhance-its-catalytic-efficiency
#18
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
https://www.readbyqxmd.com/read/26647587/-semi-rational-modification-for-improving-bond-selectivity-of-recombinant-%C3%AE-glucuronidase
#19
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
https://www.readbyqxmd.com/read/26379776/a-metabolomics-based-strategy-for-identification-of-gene-targets-for-phenotype-improvement-and-its-application-to-1-butanol-tolerance-in-saccharomyces-cerevisiae
#20
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
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