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Cofactor regeneration

Zhi-Mei Cui, Jian-Dong Zhang, Xiao-Jun Fan, Gao-Wei Zheng, Hong-Hong Chang, Wen-Long Wei
Optically pure 1-phenyl-1,2-ethanediol is a very important chiral building block and intermediate in fine chemical and pharmaceutical industries. Reduction of 2-hydroxyacetophenone provides a straightforward approach to access these important compounds. In this study, two enantiocomplementary carbonyl reductases, BDHA (2,3-butanediol dehydrogenase from Bacillus subtilis) and GoSCR (polyol dehydrogenase from Gluconobacter oxydans) were discovered for the first time to convert 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED) and (S)-1-phenyl-1,2-ethanediol ((S)-PED) with excellent stereochemical selectivity, respectively...
December 21, 2016: Journal of Biotechnology
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
Young Hoon Jung, Sooah Kim, Jungwoo Yang, Jin-Ho Seo, Kyoung Heon Kim
Furfural, one of the most common inhibitors in pre-treatment hydrolysates, reduces the cell growth and ethanol production of yeast. Evolutionary engineering has been used as a selection scheme to obtain yeast strains that exhibit furfural tolerance. However, the response of Saccharomyces cerevisiae to furfural at the metabolite level during evolution remains unknown. In this study, evolutionary engineering and metabolomic analyses were applied to determine the effects of furfural on yeasts and their metabolic response to continuous exposure to furfural...
December 8, 2016: Microbial Biotechnology
Souvik Basak, Sumanta Kumar Ghosh, Vinay Deep Punetha, Ashish N Aphale, Prabir K Patra, Nanda Gopal Sahoo
A carbonyl reductase (cr) gene from Candida glabrata CBS138 has been heterologously expressed in cofactor regenerating E. coli host to convert Ethyl-4-chloro-3-oxobutanoate (COBE) into Ethyl-4-chloro-3-hydroxybutanoate (CHBE). The CR enzyme exhibited marked velocity at substrate concentration as high as 363mM with highest turnover number (112.77±3.95s(-1)). Solitary recombineering of such catalytic cell reproduced CHBE 161.04g/L per g of dry cell weight (DCW). Introduction of combinatorially engineered crp (crp*, F136I) into this heterologous E...
February 2017: International Journal of Biological Macromolecules
Randeep K Singh, Lina Dagnino
The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation...
November 26, 2016: Oncotarget
Nina Beyer, Justyna K Kulig, Anette Bartsch, Martin A Hayes, Dick B Janssen, Marco W Fraaije
To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase...
November 29, 2016: Applied Microbiology and Biotechnology
Nicole D Paris, Andrew Soroka, Alanna Klose, Wenxuan Liu, Joe V Chakkalakal
Skeletal muscle regenerative potential declines with age, in part due to deficiencies in resident stem cells (satellite cells, SCs) and derived myogenic progenitors (MPs); however, the factors responsible for this decline remain obscure. TGFβ superfamily signaling is an inhibitor of myogenic differentiation, with elevated activity in aged skeletal muscle. Surprisingly, we find reduced expression of Smad4, the downstream cofactor for canonical TGFβ superfamily signaling, and the target Id1 in aged SCs and MPs during regeneration...
November 18, 2016: ELife
Lisa Blaschke, Wenke Wagner, Christina Werkmeister, Marion Wild, Adrian Gihring, Steffen Rupp, Susanne Zibek
Formaldehyde dismutase (FDM) is a very interesting enzyme, due to the fact that it comprises an internal cofactor regeneration mechanism. The FDM, therefore, is able to catalyze redox reactions independent of exogenous cofactor addition, rendering the enzyme powerful for industrial applications. Currently, only one enzyme of this type has been characterized enzymatically. Furthermore, only one additional DNA-sequence with high homology to FDM has been published. In this work, we identified a new variant of a formaldehyde dismutase gene (fdm) in the Pseudomonas putida J3 strain...
January 10, 2017: Journal of Biotechnology
Vijay Tejwani, Franz-Josef Schmitt, Svea Wilkening, Ingo Zebger, Marius Horch, Oliver Lenz, Thomas Friedrich
Ralstonia eutropha is a hydrogen-oxidizing ("Knallgas") bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H2-driven production of biodegradable polymers and hydrocarbons. H2 oxidation by R. eutropha takes place in the presence of O2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H2 utilization...
January 2017: Biochimica et Biophysica Acta
K Cheaib, Y Roux, C Herrero, A Trehoux, F Avenier, J-P Mahy
Dioxygen activation at copper(i) centres is of primary importance for the development of sustainable oxidation catalysis, but regeneration of copper(i) centres after each catalytic cycle remains a major problem for multi-turn-over catalysis. This work demonstrates that an artificial reductase, made of flavin cofactors incorporated into a water soluble polymer, efficiently reduces a Cu(ii)TPA complex in the presence of NADH in water.
November 4, 2016: Dalton Transactions: An International Journal of Inorganic Chemistry
C A Müller, A M Weingartner, A Dennig, A J Ruff, H Gröger, Ulrich Schwaneberg
A novel whole cell cascade for double oxidation of cyclooctane to cyclooctanone was developed. The one-pot oxidation cascade requires only a minimum of reaction components: resting E. coli cells in aqueous buffered medium (=catalyst), the target substrate and oxygen as environmental friendly oxidant. Conversion of cyclooctane was catalysed with high efficiency (50% yield) and excellent selectivity (>94%) to cyclooctanone. The reported oxidation cascade represents a novel whole cell system for double oxidation of non-activated alkanes including an integrated cofactor regeneration...
October 22, 2016: Journal of Industrial Microbiology & Biotechnology
Xiang Chen, Zhi-Qiang Liu, Chao-Ping Lin, Yu-Guo Zheng
BACKGROUND: Ethyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE) is a versatile chiral precursor for many pharmaceuticals. Although several biosynthesis strategies have been documented to convert ethyl 4-chloro-3-oxobutanoate (COBE) to (R)-CHBE, the catalytic efficiency and stereoselectivity are still too low to be scaled up for industrial applications. Due to the increasing demand of (R)-CHBE, it is essential to explore more robust biocatalyst capable of preparing (R)-CHBE efficiently. RESULTS: A stereoselective carbonyl reductase toolbox was constructed and employed into the asymmetric reduction of COBE to (R)-CHBE...
October 18, 2016: BMC Biotechnology
Gangyi Chen, Juan Dong, Yi Yuan, Na Li, Xin Huang, Xin Cui, Zhuo Tang
Nucleic acid amplification is the core technology of molecular biology and genetic engineering. Various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). However, most of these methods can only detect single stranded nucleic acid. Herein, we put forward a simple solution for opening double-stranded DNA for isothermal detection methods. The strategy employs recombination protein from E. coli (RecA) to form nucleoprotein complex with single-stranded DNA, which could scan double-stranded template for homologous sites...
September 30, 2016: Scientific Reports
Xingyu Zhou, Mingsen Li, Huaxing Huang, Keren Chen, Zhuning Yuan, Ying Zhang, Yaping Nie, Hu Chen, Xumeng Zhang, Luxi Chen, Yaosheng Chen, Delin Mo
Although the mechanism underlying modulation of transcription factors in myogenesis has been well elucidated, the function of the transcription cofactors involved in this process remains poorly understood. Here, we identified HMGB2 as an essential nuclear transcriptional co-regulator in myogenesis. HMGB2 was highly expressed in undifferentiated myoblasts and regenerating muscle. Knockdown of HMGB2 inhibited myoblast proliferation and stimulated its differentiation. HMGB2 depletion downregulated Myf5 and cyclin A2 at the protein but not mRNA level...
November 15, 2016: Journal of Cell Science
Ya-Ping Xue, Hao Zeng, Xiao-Lu Jin, Zhi-Qiang Liu, Yu-Guo Zheng
BACKGROUND: Enantiopure 2-hydroxy acids are key intermediates for the synthesis of pharmaceuticals and fine chemicals. We present an enantioselective cascade biocatalysis using recombinant microbial cells for deracemization of racemic 2-hydroxy acids that allows for efficient production of enantiopure 2-hydroxy acids. RESULTS: The method was realized by a single recombinant Escherichia coli strain coexpressing three enzymes: (S)-2-hydroxy acid dehydrogenase, (R)-2-keto acid reductase and glucose dehydrogenase...
2016: Microbial Cell Factories
Ronny Frank, Marcus Klenner, Ronny Azendorf, Manuel Bartz, Heinz-Georg Jahnke, Andrea A Robitzki
Enzymes are the most effective catalysts for a broad range of difficult chemical reactions e.g. hydroxylation of non-activated C-H Bonds and stereoselective synthesis. Nevertheless, a lot of enzymes are not accessible for the biotechnological applications or industrial use. One reason is the prerequisite of expensive cofactors. In this context, we developed a bioelectrocatalytic analysis platform for the electrochemical and photonic quantification of the direct electron transfer from the electrode to redox enzymes and therefore, bypass the need of soluble cofactors that had to be continuously exchanged or regenerated...
August 18, 2016: Biosensors & Bioelectronics
Eunice S da Silva, Vanessa Gómez-Vallejo, Zuriñe Baz, Jordi Llop, Fernando López-Gallego
Nitrogen-13 can be efficiently produced in biomedical cyclotrons in different chemical forms, and its stable isotopes are present in the majority of biologically active molecules. Hence, it may constitute a convenient alternative to Fluorine-18 and Carbon-11 for the preparation of positron-emitter-labelled radiotracers; however, its short half-life demands for the development of simple, fast, and efficient synthetic processes. Herein, we report the one-pot, enzymatic and non-carrier-added synthesis of the (13) N-labelled amino acids l-[(13) N]alanine, [(13) N]glycine, and l-[(13) N]serine by using l-alanine dehydrogenase from Bacillus subtilis, an enzyme that catalyses the reductive amination of α-keto acids by using nicotinamide adenine dinucleotide (NADH) as the redox cofactor and ammonia as the amine source...
September 12, 2016: Chemistry: a European Journal
Fen Gao, Haitao Ding, Xiaohong Xu, Yuhua Zhao
Biodegradation of triphenylmethane dyes by microorganisms is hampered by the transport barrier imposed by cell membranes. On the other hand, cell-free systems using enzyme-based biodegradation strategy are costly. Therefore, an efficient and inexpensive approach circumventing these problems is highly desirable. Here, we constructed a self-sufficient system for synthetic dye removal by coupling of spore surface-displayed triphenylmethane reductase (TMR) and glucose 1-dehydrogenase (GDH) for the first time. Display of both TMR and GDH significantly enhanced their stability under conditions of extreme pH and temperature...
August 8, 2016: Environmental Science and Pollution Research International
Quentin M Dudley, Kim C Anderson, Michael C Jewett
Cell-free metabolic engineering (CFME) is advancing a powerful paradigm for accelerating the design and synthesis of biosynthetic pathways. However, as most cell-free biomolecule synthesis systems to date use purified enzymes, energy and cofactor balance can be limiting. To address this challenge, we report a new CFME framework for building biosynthetic pathways by mixing multiple crude lysates, or extracts. In our modular approach, cell-free lysates, each selectively enriched with an overexpressed enzyme, are generated in parallel and then combinatorically mixed to construct a full biosynthetic pathway...
December 16, 2016: ACS Synthetic Biology
Aihua Liu, Ruirui Feng, Bo Liang
In the present work, NAD(+)-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD(+), and the production of NADH can be detected spectrometrically at 340nm...
September 2016: Enzyme and Microbial Technology
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