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Sam Mathew, Saravanan Prabhu Nadarajan, Uthayasuriya Sundaramoorthy, Hyunwoo Jeon, Taeowan Chung, Hyungdon Yun
OBJECTIVE: To enzymatically synthesize enantiomerically pure β-amino acids from β-keto nitriles using nitrilase and ω-transaminase. RESULTS: An enzyme cascade system was designed where in β-keto nitriles are initially hydrolyzed to β-keto acids using nitrilase from Bradyrhizobium japonicum and subsequently β-keto acids were converted to β-amino acids using ω-transaminases. Five different ω-transaminases were tested for this cascade reaction, To enhance the yields of β-amino acids, the concentrations of nitrilase and amino donor were optimized...
December 21, 2016: Biotechnology Letters
Lasse Janniche Nielsen, Peter Stuart, Martina Pičmanová, Simon Rasmussen, Carl Erik Olsen, Jesper Harholt, Birger Lindberg Møller, Nanna Bjarnholt
BACKGROUND: The important cereal crop Sorghum bicolor (L.) Moench biosynthesize and accumulate the defensive compound dhurrin during development. Previous work has suggested multiple roles for the compound including a function as nitrogen storage/buffer. Crucial for this function is the endogenous turnover of dhurrin for which putative pathways have been suggested but not confirmed. RESULTS: In this study, the biosynthesis and endogenous turnover of dhurrin in the developing sorghum grain was studied by metabolite profiling and time-resolved transcriptome analyses...
December 13, 2016: BMC Genomics
Ludmila Martínková, Lenka Rucká, Jan Nešvera, Miroslav Pátek
The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches...
January 2017: World Journal of Microbiology & Biotechnology
Zefan Li, Yuntao Zhu, Yuting Sun, Ke Qin, Weibing Liu, Wen Zhou, Xing Chen
Cell-selective protein metabolic labeling is of great interest for studying cell-cell communications and tissue homeostasis. We herein describe a nitrilase-activatable noncanonical amino acid tagging (NANCAT) strategy that exploits an exogenous nitrilase to enzymatically convert the nitrile-substituted precursors to their corresponding noncanonical amino acids (ncAAs), l-azidohomoalanine (Aha) or homopropargylglycine (Hpg), in living cells. Only cells expressing the nitrilase can generate Aha or Hpg in cellulo and metabolically incorporate them into the nascent proteins...
December 16, 2016: ACS Chemical Biology
Jizhe Sun, Huimin Yu, Jie Chen, Hui Luo, Zhongyao Shen
Rhodococcus ruber TH was selected as a parent strain to engineer for biomanufacturing of ammonium acrylate; the characteristics of this strain included accelerated growth rate, high cell tolerance and natively overexpressed nitrile hydratase (NHase). Transcriptome analysis revealed that the transcription levels of the native NHase, amidase and nitrilase were extremely high, moderate and extremely low, respectively. Through NHase-amidase double-knockout and amidase single-knockout, the engineered strains R. ruber THdAdN and R...
October 19, 2016: Journal of Industrial Microbiology & Biotechnology
Shuiqin Jiang, Lujia Zhang, Dongbin Cui, Zhiqiang Yao, Bei Gao, Jinping Lin, Dongzhi Wei
The use of halogen bond is widespread in drug discovery, design, and clinical trials, but is overlooked in drug biosynthesis. Here, the role of halogen bond in the nitrilase-catalyzed synthesis of ortho-, meta-, and para-chlorophenylacetic acid was investigated. Different distributions of halogen bond induced changes of substrate binding conformation and affected substrate selectivity. By engineering the halogen interaction, the substrate selectivity of the enzyme changed, with the implication that halogen bond plays an important role in biosynthesis and should be used as an efficient and reliable tool in enzymatic drug synthesis...
October 6, 2016: Scientific Reports
Jason M Park, Christian M Ponder, B Trevor Sewell, Michael J Benedik
Nitrilases pose attractive alternatives to the chemical hydrolysis of nitrile compounds. The activity of bacterial nitrilases towards substrate is intimately tied to the formation of large spiral-shaped oligomers. In the nitrilase CynD (cyanide dihydratase) from Bacillus pumilus, mutations in a predicted oligomeric surface region altered its oligomerization and reduced its activity. One mutant, CynD Y70C, retained uniform oligomer formation however it was inactive, unlike all other inactive mutants throughout that region all of which significantly perturbed oligomer formation...
December 28, 2016: Journal of Microbiology and Biotechnology
Mary A Crum, B Trevor Sewell, Michael J Benedik
Cyanide degrading nitrilases are noted for their potential to detoxify industrial wastewater contaminated with cyanide. However, such application would benefit from an improvement to characteristics such as their catalytic activity and stability. Following error-prone PCR for random mutagenesis, several cyanide dihydratase mutants from Bacillus pumilus were isolated based on improved catalysis. Four point mutations, K93R, D172N, A202T, and E327K were characterized and their effects on kinetics, thermostability and pH tolerance were studied...
2016: Frontiers in Microbiology
Zhiqiang Yao, Lujia Zhang, Bei Gao, Dongbing Cui, Fengqing Wang, Xiao He, John Zenghui Zhang, Dong-Zhi Wei
The discovery of unique substrate is important for developing potential applications of enzymes. However, the experimental procedures for substrate identification are laborious, time-consuming and expensive. Although in silico structure-based approaches show great promise, recent extensive studies show that these approaches remain a formidable challenge for current bio-computational methodologies. Here, we present an open-source, extensible and flexible software platform for predicting enzyme substrates called THEMIS, which performs in silico virtual screening for potential catalytic targets of enzyme based on enzymes catalysis mechanism...
August 16, 2016: Journal of Chemical Information and Modeling
Chunyan Li, Zhenlei Yue, Fengzhao Feng, Chuanwu Xi, Hailian Zang, Xuejiao An, Keran Liu
There is a great need for efficient acetonitrile removal technology in wastewater treatment to reduce the discharge of this pollutant in untreated wastewater. In this study, a nitrilase gene (nit) isolated from a nitrile-degrading bacterium (Rhodococcus rhodochrous BX2) was cloned and transformed into a biofilm-forming bacterium (Bacillus subtilis N4) that expressed the recombinant protein upon isopropylthio-β-galactoside (IPTG) induction. The recombinant bacterium (B. subtilis N4-pHT01-nit) formed strong biofilms and had nitrile-degrading capability...
October 2016: Chemosphere
Lili Fu, Meng Wang, Bingying Han, Deguan Tan, Xuepiao Sun, Jiaming Zhang
Plant myrosinases (β-thioglucoside glucohydrolases) are classified into two subclasses, Myr I and Myr II. The biological function of Myr I has been characterized as a major biochemical defense against insect pests and pathogens in cruciferous plants. However, the biological function of Myr II remains obscure. We studied the function of two Myr II member genes AtTGG4 and AtTGG5 in Arabidopsis. RT-PCR showed that both genes were specifically expressed in roots. GUS-assay revealed that both genes were expressed in the root-tip but with difference: AtTGG4 was expressed in the elongation zone of the root-tip, while AtTGG5 was expressed in the whole root-tip...
June 7, 2016: International Journal of Molecular Sciences
Takuto Kumano, Yuko Takizawa, Sakayu Shimizu, Michihiko Kobayashi
One of the nitrile-synthesizing enzymes, β-cyano-L-alanine synthase, catalyzes β-cyano-L-alanine (β-CNAla) from potassium cyanide and O-acetyl-L-serine or L-cysteine. We have identified this enzyme from Pseudomonas ovalis No. 111. In this study, we cloned the β-CNAla synthase gene and expressed it in Escherichia coli and Rhodococcus rhodochrous. Furthermore, we carried out co-expression of β-CNAla synthase with nitrilase or nitrile hydratases in order to synthesize aspartic acid and asparagine from KCN and O-acetyl-L-serine...
September 12, 2016: Journal of General and Applied Microbiology
Du Seok Choi, Chae Woo Lim, Byung Kook Hwang
Proteomics and functional analyses of the Arabidopsis - Pseudomonas syringae pv. tomato interactions reveal that Arabidopsis nitrilases are required for plant defense and R gene-mediated resistant responses to microbial pathogens. A high-throughput in planta proteome screen has identified Arabidopsis nitrilase 2 (AtNIT2), which was de novo-induced by Pseudomonas syringae pv. tomato (Pst) infection. The AtNIT2, AtNIT3, and AtNIT4 genes, but not AtNIT1, were distinctly induced in Arabidopsis leaves by Pst infection...
August 2016: Planta
Bartosz Sekula, Milosz Ruszkowski, Maura Malinska, Zbigniew Dauter
Putrescine, 1,4-diaminobutane, is an intermediate in the biosynthesis of more complexed polyamines, spermidine and spermine. Unlike other eukaryotes, plants have evolved a multistep pathway for putrescine biosynthesis that utilizes arginine. In the final reaction, N-carbamoylputrescine is hydrolyzed to putrescine by N-carbamoylputrescine amidohydrolase (CPA, EC During the hydrolysis, consecutive nucleophilic attacks on the substrate by Cys158 and water lead to formation of putrescine and two by-products, ammonia and carbon dioxide...
2016: Frontiers in Plant Science
Daniel Wibberg, Andreas Bremges, Tanja Dammann-Kalinowski, Irena Maus, M Isabel Igeño, Ralph Vogelsang, Christoph König, Víctor M Luque-Almagro, M Dolores Roldán, Alexander Sczyrba, Conrado Moreno-Vivián, Rafael Blasco, Alfred Pühler, Andreas Schlüter
Pseudomonas pseudoalcaligenes CECT5344 tolerates cyanide and is also able to utilize cyanide and cyano-derivatives as a nitrogen source under alkaline conditions. The strain is considered as candidate for bioremediation of habitats contaminated with cyanide-containing liquid wastes. Information on the genome sequence of the strain CECT5344 became available previously. The P. pseudoalcaligenes CECT5344 genome was now resequenced by applying the single molecule, real-time (SMRT(®)) sequencing technique developed by Pacific Biosciences...
August 20, 2016: Journal of Biotechnology
Zhe-Ming Wu, Ren-Chao Zheng, Yu-Guo Zheng
Amidases can be assigned into two families according to their amino acid sequences. Three amidases (Dt-Amis) were mined and identified from genome of Delftia tsuruhatensis. Homology analysis demonstrated that Dt-Ami 2 and Dt-Ami 6 belonged to amidase signature (AS) family, while Dt-Ami 7 belonged to nitrilase superfamily. AS amidases were shown to hydrolyze a wide spectrum of amides. Kinetic analysis demonstrated that the extension of chain length of aliphatic amides considerably decreased the Km values, and the turnover numbers (kcat) were high with linear aliphatic amides as substrates...
May 2016: Enzyme and Microbial Technology
Ramon Terrado, Adam Monier, Robyn Edgar, Connie Lovejoy
In an effort to better understand the diversity of genes coding for nitrogen (N) uptake and assimilation pathways among microalgae, we analyzed the transcriptomes of five phylogenetically diverse single celled algae originally isolated from the same high arctic marine region. The five photosynthetic flagellates (a pelagophyte, dictyochophyte, chrysoph-yte, cryptophyte and haptophyte) were grown on standard media and media with only urea or nitrate as a nitrogen source; cells were harvested during late exponential growth...
June 2015: Journal of Phycology
Geraldine V Dennett, Jenny M Blamey
Several environmental samples from Antarctica were collected and enriched to search for microorganisms with nitrilase activity. A new thermostable nitrilase from a novel hyperthermophilic archaea Pyrococcus sp. M24D13 was purified and characterized. The activity of this enzyme increased as the temperatures rise from 70 up to 85°C. Its optimal activity occurred at 85°C and pH 7.5. This new enzyme shows a remarkable resistance to thermal inactivation retaining more than 50% of its activity even after 8 h of incubation at 85°C...
2016: Frontiers in Bioengineering and Biotechnology
Yong Antican Wang, Yunguang Sun, Justin M Le Blanc, Charalambos Solomides, Tingting Zhan, Bo Lu
Uncovering novel growth modulators for non-small cell lung cancer (NSCLC) may lead to new therapies for these patients. Previous studies suggest Nit1 suppresses chemically induced carcinogenesis of the foregut in a mouse model. In this study we aimed to determine the role of Nit1 in a transgenic mouse lung cancer model driven by a G12D Kras mutation. Nit1 knockout mice (Nit1-/-) were crossed with KrasG12D/+ mice to investigate whether a G12D Kras mutation and Nit1 inactivation interact to promote or inhibit the development of NSCLC...
April 19, 2016: Oncotarget
Masako Matsumoto, Yoshiteru Hashimoto, Yuki Saitoh, Takuto Kumano, Michihiko Kobayashi
An inducible expression vector, pSH19, which harbors regulatory expression system PnitA-NitR, for streptomycetes was constructed previously. Here, we have modified pSH19 to obtain shuttle vectors for Streptomyces-E. coli by introducing the replication origin of a plasmid for E. coli (ColE1) and an antibiotic-resistant gene. Six inducible shuttle vectors, pESH19cF, pESH19cR, pESH19kF, pESH19kR, pESH19aF, and pESH19aR, for Streptomyces-E. coli, were successfully developed. The stability of these vectors was examined in five different E...
June 2016: Bioscience, Biotechnology, and Biochemistry
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