The Experts below are selected from a list of 2118 Experts worldwide ranked by ideXlab platform
Yu-guo Zheng - One of the best experts on this subject based on the ideXlab platform.
-
Engineering a Pichia pastoris Nitrilase whole cell catalyst through the increased Nitrilase gene copy number and co-expressing of ER oxidoreductin 1.
Applied microbiology and biotechnology, 2020Co-Authors: Qi Shen, Ya-ping Xue, Zhi-qiang Liu, Neng Xiong, Shu-ping Zou, Yu-guo ZhengAbstract:1-Cyanocyclohexaneacetic acid (1-CHAA) is a critical intermediate for the synthesis of the antiepileptic agent gabapentin. Previously, our group has established a novel manufacturing route for 1-CHAA through bioconversion catalyzed by an Escherichia coli (E. coli) Nitrilase whole cell catalyst. However, the Nitrilase expressed in E. coli has several drawbacks such as a low level of reusability, which hampered its industrial application. Herein, we investigated the potential of using the methylotrophic yeast Pichia pastoris (P. pastoris) for producing the Nitrilase whole cell catalyst. To achieve strains with high catalytic activities, we investigated the effects of the promoter choice, expressing cassette copy number, and co-expression of chaperone on the production of Nitrilase. Our results demonstrated that the strain harboring the multicopy integrations of Nitrilase gene under the control of the alcohol oxidase 1 (AOX1) promoter and co-expressing of ER oxidoreductin 1 (ERO1) exhibited an 18-fold enhancement in the Nitrilase activity compared with the strain containing a single integration of Nitrilase gene under the control of glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter. This optimized P. pastoris strain, compared with the E. coli Nitrilase whole cell catalyst, shows greatly improved levels of reusability and thermostability while has a similar high-substrate tolerance.
-
Highly efficient conversion of 1-cyanocycloalkaneacetonitrile using a "super Nitrilase mutant".
Bioprocess and biosystems engineering, 2018Co-Authors: Neng Xiong, Ya-ping Xue, Zhi-qiang Liu, Shu-ping Zou, Yu-xiao Liu, Yu-guo ZhengAbstract:Nitrilase is the member of carbon-nitrogen hydrogen hydrolase superfamily, which has been widely used for the hydrolysis of nitriles into corresponding carboxylic acids. But most Nitrilases are plagued by product inhibition in the industrial application. In this study, a "super Nitrilase mutant" of Nitrilase with high activity, thermostability and improved product tolerance from Acidovorax facilis ZJB09122 was characterized. Then, an efficient process was developed by employing the whole cell of recombinant E. coli for the conversion of high concentration of 1-cyanocyclohexylacetonitrile-to-1-cyanocyclohexaneacetic acid, an important intermediate of gabapentin. Under the optimized conditions, the higher substrate concentrations such as 1.3 M, 1.5 M and 1.8 M could be hydrolyzed by 13.58 g DCW/L with outstanding productivity (> 740 g/L/day). This study developed a highly efficient bioprocess for the preparation of 1-cyanocyclohexaneacetic acid which has the great potential for industrial application.
-
Engineering the residues on "A" surface and C-terminal region to improve thermostability of Nitrilase.
Enzyme and microbial technology, 2018Co-Authors: Ting Cai, Ya-ping Xue, Neng Xiong, Shu-ping Zou, Yu-guo ZhengAbstract:Nitrilases can hydrolyze nitriles to corresponding carboxylic acids in one single step, which have great potential as valuable biocatalysts for chemical synthesis. However, the poor thermostability of the Nitrilases restrict their applications in industry. In this work, error-prone PCR and site-directed mutagenesis were utilized to improve the thermostability of Nitrilases. Several mutants (AcN-Q339K, AcN-Q343K, AcN-T201F, AcN-T201W, AcN-T201L, AcN-T201I) were obtained with dramatically improved thermostability. The best mutant AcN-T201F/Q339K/Q343K exhibited about 14-fold longer half-life at 45 °C. The result of homology modeling suggested that the site 201, which was located on the "A" surface (the dimer interaction), played an important role in the oligomerization of Nitrilase and the stabilization of substrate binding pocket. The Phe substitution on site 201 was selected in protein engineering of Nitrilase LNIT5, which also demonstrated an improvement of thermostability. In addition, lysine substitution on Q339 and Q343 which brought positive charges to the α helix in the C-terminal region stabilized the surface.
-
High-throughput screening methods for Nitrilases
Applied microbiology and biotechnology, 2016Co-Authors: Ya-ping Xue, Yue-kai Yang, Zhi-qiang Liu, Yu-guo ZhengAbstract:Nitrilases have been widely acknowledged as important alternatives to chemical catalysts, as they have been proved to transform an immense variety of nitriles under mild conditions and often in a stereoselective or regioselective manner. In the discovery of new Nitrilases to establish viable industrial processes, screening plays an important role in identifying which subset of candidates contains a Nitrilase of interest from a collection of organisms, clone banks, or enzyme libraries. However, the traditional methods for evaluating the Nitrilases are a time-consuming, laborious, and costly process and have been regarded as a bottleneck in developing these Nitrilases as industrial biocatalysts. In the past few years, a number of high-throughput screening methods have been developed for rapid evaluation and identification of Nitrilases. Here, we review the various methodologies developed for high-throughput screening of Nitrilases and focus on their advantages and limitations.
-
improvement of alcaligenes faecalis Nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of r mandelic acid
Journal of Agricultural and Food Chemistry, 2014Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xinhong Zhang, Yu-guo ZhengAbstract:Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic acids. To improve the activity, the Nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis Nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 7.5 h of conversion reached 693 mM w...
Ludmila Martinkova - One of the best experts on this subject based on the ideXlab platform.
-
Genetic and Functional Diversity of Nitrilases in Agaricomycotina.
International journal of molecular sciences, 2019Co-Authors: Lenka Rucká, Helena Pelantová, Miroslav Pátek, Martin Chmátal, Natalia Kulik, Lucie Petrásková, Petr Novotný, Romana Příhodová, Ludmila MartinkovaAbstract:Nitrilases participate in the nitrile metabolism in microbes and plants. They are widely used to produce carboxylic acids from nitriles. Nitrilases were described in bacteria, Ascomycota and plants. However, they remain unexplored in Basidiomycota. Yet more than 200 putative Nitrilases are found in this division via GenBank. The majority of them occur in the subdivision Agaricomycotina. In this work, we analyzed their sequences and classified them into phylogenetic clades. Members of clade 1 (61 proteins) and 2 (25 proteins) are similar to plant Nitrilases and Nitrilases from Ascomycota, respectively, with sequence identities of around 50%. The searches also identified five putative cyanide hydratases (CynHs). Representatives of clade 1 and 2 (NitTv1 from Trametes versicolor and NitAg from Armillaria gallica, respectively) and a putative CynH (NitSh from Stereum hirsutum) were overproduced in Escherichia coli. The substrates of NitTv1 were fumaronitrile, 3-phenylpropionitrile, β-cyano-l-alanine and 4-cyanopyridine, and those of NitSh were hydrogen cyanide (HCN), 2-cyanopyridine, fumaronitrile and benzonitrile. NitAg only exhibited activities for HCN and fumaronitrile. The substrate specificities of these Nitrilases were largely in accordance with substrate docking in their homology models. The phylogenetic distribution of each type of Nitrilase was determined for the first time.
-
Biocatalytic production of mandelic acid and analogues: a review and comparison with chemical processes
Applied Microbiology and Biotechnology, 2018Co-Authors: Ludmila MartinkovaAbstract:The aim of this study is to summarize the current progress in the design of biocatalytic processes applicable for the production of optically pure mandelic acids and their analogues. These compounds are used as building blocks for pharmaceutical chemistry and as chiral resolving agents. Their enzymatic syntheses mainly employed nitrile hydrolysis with Nitrilases, ester hydrolysis, ammonolysis or esterification with lipases or esterases, and ketone reduction or alcohol oxidation with dehydrogenases. Each of these methods will be characterized in terms of its product concentrations, enantioselectivities, and the types of catalysts used. This review will focus on the dynamic kinetic resolution of mandelonitrile and analogues by Nitrilases resulting in the production of high concentrations of ( R )-mandelic acid or ( R )-2-chloromandelic acid with excellent e.e. Currently, there is no comparable process for ( S )-mandelic acids. However, the coupling of the S -selective cyanation of benzaldehyde with the enantioretentive hydrolysis of ( S )-mandelonitrile thus obtained is a promising strategy. The major product can be changed from ( S )-acid to ( S )-amide using Nitrilase mutants. The competitiveness of the biocatalytic and chemical processes will be assessed. This review covers the literature published within 2003–2017.
-
Recent advances and challenges in the heterologous production of microbial Nitrilases for biocatalytic applications
World Journal of Microbiology and Biotechnology, 2016Co-Authors: Ludmila Martinkova, Lenka Rucká, Jan Nešvera, Miroslav PátekAbstract: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. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of Nitrilases. By exploiting this resource, the number of experimentally verified Nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type Nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of Nitrilases are classified according to their target products (α-hydroxy acids, α- and β-amino acids, cyano acids, amides). The emerging uses of Nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of Nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications. Graphical Abstract
-
Bringing Nitrilase sequences from databases to life: the search for novel substrate specificities with a focus on dinitriles.
Applied Microbiology and Biotechnology, 2015Co-Authors: Alicja B. Veselá, Lenka Rucká, Ondřej Kaplan, Helena Pelantová, Jan Nešvera, Miroslav Pátek, Ludmila MartinkovaAbstract:The aim of this study was to discover new Nitrilases with useful activities, especially towards dinitriles that are precursors of high-value cyano acids. Genes coding for putative Nitrilases of different origins (fungal, plant, or bacterial) with moderate similarities to known Nitrilases were selected by mining the GenBank database, synthesized artificially and expressed in Escherichia coli. The enzymes were purified, examined for their substrate specificities, and classified into subtypes (aromatic Nitrilase, arylacetoNitrilase, aliphatic Nitrilase, cyanide hydratase) which were largely in accordance with those predicted from bioinformatic analysis. The catalytic potential of the Nitrilases for dinitriles was examined with cyanophenyl acetonitriles, phenylenediacetonitriles, and fumaronitrile. The Nitrilase activities and selectivities for dinitriles and the reaction products (cyano acid, cyano amide, diacid) depended on the enzyme subtype. At a preparative scale, all the examined dinitriles were hydrolyzed into cyano acids and fumaronitrile was converted to cyano amide using E. coli cells producing arylacetoNitrilases and an aromatic Nitrilase, respectively.
-
A Comparative Study of Nitrilases Identified by Genome Mining
Molecular Biotechnology, 2013Co-Authors: Ondřej Kaplan, Alena Petřickova, Martina Pičmanová, Anna Rinágelová, Alicja B. Veselá, Miroslav Pátek, Fabrizia Pasquarelli, Tek Chand Bhalla, Ludmila MartinkovaAbstract:Escherichia coli strains expressing different Nitrilases transformed nitriles or KCN. Six Nitrilases (from Aspergillus niger (2), A. oryzae, Neurospora crassa, Arthroderma benhamiae, and Nectria haematococca) were arylacetoNitrilases, two enzymes (from A. niger and Penicillium chrysogenum) were cyanide hydratases and the others (from P. chrysogenum, P. marneffei, Gibberella moniliformis, Meyerozyma guilliermondi, Rhodococcus rhodochrous, and R. ruber) preferred (hetero)aromatic nitriles as substrates. Promising Nitrilases for the transformation of industrially important substrates were found: the Nitrilase from R. ruber for 3-cyanopyridine, 4-cyanopyridine and bromoxynil, the Nitrilases from N. crassa and A. niger for (R,S)-mandelonitrile, and the cyanide hydratase from A. niger for KCN and 2-cyanopyridine.
Markus Piotrowski - One of the best experts on this subject based on the ideXlab platform.
-
Substrate specificity of plant Nitrilase complexes is affected by their helical twist
Communications biology, 2018Co-Authors: Jeremy D. Woodward, B. Trevor Sewell, Inga Trompetter, Markus PiotrowskiAbstract:Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important enzymes are often specific for certain substrates, but directed attempts at modifying their substrate specificities by exchanging binding pocket residues have been largely unsuccessful. Thus, the basis for their selectivity is still unknown. Here we show, based on work with two highly similar Nitrilases from the plant Capsella rubella, that modifying Nitrilase helical twist, either by exchanging an interface residue or by imposing a different twist, without altering any binding pocket residues, changes substrate preference. We reveal that helical twist and substrate size correlate and when binding pocket residues are exchanged between two Nitrilases that show the same twist but different specificities, their specificities change. Based on these findings we propose that helical twist influences the overall size of the binding pocket.
-
Evolution of Nitrilases in glucosinolate-containing plants.
Phytochemistry, 2009Co-Authors: Tim Janowitz, Inga Trompetter, Markus PiotrowskiAbstract:Nitrilases, enzymes that catalyze the hydrolysis of organic cyanides, are ubiquitous in the plant kingdom. The typical plant Nitrilase is a Nitrilase 4 homolog which is involved in the cyanide detoxification pathway. In this pathway, Nitrilase 4 converts beta-cyanoalanine, the intermediate product of cyanide detoxification, into asparagine, aspartic acid and ammonia. In the Brassicaceae, a new family of Nitrilases has evolved, the Nitrilase 1 homologs. These enzymes are not able to use beta-cyanoalanine as a substrate. Instead, they display rather broad substrate specificities and are able to hydrolyze nitriles that result from the decomposition of glucosinolates, the typical secondary metabolites of the Brassicaceae. Here we summarize and discuss data indicating that Nitrilase 1 homologs have evolved to function in glucosinolate catabolism.
-
Primary or secondary? Versatile Nitrilases in plant metabolism.
Phytochemistry, 2008Co-Authors: Markus PiotrowskiAbstract:Abstract The potential of plant Nitrilases to convert indole-3-acetonitrile into the plant growth hormone indole-3-acetic acid has earned them the interim title of “key enzyme in auxin biosynthesis”. Although not widely recognized, this view has changed considerably in the last few years. Recent work on plant Nitrilases has shown them to be involved in the process of cyanide detoxification, in the catabolism of cyanogenic glycosides and presumably in the catabolism of glucosinolates. All plants possess at least one Nitrilase that is homologous to the Nitrilase 4 isoform of Arabidopsis thaliana . The general function of these Nitrilases lies in the process of cyanide detoxification, in which they convert the intermediate detoxification product β-cyanoalanine into asparagine, aspartic acid and ammonia. Cyanide is a metabolic by-product in biosynthesis of the plant hormone ethylene, but it may also be released from cyanogenic glycosides, which are present in a large number of plants. In Sorghum bicolor , an additional Nitrilase isoform has been identified, which can directly use a catabolic intermediate of the cyanogenic glycoside dhurrin, thus enabling the plant to metabolize its cyanogenic glycoside without releasing cyanide. In the Brassicaceae, a family of Nitrilases has evolved, the members of which are able to hydrolyze catabolic products of glucosinolates, the predominant secondary metabolites of these plants. Thus, the general theme of Nitrilase function in plants is detoxification and nitrogen recycling, since the valuable nitrogen of the nitrile group is recovered in the useful metabolites asparagine or ammonia. Taken together, a picture emerges in which plant Nitrilases have versatile functions in plant metabolism, whereas their importance for auxin biosynthesis seems to be minor.
-
Enantioselective Nitrilase from Pseudomonas putida: Cloning, Heterologous Expression, and Bioreactor Studies
Molecular biotechnology, 2008Co-Authors: Anirban Banerjee, Markus Piotrowski, Praveen Kaul, Sachin Dubey, Brajesh Barse, Uttam Chand BanerjeeAbstract:Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective Nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The Nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of Nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(-)-mandelic acid.
-
the Nitrilase zmnit2 converts indole 3 acetonitrile to indole 3 acetic acid
Plant Physiology, 2003Co-Authors: Woong June Park, Markus Piotrowski, Verena Kriechbaumer, Axel Müller, Robert B. Meeley, Alfons Gierl, Erich GlawischnigAbstract:We isolated two Nitrilase genes, ZmNIT1 and ZmNIT2, from maize (Zea mays) that share 75% sequence identity on the amino acid level. Despite the relatively high homology to Arabidopsis NIT4, ZmNIT2 shows no activity toward β-cyano-alanine, the substrate of Arabidopsis NIT4, but instead hydrolyzes indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). ZmNIT2 converts IAN to IAA at least seven to 20 times more efficiently than AtNIT1/2/3. Quantitative real-time polymerase chain reaction revealed the gene expression of both Nitrilases in maize kernels where high concentrations of IAA are synthesized tryptophan dependently. Nitrilase protein and endogenous Nitrilase activity are present in maize kernels together with the substrate IAN. These results suggest a role for ZmNIT2 in auxin biosynthesis.
Ya-ping Xue - One of the best experts on this subject based on the ideXlab platform.
-
Engineering a Pichia pastoris Nitrilase whole cell catalyst through the increased Nitrilase gene copy number and co-expressing of ER oxidoreductin 1.
Applied microbiology and biotechnology, 2020Co-Authors: Qi Shen, Ya-ping Xue, Zhi-qiang Liu, Neng Xiong, Shu-ping Zou, Yu-guo ZhengAbstract:1-Cyanocyclohexaneacetic acid (1-CHAA) is a critical intermediate for the synthesis of the antiepileptic agent gabapentin. Previously, our group has established a novel manufacturing route for 1-CHAA through bioconversion catalyzed by an Escherichia coli (E. coli) Nitrilase whole cell catalyst. However, the Nitrilase expressed in E. coli has several drawbacks such as a low level of reusability, which hampered its industrial application. Herein, we investigated the potential of using the methylotrophic yeast Pichia pastoris (P. pastoris) for producing the Nitrilase whole cell catalyst. To achieve strains with high catalytic activities, we investigated the effects of the promoter choice, expressing cassette copy number, and co-expression of chaperone on the production of Nitrilase. Our results demonstrated that the strain harboring the multicopy integrations of Nitrilase gene under the control of the alcohol oxidase 1 (AOX1) promoter and co-expressing of ER oxidoreductin 1 (ERO1) exhibited an 18-fold enhancement in the Nitrilase activity compared with the strain containing a single integration of Nitrilase gene under the control of glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter. This optimized P. pastoris strain, compared with the E. coli Nitrilase whole cell catalyst, shows greatly improved levels of reusability and thermostability while has a similar high-substrate tolerance.
-
Highly efficient conversion of 1-cyanocycloalkaneacetonitrile using a "super Nitrilase mutant".
Bioprocess and biosystems engineering, 2018Co-Authors: Neng Xiong, Ya-ping Xue, Zhi-qiang Liu, Shu-ping Zou, Yu-xiao Liu, Yu-guo ZhengAbstract:Nitrilase is the member of carbon-nitrogen hydrogen hydrolase superfamily, which has been widely used for the hydrolysis of nitriles into corresponding carboxylic acids. But most Nitrilases are plagued by product inhibition in the industrial application. In this study, a "super Nitrilase mutant" of Nitrilase with high activity, thermostability and improved product tolerance from Acidovorax facilis ZJB09122 was characterized. Then, an efficient process was developed by employing the whole cell of recombinant E. coli for the conversion of high concentration of 1-cyanocyclohexylacetonitrile-to-1-cyanocyclohexaneacetic acid, an important intermediate of gabapentin. Under the optimized conditions, the higher substrate concentrations such as 1.3 M, 1.5 M and 1.8 M could be hydrolyzed by 13.58 g DCW/L with outstanding productivity (> 740 g/L/day). This study developed a highly efficient bioprocess for the preparation of 1-cyanocyclohexaneacetic acid which has the great potential for industrial application.
-
Engineering the residues on "A" surface and C-terminal region to improve thermostability of Nitrilase.
Enzyme and microbial technology, 2018Co-Authors: Ting Cai, Ya-ping Xue, Neng Xiong, Shu-ping Zou, Yu-guo ZhengAbstract:Nitrilases can hydrolyze nitriles to corresponding carboxylic acids in one single step, which have great potential as valuable biocatalysts for chemical synthesis. However, the poor thermostability of the Nitrilases restrict their applications in industry. In this work, error-prone PCR and site-directed mutagenesis were utilized to improve the thermostability of Nitrilases. Several mutants (AcN-Q339K, AcN-Q343K, AcN-T201F, AcN-T201W, AcN-T201L, AcN-T201I) were obtained with dramatically improved thermostability. The best mutant AcN-T201F/Q339K/Q343K exhibited about 14-fold longer half-life at 45 °C. The result of homology modeling suggested that the site 201, which was located on the "A" surface (the dimer interaction), played an important role in the oligomerization of Nitrilase and the stabilization of substrate binding pocket. The Phe substitution on site 201 was selected in protein engineering of Nitrilase LNIT5, which also demonstrated an improvement of thermostability. In addition, lysine substitution on Q339 and Q343 which brought positive charges to the α helix in the C-terminal region stabilized the surface.
-
High-throughput screening methods for Nitrilases
Applied microbiology and biotechnology, 2016Co-Authors: Ya-ping Xue, Yue-kai Yang, Zhi-qiang Liu, Yu-guo ZhengAbstract:Nitrilases have been widely acknowledged as important alternatives to chemical catalysts, as they have been proved to transform an immense variety of nitriles under mild conditions and often in a stereoselective or regioselective manner. In the discovery of new Nitrilases to establish viable industrial processes, screening plays an important role in identifying which subset of candidates contains a Nitrilase of interest from a collection of organisms, clone banks, or enzyme libraries. However, the traditional methods for evaluating the Nitrilases are a time-consuming, laborious, and costly process and have been regarded as a bottleneck in developing these Nitrilases as industrial biocatalysts. In the past few years, a number of high-throughput screening methods have been developed for rapid evaluation and identification of Nitrilases. Here, we review the various methodologies developed for high-throughput screening of Nitrilases and focus on their advantages and limitations.
-
improvement of alcaligenes faecalis Nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of r mandelic acid
Journal of Agricultural and Food Chemistry, 2014Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xinhong Zhang, Yu-guo ZhengAbstract:Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic acids. To improve the activity, the Nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis Nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 7.5 h of conversion reached 693 mM w...
B. Trevor Sewell - One of the best experts on this subject based on the ideXlab platform.
-
Substrate specificity of plant Nitrilase complexes is affected by their helical twist
Communications biology, 2018Co-Authors: Jeremy D. Woodward, B. Trevor Sewell, Inga Trompetter, Markus PiotrowskiAbstract:Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important enzymes are often specific for certain substrates, but directed attempts at modifying their substrate specificities by exchanging binding pocket residues have been largely unsuccessful. Thus, the basis for their selectivity is still unknown. Here we show, based on work with two highly similar Nitrilases from the plant Capsella rubella, that modifying Nitrilase helical twist, either by exchanging an interface residue or by imposing a different twist, without altering any binding pocket residues, changes substrate preference. We reveal that helical twist and substrate size correlate and when binding pocket residues are exchanged between two Nitrilases that show the same twist but different specificities, their specificities change. Based on these findings we propose that helical twist influences the overall size of the binding pocket.
-
Probing an Interfacial Surface in the Cyanide Dihydratase from Bacillus pumilus, A Spiral Forming Nitrilase.
Frontiers in microbiology, 2016Co-Authors: Jason M. Park, Andani E. Mulelu, B. Trevor Sewell, Michael J. BenedikAbstract:Nitrilases are of significant interest both due to their potential for industrial production of valuable products as well as degradation of hazardous nitrile-containing wastes. All known functional members of the Nitrilase superfamily have an underlying dimer structure. The true Nitrilases expand upon this basic dimer and form large spiral or helical homo-oligomers. The formation of this larger structure is linked to both the activity and substrate specificity of these Nitrilases. The sequences of the spiral Nitrilases differ from the non-spiral forming homologs by the presence of two insertion regions. Homology modeling suggests that these regions are responsible for associating the Nitrilase dimers into the oligomer. Here we used cysteine scanning across these two regions, in the spiral forming Nitrilase cyanide dihydratase from Bacillus pumilus (CynD), to identify residues altering the oligomeric state or activity of the Nitrilase. Several mutations were found to cause changes to the size of the oligomer as well as reduction in activity. Additionally one mutation, R67C, caused a partial defect in oligomerization with the accumulation of smaller oligomer variants. These results support the hypothesis that these insertion regions contribute to the unique quaternary structure of the spiral microbial Nitrilases.
-
Structural and biochemical characterization of a Nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8
Applied microbiology and biotechnology, 2010Co-Authors: Dael S. Williamson, Kyle C. Dent, Brandon Weber, Arvind Varsani, Joni Frederick, Robert N. Thuku, Rory A. Cameron, Johan H. Van Heerden, Don A. Cowan, B. Trevor SewellAbstract:Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible Nitrilase was located and cloned using degenerate primers coding for well-conserved Nitrilase sequences, coupled with inverse PCR. The Nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of approximately 600 kDa by size exclusion chromatography. Similar to several plant Nitrilases and some bacterial Nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the Nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, "c-shaped", circular and "figure-8" shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.