Nitrilase Enzyme Research Articles

Cloning and heterologous expression of Fusarium oxysporum nitrilase gene in Escherichia coli and evaluation in cyanide degradation

Cyanide is widely utilized in the extraction of precious metal extraction even though it has been deemed as the most toxic compound. Fusarium oxysporum has been shown to degrade cyanide through the activity of the Nitrilase enzyme. In this study, the coding sequence of nitrilase gene from F. oxysporum genomic DNA was optimized for cloning and expression in E. coli. The pUC57 containing synthetic optimized nitrilase gene was transferred into E. coli DH5α strain. This nitrilase gene was sub-cloned into pET26b (+) expression vector containing an in-built His-tag at the C-terminal end to facilitate its purification. The recombinant plasmid, pETAM1, was confirmed by PCR, digestion pattern, and sequencing. The recombinant protein was overproduced in E. coli BL21 (DE3). The results of the SDS-PAGE pattern and Western blot analysis confirmed the expression of the expected recombinant protein. For expression optimization of Nitrilase protein, M16 orthogonal experimental design of the Taguchi method was used. The effect of induction time, temperature and IPTG concentration were examined using four levels for each factors. Estimation of the amount of the expressed protein was calculated via densitometry on SDS-PAGE. The enzyme activity and expression in E. coli proved to be successful since there was ammonia production when potassium cyanide and acrylonitrile were used as substrates while the highest enzyme activity of 88% was expressed at 30 °C. The Km and Vm values of the expressed Nitrilase enzyme were determined to be 0.68 mM and 0.48 mM/min respectively.

NIT24 and NIT29-mediated IAA synthesis of Xanthomonas oryzae pv. oryzicola suppresses immunity and boosts growth in rice.

Auxin plays a pivotal role in the co-evolution of plants and microorganisms. Xanthomonas oryzae pv. oryzicola (Xoc) stands as a significant factor that affects rice yield and quality. However, the current understanding of Xoc's capability for indole 3-acetic acid (IAA) synthesis and its mechanistic implications remains elusive. In this study, we performed a comprehensive genomic analysis of Xoc strain RS105, leading to the identification of two nitrilase enzyme family (NIT) genes, designated as AKO15524.1 and AKO15829.1, subsequently named NIT24 and NIT29, respectively. Our investigation unveiled that the deletion of NIT24 and NIT29 resulted in a notable reduction in IAA synthesis capacity within RS105, thereby impacting extracellular polysaccharide production. This deficiency was partially ameliorated through exogenous IAA supplementation. The study further substantiated that NIT24 and NIT29 have nitrilase activity and the ability to catalyse IAA production invitro. The lesion length and bacterial population statistics experiments confirmed that NIT24 and NIT29 positively regulated the pathogenicity of RS105, suggesting that NIT24 and NIT29 may regulate Xoc invasion by affecting IAA synthesis. Furthermore, our analysis corroborated mutant strains, RS105_ΔNIT24 and RS105_ΔNIT29, which elicited the outbreak of reactive oxygen species, the deposition of callose and the upregulation of defence-related gene expression in rice. IAA exerted a significant dampening effect on the immune responses incited by these mutant strains in rice. In addition, the absence of NIT24 and NIT29 affected the growth-promoting effect of Xoc on rice. This implies that Xoc may promote rice growth by secreting IAA, thus providing a more suitable microenvironment for its own colonization. In summary, our study provides compelling evidence for the existence of a nitrilase-dependent IAA biosynthesis pathway in Xoc. IAA synthesis-related genes promote Xoc colonization by inhibiting rice immune defence response and affecting rice growth by increasing IAA content in Xoc.

Enantioselective Synthesis of α-Thiocarboxylic Acids by Nitrilase Biocatalysed Dynamic Kinetic Resolution of α-Thionitriles.

The enantioselective synthesis of α‐thiocarboxylic acids by biocatalytic dynamic kinetic resolution (DKR) of nitrile precursors exploiting nitrilase enzymes is described. A panel of 35 nitrilase biocatalysts were screened and enzymes Nit27 and Nit34 were found to catalyse the DKR of racemic α‐thionitriles under mild conditions, affording the corresponding carboxylic acids with high conversions and good‐to‐excellent ee. The ammonia produced in situ during the biocatalytic transformation favours the racemization of the nitrile enantiomers and, in turn, the DKR without the need of any external additive base.

Computational modeling for mutational analysis of nitrilase enzyme towards enhancement of binding empathy

Nitrilase enzyme (a green catalyst) is an industrially important enzyme which hydrolyses various nitrile compounds (containing -CN functional group) into amides and corresponding carboxylic acids. The current study explored the binding affinity and a method to enhance the catalysis activity of the enzyme using computational approaches. Four mutants were generated using sequential site-directed mutagenesis aiming that an increase in hydrogen bonds that will further increase binding efficiency towards the ligand. Molecular dynamics simulation was rigorously performed to check the stability of those mutants followed by docking to verify its interaction with the ligand. Various statistical dynamics analyses were performed to validate the structure. All the studies predict that built mutants are stable. Mutants 2 and 3 showed a better affinity towards acrylamide by forming the highest number of hydrogen bonds implying better catalysis. The binding affinity values of the Mutant 2 and Mutant 3 with acrylamide are −7.44 kcal/mol and −7.17 kcal/mol, respectively. This study may prove useful for the industry to develop efficient nitrilase enzymes with improved catalytic activity. Communicated by Ramaswamy H. Sarma

The helical arrangement of nitrilase enzymes determines their substrate size

Nitrilases are commercially important biocatalysts found in diverse taxa that catalyse the hydrolysis of nitriles to amides and acids. In vitro they form rings, terminating spirals or long filaments that seem to be necessary for activity. In vivo, NIT1 assemblies have been observed in Arabidopsis. Substrate size is correlated with the helical twist of nitrilase filaments and can be altered by changing a single residue by site‐directed mutagenesis or by mixing with an excess of enzyme in a different helical conformation. We have determined the first close‐to‐atomic resolution structure of an active nitrilase by cryo‐EM and identified substrate‐specifying residues for site saturation directed evolution. In silico docking of the substrate, β‐L‐cyanoalanine, into the binding pocket led to the identification of a loop that limits the maximum length of a bound substrate and shifts position as a function of helical twist. We propose that the overall size of the binding pocket is specified by this loop, while binding pocket residues alter selectivity for different functional groups. We propose that this mechanism allows nitrilases to rapidly neofunctionalise according to cellular requirements.Support or Funding InformationWe acknowledge Diamond for access and support of the Cryo‐EM facilities at the UK national electron bio‐imaging centre (eBIC), (proposal EM20975‐1), funded by the Wellcome Trust, MRC, and BBSRC. Y. Qayiya, N. Arendse, M. Jaffer, B. Weber, and S. Neumann for technical assistance. This work was funded by the South Africa/Germany Cooperation in Science and Technology Joint Research Grant, the Deutsche Forschungsgemeinschaft (PI 424/1, PI 424/3), Research Career Advancement Fellowship from the National Research Foundation of South Africa (92556) and GCRF‐START (UK Science and Technology Facilities Council grant ref. ST/R002754/1).

Efficacy of spectroscopic optical coherence tomography (sOCT) for instantaneous study of nitrilase enzyme kinetics

We present monitoring of nitrilase enzyme assay in real-time by means of Spectroscopic Optical Coherence Tomography (sOCT). Combination of principal component analysis (PCA) algorithm with Swept Source Optical Coherence Tomography (SSOCT), to extract the absorption spectra of the sample, termed as sOCT. Spectroscopic information of nitrilase enzyme obtained from sOCT is used to investigate dynamic enzyme kinetic reactions. Different principle components give complete information regarding the composition of the samples. Further, Fourier Transform Infrared Spectroscopy (FTIR) analysis has been also performed to validate the PCA absorption spectra. Samples were prepared in controlled condition and evaluated at a regular time interval of the incubation period. The developed algorithm and system are combinedly capable of in-vitro real-time, study of enzyme kinetics with respect to time as biosensor.

A comparative multivariate analysis of nitrilase enzymes: An ensemble based computational approach

Nitrilases, member of nitrilase superfamily catalyse the hydrolysis of different nitriles to corresponding amides and acids. In this article, we demonstrate two-fold computational comparative analysis on coding gene sequences, amino acid sequences, three-dimensional structure of the nitrilases from different species and discovered conserved motifs linked with related species. A large ensemble-based dataset was utilized from bacteria, fungi, plants and animals. Here, we used comparative genomics, motif analyses and Bayesian phylogenetic analyses in combination with structural analyses [molecular dynamics simulation, principal component analysis (PCA), dynamic cross correlation (DCCM), root mean squared inner product (RMSIP), free energy surface (FES)] to investigate the evolution, ecological relationship and structure-function association of nitrilase family. The inferred evolutionary tree displayed nitrilase gene clusters to be shared among bacteria, fungi and plants. Structural analysis revealed that the folding of catalytic sites is similar among species; however, the loop region varies. We provide evidence based on PCA that the nitrilases are clustered into different clades due to variation in side chains. Numerous of significant correlations were found between sequence clades and the structural discriminating properties of nitrilases originating from different species. The results are consistent with the hypothesis that bacterial nitrilases are in ecological and evolutionary relationships with fungi and plants during plant-pathogen interaction to large extent. This compact and detail results also open new dimensions for further studying and improvement of industrially important nitrilase enzymes.

Dynamic Modeling of Indole Glucosinolate Hydrolysis and Its Impact on Auxin Signaling.

Plants release chemicals to deter attackers. Arabidopsis thaliana relies on multiple defense compounds, including indol-3-ylmethyl glucosinolate (I3G), which upon hydrolysis initiated by myrosinase enzymes releases a multitude of bioactive compounds, among others, indole-3-acetonitrile and indole-3-acetoisothiocyanate. The highly unstable isothiocyanate rapidly reacts with other molecules. One of the products, indole-3-carbinol, was reported to inhibit auxin signaling through binding to the TIR1 auxin receptor. On the contrary, the nitrile product of I3G hydrolysis can be converted by nitrilase enzymes to form the primary auxin molecule, indole-3-acetic acid, which activates TIR1. This suggests that auxin signaling is subject to both antagonistic and protagonistic effects of I3G hydrolysis upon attack. We hypothesize that I3G hydrolysis and auxin signaling form an incoherent feedforward loop and we build a mathematical model to examine the regulatory network dynamics. We use molecular docking to investigate the possible antagonistic properties of different I3G hydrolysis products by competitive binding to the TIR1 receptor. Our simulations reveal an uncoupling of auxin concentration and signaling, and we determine that enzyme activity and antagonist binding affinity are key parameters for this uncoupling. The molecular docking predicts that several I3G hydrolysis products strongly antagonize auxin signaling. By comparing a tissue disrupting attack – e.g., by chewing insects or necrotrophic pathogens that causes rapid release of I3G hydrolysis products – to sustained cell-autonomous I3G hydrolysis, e.g., upon infection by biotrophic pathogens, we find that each scenario gives rise to distinct auxin signaling dynamics. This suggests that plants have different defense versus growth strategies depending on the nature of the attack.

KERAGAMAN BAKTERI PENGHASIL ENZIM PENGHIDROLISIS NITRIL DI PULAU ENGGANO BENGKULU

Potential nitrile degrading microbes have been isolated from marine sponge, marine water and soil in Enggano Island. Nitrilase enzyme has a function in degrading nitrile compund. Nitrilases are important industrial enzymes because of their ability to produce biologically active to degrade enantiomers, such as S-(+)-1-(4’-isobutylphenyl) propionic acid (S-(+)-ibuprofen) and R-(-) mandelic acid. Mandelic acids, which are important as pharmaceutical intermediates, can be produced in enantiomerically pure form by the hydrolysis of their corresponding nitrile. The aim of the study was to explore the diversity of nitrile degrading bacteria in Enggano Island, and their ability to utilize nitrile as a substrate growth. Screening of such microbes were carried out by using microtitter plate method based on growth ability tested by INT (Iodonitrotetrazoliumchloride). Degradation product was determined by High Performance Liquid Chromatography (HPLC). Seventy nine bacteria were able to grow on acetamide, acetonitrile, benzonitrile, adiponitrile, mandelonitrile, succinonitrile, lactonitrile, dan benzilcyanide as the sole source of carbon and nitrogen. Two isolates, YIM 56238 and PO69, have shown to enantioselectively hydrolyze racemic mandelonitrile to mandelic acid. Based on 16S rRNA gene identification, these bacteria have the highest sequence similarity to Microccous endophyticus strain YIM 56238 and Rhodococcus pyridinivorans strain PO69.

Improving stability and reusability of Rhodococcus pyridinivorans NIT-36 nitrilase by whole cell immobilization using chitosan

Immobilized enzymes have great significance for industrial processes. In this study whole cell immobilization of Rhodococcus pyridinivorans NIT-36 has been undertaken using chitosan microspheres as an immobilized matrix. R. pyridinivorans NIT-36 harbors a significant intracellular enzyme nitrilase. Chitosan microspheres were generated by supplementing chitosan with glutaraldehyde and results, supported the porous microsphere structure via SEM. The resultant microspheres exhibited cell immobilization capacity of 450mg/g. The immobilized cells exhibited a considerable increase in temperature tolerance at 60°C as compared to free cells. The immobilized microspheres also demonstrated higher substrate tolerance. The immobilized nitrilase retained 80% activity when stored at 4°C for 10 days and retained 50% activity after 7 reuse cycles. It may be concluded that chitosan microspheres are a novel immobilization agent for whole cell immobilization which enhances the stability and reusability of nitrilase enzyme.

Bench scale synthesis of p-hydroxybenzoic acid using whole-cell nitrilase of Gordonia terrae mutant E9.

Mutants of Gordonia terrae were generated using chemical mutagens for better activity, stability and higher substrate/product tolerance of its nitrilase enzyme. Mutant E9 showed two-time increase in activity and tolerated p-hydroxybenzonitrile (p-HBN) up to 50 mM. Response surface methodology and inducer mediation approach further enhanced the production of enzyme to 2.5-fold. The bench scale production of p-hydroxybenzoic acid (p-HBA) was carried out in a fed-batch reaction (500-mL scale) using whole-cell nitrilase of mutant E9 in 0.1 M potassium phosphate buffer (pH 8.0) at 40 °C. Total six feedings each at an interval of 45 min resulted in accumulation of 360 mM (21.6 g) of p-HBA with a purity of 99%. The catalytic and volumetric productivity of bioprocess using mutant G. terrae was improved to 1.8 g h(-1) g DCW (-1) and 43.2 g L(-1), respectively, from 0.78 g h(-1) g DCW (-1) and 28.8 g L(-1) using resting cells of wild strain. K m and V max of purified nitrilase from mutant E9 were 55 U mg(-1) and 1.8 mM for p-HBN with a higher turnover number of 36 s(-1) × 10(-3).

Heterologous expression of mitochondria-targeted microbial nitrilase enzymes increases cyanide tolerance in Arabidopsis.

Anthropogenic activities have resulted in cyanide (CN) contamination of both soil and water in many areas of the globe. While plants possess a detoxification pathway that serves to degrade endogenously generated CN, this system is readily overwhelmed, limiting the use of plants in bioremediation. Genetic engineering of additional CN degradation pathways in plants is one potential strategy to increase their tolerance to CN. Here we show that heterologous expression of microbial nitrilase enzymes targeted to the mitochondria increases CN tolerance in Arabidopsis. Root length in seedlings expressing either a CN dihydratase from Bacillus pumilis or a CN hydratase from Neurospora crassa was increased by 45% relative in wild-type plants in the presence of 50μm KCN. We also demonstrate that in contrast to its strong inhibitory effects on seedling establishment, seed germination of the Col-0 ecotype of Arabidopsis is unaffected by CN.

Effect of physicochemical parameters on nitrile-hydrolyzing potentials of newly isolated nitrilase of Fusarium oxysporum f. sp. lycopercisi ED-3.

In recent years, nitrilases from fungus have received increasing attention, and most of the studies are performed on nitrilases of bacterial origin. Frequently used methods are based on analytical methods such as high-performance liquid chromatography, liquid chromatography-mass spectrometry, and gas chromatography; therefore, an efficient, user friendly, and rapid method has been developed to screen nitrilase enzyme based on the principle of color change of a pH indicator. Phenol red amended with the minimal medium appears light yellow at neutral pH, which changes into pink with the formation of ammonia, indicating nitrilase activity in the reaction medium. A highly potent strain ED-3 identified as Fusarium oxysporum f. sp. lycopercisi (specific activity 17.5 µmol/Min/mg dcw) was isolated using this method. The nitrilase activity of F. oxysporum f. sp. lycopercisi ED-3 strain showed wide substrate specificity toward aliphatic nitriles, aromatic nitriles, and orthosubstituted heterocyclic nitriles. 4-Aminobenzonitrile was found to be a superior substrate among all the nitriles used in this study. This nitrilase was active within pH 5-10 and temperature ranging from 25 to 60 °C with optimal at pH 7.0 and temperature at 50 °C. The nitrilase activity was enhanced to several folds through optimization of culture and biotransformation conditions from 1,121 to 1,941 µmol/Min.

Erratum to: Understanding nitrile-degrading enzymes: classification, biocatalytic nature and current applications

AbstractNitrile-degrading enzymes commonly known as nitrilase enzymes are able to metabolize nitrile-substituent compounds and they have several industrial applications, for example: in drugs synthesis. It is also common to observe their exploitation for obtaining chemical compounds with commercial interests related to cosmetics production, paints and additives. In addition, these are frequently used in the active metabolites synthesis of pesticides. Due to the catalytic nature of such proteins, it is possible to take advantage of their biotechnological potential to be applied in various scientific fields including synthetic biocatalysis and environmental remediation, since they have been successfully used for soils nitrile-wastes decontamination such as cyanide, bromoxynil and benzonitrile. On the other hand, these enzymes are considered very important intermediaries of metabolic pathways related to indolic compounds that are produced by bacteria, plants and superior fungi, acting in most cases as vegetal growth hormones. Given the fact that indole-derivative molecules play an important role in physiological responses in superior organisms, nitrilase enzymes may be considered as important part of unknown multi-enzymatic secondary metabolites pathways. In light of the above considerations, this review attempts to summarize the current status of nitrilase research and describing in detail the main characteristics of nitrile-converting enzymes with emphasis on fungal proteins, including their function and catalytic selectivity. Likewise, their relationship with plant metabolism and biotechnological importance in bioremediation processes is discussed.

Understanding nitrile-degrading enzymes: classification, biocatalytic nature and current applications

AbstractNitrile-degrading enzymes commonly known as nitrilase enzymes are able to metabolize nitrile-substituent compounds and they have several industrial applications, for example: in drugs synthesis. It is also common to observe their exploitation for obtaining chemical compounds with commercial interests related to cosmetics production, paints and additives. In addition, these are frequently used in the active metabolites synthesis of pesticides. Due to the catalytic nature of such proteins, it is possible to take advantage of their biotechnological potential to be applied in various scientific fields including synthetic biocatalysis and environmental remediation, since they have been successfully used for soils nitrile-wastes decontamination such as cyanide, bromoxynil and benzonitrile. On the other hand, these enzymes are considered very important intermediaries of metabolic pathways related to indolic compounds that are produced by bacteria, plants and superior fungi, acting in most cases as vegetal growth hormones. Given the fact that indole-derivative molecules play an important role in physiological responses in superior organisms, nitrilase enzymes may be considered as important part of unknown multi-enzymatic secondary metabolites pathways. In light of the above considerations, this review attempts to summarize the current status of nitrilase research and describing in detail the main characteristics of nitrile-converting enzymes with emphasis on fungal proteins, including their function and catalytic selectivity. Likewise, their relationship with plant metabolism and biotechnological importance in bioremediation processes is discussed.

A high throughput screening strategy for the assessment of nitrile-hydrolyzing activity towards the production of enantiopure β-hydroxy acids

Abstract Nitrile hydrolysing enzymes have found wide use in the pharmaceutical industry for the production of fine chemicals. This work presents a strategy that facilitates the rapid identification of bacterial isolates demonstrating nitrile hydrolysing activity. The strategy incorporates toxicity, starvation and induction studies along with subsequent colorimetric screening for activity, further focusing the assessment towards the substrates of interest. This high-throughput strategy uses a 96 well plate system, and has enabled the rapid biocatalytic screening of 256 novel bacterial isolates towards β-hydroxynitriles. Results demonstrate the strategy's potential to rapidly assess a variety of β-hydroxynitriles including aliphatic, aromatic and dinitriles. A whole cell catalyst Rhodococcus erythropolis SET1 was identified and found to catalyse the hydrolysis of 3-hydroxybutyronitrile with remarkably high enantioselectivity under mild conditions, to afford (S)-3-hydroxybutyric acid in 42% yield and >99.9% ee. The biocatalytic capability of this strain including the variation of parameters such as temperature and time were further investigated and all results indicate the presence of a highly enantioselective if not enantiospecific nitrilase enzyme within the microbial whole cell.

Understanding nitrile-degrading enzymes: classification, biocatalytic nature and current applications

AbstractNitrile-degrading enzymes commonly known as nitrilase enzymes are able to metabolize nitrile-substituent compounds and they have several industrial applications, for example: in drugs synthesis. It is also common to observe their exploitation for obtaining chemical compounds with commercial interests related to cosmetics production, paints and additives. In addition, these are frequently used in the active metabolites synthesis of pesticides. Due to the catalytic nature of such proteins, it is possible to take advantage of their biotechnological potential to be applied in various scientific fields including synthetic biocatalysis and environmental remediation, since they have been successfully used for soils nitrile-wastes decontamination such as cyanide, bromoxynil and benzonitrile. On the other hand, these enzymes are considered very important intermediaries of metabolic pathways related to indolic compounds that are produced by bacteria, plants and superior fungi, acting in most cases as vegetal growth hormones. Given the fact that indole-derivative molecules play an important role in physiological responses in superior organisms, nitrilase enzymes may be considered as important part of unknown multi-enzymatic secondary metabolites pathways. In light of the above considerations, this review attempts to summarize the current status of nitrilase research and describing in detail the main characteristics of nitrile-converting enzymes with emphasis on fungal proteins, including their function and catalytic selectivity. Likewise, their relationship with plant metabolism and biotechnological importance in bioremediation processes is discussed.

Highlights of Recent Articles on Data Mining in Genomics and Proteomics

Advancements in ‘Omics’ technologies continue to enhance research to solve the world’s medical and agricultural problems. The 2012 issues of Data Mining in Genomics & Proteomics solicited contributions from leading authors covering various aspects of data mining technologies for genomics and proteomics, and the application of these technologies to biological sciences. Zhou et al. [1] developed a computational platform called OmicsMiner to facilitate the design of customized pipelines to process multiple biological datasets. The platform is adaptable and can easily be expanded by integrating new algorithms and protocols. OmicsMiner has a well-designed graphical interface for data processing that utilizes Java protocols, which do not require additional sub-protocol installations. Gomase and Chitlange [2] describe a microbial proteomic approach to predict the binding ability of antigen protein for selecting nonamers for use in rational vaccine design and increase understanding of the roles of the immune system in infectious diseases. They demonstrated the utility of this approach in a project to examine the tapeworm parasite of sheep, Taenia ovis, which causes Sheep Measles. The authors used position specific scoring matrices (PSSM) for antigen design. Prediction of antigen binding ability is important in vaccine development for Sheep Measles and was obtained by using the support vector machine (SVM) based method. A proteomic approach employing 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used by Yang et al. [3] to separate the membrane proteins of the Chinese Brucella melitensis M5 strain that causes livestock and human disease. Characterization and identification of immunogenic proteins were accomplished using protein blots, mass spectrometry and bioinformatics. Using this approach, they were able to identify immune reactive proteins that show promise as vaccine candidates. In another protein related article, Sharma and Bhalla [4] reported on the computational analysis of nitrilase enzymes to define unique motifs, and correlated physiochemical and phylogenetic analyses. The results can be applied to the prediction and differentiation of these enzymes based on their substrate specificity and to find new sources of nitrilase across various databases. Several approaches to disease control and prevention were discussed in articles addressing the association of increased B-cell activating factor (BAFF) expression with disease markers in patients with Lupus Nephritis (LN), the potential advantages of retinoids treatment in LN, and the role of glycoprotein M64 (GPM6A) in human thyroid cancer. Eilertsen et al. [5] utilized quantitative RT-PCR to analyze gene expression in systemic lupus erythematosus patients and further investigated their reaction with clinical, histological and acute phase protein. They found increased BAFF mRNA expression and concluded that the increase is not reflective of histological disease severity. A review article by Khalid et al. [6] presents an overview of therapeutic use of retinoids, a group of natural and synthetic derivatives of vitamin A for LN. The review discusses the details of LN pathogenesis and severity in humans, and current and new agents for the treatment.

Kinetika Enzim Nitrilase Dari Sel Utuh Rhodococcus spp Pada Biotransformasi Mandelonitril

Rhodococcus spp can be used as mandelonitrile substrate for carbon and nitrogen source in life cycle of metabolism. They have potential of activity biotransformation for produced (R/S)-mandelic acid from mandelonitrile. (R/S)-mandelic acid is an important biotransformation product for production of pharmaceuticals such as semisynthetic penicillins, cephalosporins, antitumor agents, antiobesity agents and antiinflamation agents. This research was conducted to determine the enzyme kinetics (Km and Vmax) of nitrilase from Rhodococcus TPIK, LP3, GLB5 (induction acetonitrile 1000 mM - mandelonitrile 100 mM) with florometric methods, spectrophotometer analysis (λ = 413 nm). This research was carried out by assaying nitrilase enzyme activities in various concentration of mandelonitrile subtrates which were between 10 mM – 100 mM with 10 mM interval. The result showed that the enzyme kinetics of nitrilase from Rhodococcus TPIK (Km was 72.303 mM and Vmax was 2.075 mM/ml cell/minute), Rhodococcus LP3 (Km was 47.048 mM and Vmax was 1.942 mM/ml cell/minute), Rhodococcus GLB5 (Km was 34.375 mM and Vmax was 2.083 mM/ml cell/minute). Nitrilase enzyme from Rhodococcus GLB5 have smallest Km value. So we can interpretationed, this enzyme have good complexity Enzyme-Substrate, high affinity with substrate, and high speed reaction for forms product mandelic acid.

Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity

Microbial nitrilases are biocatalysts of interest and the enzyme produced using various inducers exhibits altered substrate specificity, which is of great interest in bioprocess development. The aim of the present study is to investigate the nitrilase-producing Alcaligenes faecalis MTCC 10757 (IICT-A3) for its ability to transform various nitriles in the presence of different inducers after optimization of various parameters for maximum enzyme production and activity. The production of A. faecalis MTCC 10757 (IICT-A3) nitrilase was optimum with glucose (1.0%), acrylonitrile (0.1%) at pH 7.0. The nitrilase activity of A. faecalis MTCC 10757 (IICT-A3) was optimum at 35 °C, pH 8.0 and the enzyme was stable up to 6 h at 50 °C. The nitrilase enzyme produced using different inducers was investigated for substrate specificity. The enzyme hydrolyzed aliphatic, heterocyclic and aromatic nitriles with different substitutions. Acrylonitrile was the most preferred substrate (~40 U) as well as inducer. Benzonitrile was hydrolyzed with almost twofold higher relative activity than acrylonitrile when it was used as an inducer. The versatile nitrilase-producing A. faecalis MTCC 10757 (IICT-A3) exhibits efficient conversion of both aliphatic and aromatic nitriles. The aromatic nitriles, which show not much or no affinity towards nitrilase from A. faecalis, are hydrolyzed effectively with this nitrilase-producing organism. Studies are in progress to exploit this organism for synthesis of industrially important compounds.