Plants produce several hundreds of thousands of secondary metabolites that are important for adaptation to various environmental conditions. Although different groups of secondary metabolites are synthesized through unique biosynthetic pathways, plants must orchestrate their production simultaneously. Phenylpropanoids and glucosinolates are two classes of secondary metabolites that are synthesized through apparently independent biosynthetic pathways. Genetic evidence has revealed that the accumulation of glucosinolate intermediates limits phenylpropanoid production in a Mediator Subunit 5 (MED5)-dependent manner. To elucidate the molecular mechanism underlying this process, we analyzed the transcriptomes of a suite of Arabidopsis thaliana glucosinolate-deficient mutants using RNAseq and identified misregulated genes that are rescued by the disruption of MED5. The expression of a group of Kelch Domain F-Box genes (KFBs) that function in PAL degradation is affected in glucosinolate biosynthesis mutants and the disruption of these KFBs restores phenylpropanoid deficiency in the mutants. Our study suggests that glucosinolate/phenylpropanoid metabolic crosstalk involves the transcriptional regulation of KFB genes that initiate the degradation of the enzyme phenylalanine ammonia-lyase, which catalyzes the first step of the phenylpropanoid biosynthesis pathway. Nevertheless, KFB mutant plants remain partially sensitive to glucosinolate pathway mutations, suggesting that other mechanisms that link the two pathways also exist.

https://nph.onlinelibrary.wiley.com/doi/epdf/10.1111/nph.16108

Glucosinolate and phenylpropanoid biosynthesis are linked by proteasome‐dependent degradation of PAL

Abstract(R)-mandelic acid was produced from racemic mandelonitrile using free and immobilized cells of Pseudomonas putida MTCC 5110 harbouring a stereoselective nitrilase. In addition to the optimization of culture conditions and medium components, an inducer feeding approach is suggested to achieve enhanced enzyme production and therefore higher degree of conversion of mandelonitrile. The relationship between cell growth periodicity and enzyme accumulation was also studied, and the addition of the inducer was delayed by 6 h to achieve maximum nitrilase activity. The nitrilase expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. P. putida MTCC 5110 cells were further immobilized in calcium alginate, and the immobilized biocatalyst preparation was used for the enantioselective hydrolysis of mandelonitrile. The immobilized system was characterized based on the Thiele modulus (ϕ). Efficient biocatalyst recycling was achieved as a result of immobilization with immobilized cells exhibiting 88% conversion even after 20 batch recycles. Finally, a fed batch reaction was set up on a preparative scale to produce 1.95 g of (R)-(-)-mandelic acid with an enantiomeric excess of 98.8%.

Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis (vol 72, pg 77, 2006)

Nitrile metabolizing enzymes, i.e., aldoxime dehydratase, hydroxynitrile lyase, nitrilase, nitrile hydratase, and amidase, are the key catalysts in carbon nitrogen triple bond anabolism and catabolism. Over the past several years, these enzymes have drawn considerable attention as prominent biocatalysts in academia and industries because of their wide applications. Research on various aspects of these biocatalysts, i.e., sources, screening, function, purification, molecular cloning, structure, and mechanisms, has been conducted, and bioprocesses at various scales have been designed for the synthesis of myriads of useful compounds. This review is focused on the potential of nitrile metabolizing enzymes in the production of commercially important fine chemicals such as nitriles, carboxylic acids, and amides. A number of opportunities and challenges of nitrile metabolizing enzymes in bioprocess development for the production of bulk and fine chemicals are discussed.

Nitrile Metabolizing Enzymes in Biocatalysis and Biotransformation.

The bacterial genus Gordonia encompasses a variety of versatile species that have been isolated from a multitude of environments. Gordonia was described as a genus about 20 years ago, and to date, ...

An insight into the ecology, diversity and adaptations of Gordonia species.

Among the many biological entities employed in the development of biosensors, enzymes have attracted the most attention. Nanotechnology has been fostering excellent prospects in the development of enzymatic biosensors, since enzyme immobilization onto conductive nanostructures can improve characteristics that are crucial in biosensor transduction, such as surface-to-volume ratio, signal response, selectivity, sensitivity, conductivity, and biocatalytic activity, among others. These and other advantages of nanomaterial-based enzymatic biosensors are discussed in this work via the compilation of several reports on their applications in different industrial segments. To provide detailed insights into the state of the art of this technology, all the relevant concepts around the topic are discussed, including the properties of enzymes, the mechanisms involved in their immobilization, and the application of different enzyme-derived biosensors and nanomaterials. Finally, there is a discussion around the pressing challenges in this technology, which will be useful for guiding the development of future research in the area.

https://www.mdpi.com/2673-3293/2/1/12/pdf

Designing of Nanomaterials-Based Enzymatic Biosensors: Synthesis, Properties, and Applications

Aldoxime–nitrile pathway is one of the important routes of carbon and nitrogen metabolism in many life forms and a key interface for plant–microbe interactions. This pathway starts with transformation of amino acids to aldoximes, which are converted to nitriles and the later are ultimately hydrolyzed to acids and ammonia. Understanding and engineering of the enzymes involved in this pathway viz. cytochrome P450/CYP79, aldoxime dehydratase, nitrilase, nitrile hydratase, amidase and hydroxynitrile lyase, presents unprecedented opportunities in biocatalysis and green chemistry. Co-expressing these enzymes in prokaryotic and eukaryotic microbial hosts and tailoring their properties i.e. activity, specificity, stability and enantioselectivity may lead to develop sustainable bioprocesses for the synthesis of industrially important nitriles, amides and acids.

Enzymes of aldoxime–nitrile pathway for organic synthesis

The discharge of cyanide-containing effluents into the environment contaminates water bodies and soil. Effective methods of treatment which can detoxify cyanide are the need of the hour. The aim of the present study is to develop a bioreactor for complete degradation of cyanide using immobilized cells of Serratia marcescens RL2b. Alginate-entrapped cells of S. marcescens RL2b were used for complete degradation of cyanide in a packed bed reactor (PBR). Cells grown in minimal salt medium (pH 6.0) were harvested after 20 h and exhibited 0.4 U mg−1 dcw activity and 99 % cyanide degradation in 10 h. These resting cells were entrapped using 3 % alginate beads and packed in a column reactor (20 × 1.7 cm). Simulated cyanide (12 mmol l−1)-containing wastewater was loaded and fractions were collected after different time intervals at various flow rates. Complete degradation of 12 m mmol l−1 (780 mg l−1) cyanide in 10 h was observed at a flow rate of 1.5 ml h−1. The degradation of cyanide in PBR showed direct dependence on retention time. The retention time of cyanide in the reactor was 9.27 h. The PBR can degrade 1.2 g of cyanide completely in 1 day.

/pdf/packed-bed-reactor-for-degradation-of-simulated-cyanide-2ksz01zvyh.pdf

Packed bed reactor for degradation of simulated cyanide-containing wastewater

A potentiometric biosensor for cyanide detection has been developed using whole cell cyanide dihydratase activity of Flavobacterium indicum coupled to an ammonium ion selective electrode. The cyanide dihydratase activity was optimum when 2% agar and cells in the amount of 0.175 mg/mL dry cell weight (dcw) were used for immobilization. The free and immobilized whole cell cyanide dihydratase of F. indicum exhibited maximum activity (0.192 U/mg dcw and 0.175 U/mg dcw, respectively) in 0.1 M sodium phosphate buffer (pH 8.0) at 35°C with 5 mM potassium cyanide. KM and Vmax for free and immobilized whole cell cyanide dihydratase were 5.25 mM, 0.33 U/mg dcw and 6.25 mM, 0.25 U/mg, respectively. The agar immobilized whole cell cyanide dihydratase was used as a biocomponent coupled to a potentiometer. A linear relationship was observed between response time and cyanide concentration. The cyanide concentration of 0.06 ppm could be detected using this potentiometric biosensor with a response time of 2 min. The developed potentiometric biosensor could be used for the detection of cyanide in industrial wastewater and food samples.

A Potentiometric Biosensor for Cyanide Detection using Immobilized Whole Cell Cyanide Dihydratase of Flavobacterium indicum MTCC 6936

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.

Virender Kumar

Papers

Nitrile Metabolizing Enzymes in Biocatalysis and Biotransformation.

Enzymes of aldoxime–nitrile pathway for organic synthesis

Packed bed reactor for degradation of simulated cyanide-containing wastewater

A Potentiometric Biosensor for Cyanide Detection using Immobilized Whole Cell Cyanide Dihydratase of Flavobacterium indicum MTCC 6936

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