Validation of Lon Gene Disruption using Linear DNA Cassette by Crelox Mechanism in E. coli Strains: To Achieve Better Solubility of Putrescine Monooxygenase.

Numerous expression systems, engineered strains, and cultivation systems have been developed globally but producing recombinant proteins in the soluble form continues to remain a challenge.  Escherichia coli, a preferred host for the recombinant production of biopharmaceuticals and other proteins. Up to 75% of human proteins expressed in E. coli have only 25% in an active soluble form. The proteolytic activity of Lon encoded protease triggers the inclusion bodies leading to heterogenous secreted proteins thereby hampering downstream processing and isolation. Putrescine monooxygenases are versatile with applications in iron acquisition, pathogen control, biotransformation, bio-remediation and redox reaction are still isolated from plant and microbial sources at low yields. As a prerequisite to developing protease knockout E. coli strains, using the Cre-loxP recombination strategy we have built a full-length Lon disruption cassette (5'lon-lox66-cre-KanR-lox71-3'lon) (3368 bp) consisting of upstream and downstream regions of Lon, loxP sites, and Cre gene driven by T7 promoter to the expression of Cre recombinase and a selectable kanamycin resistance gene. Here, after the integration of the knock-out cassette into the host genome, we show the production of homogeneous protein species of recombinant Putrescine monooxygenase by using an E. coli platform strain in which Lon gene is deleted. This Lon knock-out strain secreted more homogeneous protein at a volumetric yield of 60% of the wild-type strain. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01056-x.

Determination of chlorantraniliprole 18.5% SC in the paddy ecosystem and its risk assessment.

Chlorantraniliprole belongsto theanthranilic diamide group is widely used against broad range of lepidopteron pests in a variety of vegetable and rice pests includingyellow rice stem borer and leaf folder. Supervised field trials were conducted duringRabi (2018-2019) and Kharif (2019) to evaluate the dissipation pattern and risk assessment of chlorantraniliprole 18.5% SC in paddy ecosystem following foliar application at 30 and 60 g a.i. ha-1 in two different cropping seasons.Modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) technique was used for the extraction of CAP residues with acetonitrile and determined by LC-MS/MS (ESI +).The limit of quantification (LOQ) was 0.01 µg g-1 for paddy leaf, straw, husk, and brown rice, respectively and 0.005 µg g-1 for soil. The average recoveries obtained were 84.30-88.92% from paddy leaf, 94.25-97.81% from straw, 90.21-93.38% from husk, 93.57-96.40% from brown rice and 89.93-91.14% from soil. The residues in paddy leaf dissipated within 35-40 days with a half-life of 4.33-5.07 days in Rabi and 3.92-4.86 days in Kharif at 30 and 60 g a.i. ha-1, respectively. The residues in soil dissipated within 15-21 days with a half-life of 14.44-15.75 days in Rabi and 13.33-14.44 days in Kharif at respective doses. At harvest chlorantraniliprole residues were not detected in straw, husk, and brown rice. The dietary risk of paddy leaf (green fodder) for cattle was found safe for consumption as the hazard index is less than one. Soil ecological risk assessment was found to be less than one (RQ < 0.1) for earthworms (Eisenia foetida) and arthropods (Aphidiusrhopalosiphi). The presentmethod could be useful inthe analysis ofchlorantraniliproleresidues in different cereals and vegetable crop ecosystems and application at recommended dose is safe for the final produce at harvest.

In silico structural, phylogenetic and drug target analysis of putrescine monooxygenase from Shewanella putrefaciens-95.

BACKGROUND: The enormous and irresponsible use of antibiotics has led to the emergence of resistant strains of bacteria globally. A new approach to combat this crisis has been nutritional immunity limiting the availability of nutrients to pathogens. Targeting the siderophore biosynthetic pathway that helps in iron acquisition, an essential microelement in the bacterial system has been the topic of interest in recent days that backs the concept of nutritional immunity. Supporting this view, we have chosen to study a key enzyme in the biosynthetic pathway of putrebactin called putrescine monooxygenase (SpPMO) from Shewanella putrefaciens. In our previous study, we co-expressed putrescine monooxygenase recombinantly in Escherichia coli BL21 Star (DE3). The bioinformatic analysis and screening of inhibitors will broaden the scope of SpPMO as a drug target. RESULTS: In the present study, we have analysed the physicochemical properties of the target enzyme and other N-hydroxylating monooxygenases (NMOs) using ExPASy server. The target enzyme SpPMO and most of the selected NMOs have a slightly acidic isoelectric point and are medially thermostable and generally insoluble. The multiple sequence alignment identified the GXGXX(N/A), DXXXFATGYXXXXP motives and conserved amino acids involved in FAD binding, NADP binding, secondary structure formation and substrate binding. The phylogenetic analysis indicated the distribution of the monooxygenases into different clades according to their substrate specificity. Further, a 3D model of SpPMO was predicted using I-TASSER online tool with DfoA from Erwinia amylovora as a template. The model was validated using the SAVES server and deposited to the Protein Model Database with the accession number PM0082222. The molecular docking analysis with different substrates revealed the presence of a putrescine binding pocket made of conserved amino acids and another binding pocket present on the surface of the protein wherein all other ligands interact with high binding affinity. The molecular docking of naturally occurring inhibitor molecules with SpPMO 3D model identified curcumin and niazirin with 1.83 and 2.81 µM inhibition constants as two promising inhibitors. Further studies on kinetic parameters of curcumin and niazirin inhibitors in vitro determined the Ki to be 2.6±0.0036 µM and 18.38±0.008 µM respectively. CONCLUSION: This analysis will help us understand the structural, phylogenetic and drug target aspects of putrescine monooxygenase from Shewanella putrefaciens-95 in detail. It sheds light on the precautionary measures that can be developed to inhibit the enzyme and thereby the secondary infections caused by them.

Determination of 77 Multiclass Pesticides and Their Metabolitesin Capsicum and Tomato Using GC-MS/MS and LC-MS/MS.

A quick, sensitive, and reproducible analytical method for the determination of 77 multiclass pesticides and their metabolites in Capsicum and tomato by gas and liquid chromatography tandem mass spectrometry was standardized and validated. The limit of detection of 0.19 to 10.91 and limit of quantification of 0.63 to 36.34 µg·kg-1 for Capsicum and 0.10 to 9.55 µg·kg-1 (LOD) and 0.35 to 33.43 µg·kg-1 (LOQ) for tomato. The method involves extraction of sample with acetonitrile, purification by dispersive solid phase extraction using primary secondary amine and graphitized carbon black. The recoveries of all pesticides were in the range of 75 to 110% with a relative standard deviation of less than 20%. Similarly, the method precision was evaluated interms of repeatability (RSDr) and reproducibility (RSDwR) by spiking of mixed pesticides standards at 100 µg·kg-1 recorded anRSD of less than 20%. The matrix effect was acceptable and no significant variation was observed in both the matrices except for few pesticides. The estimated measurement uncertainty found acceptable for all the pesticides. This method found suitable for analysis of vegetable samples drawn from market and farm gates.

Expression and purification of codon-optimized cre recombinase in E. coli.

The presence of antibiotic resistance genes in genetically modified bacteria raises a regulatory concern in the production of therapeutic proteins and additionally reduces the number of plasmids available for propagation in a cell. Cre recombinase from bacteriophage P1, involved in Cre/loxP mechanism is one of the widely used systems for selectable marker gene removal. We have overexpressed codon-optimized cre gene in pColdIV and pET28a(+) vector systems and purified His6-Cre recombinase by immobilized metal affinity chromatography. N-terminal His6 tagged Cre recombinase obtained was approximately 26 fold purified and promoted the site-specific recombination of two loxP sites of linearized pLox2+ vector allowing the excision of a re-circularized plasmid and a short stretch of DNA containing the recombined loxP site. The results of the expression using two vectors, purification and activity assessment of His6 tagged Cre recombinase is presented here.

Advances in cloning, structural and bioremediation aspects of nitrile hydratases.

Nitrile hydratase (NHase) is a prominent enzyme in many microorganisms for its nitrile metabolism. The potentiality in the bioconversion of nitriles to its high-value amides has been extensively used in industries for the production of acrylamide and nicotinamide which are essential chemicals. Enzymologists are still considering NHases for its potential biotechnological applications including biotransformations and bioremediations. But most of the nitrile hydratases have limitations like the low expression, low thermostability and enantioselectivity. Though considerable data has been generated in the area of gene configuration, crystal structure, kinetic mechanism and photoreactivity of NHases, there is a need for constant improvement to develop a robust biocatalyst for bioremediation of toxic nitriles. With these considerations, in the present review, we report advances with the main focus to structure, catalytic mechanism, cloning strategy, gene expression, bioinformatic tools, metagenomics, thermostability and current bioremediation applications of NHases.

Chaperone-assisted expression, purification, and characterization of recombinant nitrile hydratase NI1 from Comamonas testosteroni.

Nitrile hydratases (NHases) are industrially important iron- and cobalt-containing enzymes that are used in the large-scale synthesis of acrylamide. Heterologous expression of NHases has been complicated by the fact that other proteins (activators or metallochaperones) appear to be required to produce NHases in their catalytically active form. We report a novel heterologous system for the expression of catalytically active iron-containing NI1 NHase in Escherichia coli, involving coexpression with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be highly similar to the enzyme purified from Comamonas testosteroni according to its spectroscopic features, catalytic properties with various substrates, and post-translational modifications. In addition, we report a rapid and convenient spectrophotometric method to monitor the activity of NI1 NHase during purification.