Arindam Ghatak

Alcohol dehydrogenases (ADHs) are popular catalysts for synthesizing chiral synthons a vital step for active pharmaceutical intermediate (API) production. They are grouped into three superfamilies namely, medium-chain (MDRs), short-chain dehydrogenase/reductases (SDRs), and iron-containing alcohol dehydrogenases. The former two are used extensively for producing various chiral synthons. Many studies screen multiple enzymes or engineer a specific enzyme for catalyzing a substrate of interest… Show more

Alcohol dehydrogenases (ADHs) are popular catalysts for synthesizing chiral synthons a vital step for active pharmaceutical intermediate (API) production. They are grouped into three superfamilies namely, medium-chain (MDRs), short-chain dehydrogenase/reductases (SDRs), and iron-containing alcohol dehydrogenases. The former two are used extensively for producing various chiral synthons. Many studies screen multiple enzymes or engineer a specific enzyme for catalyzing a substrate of interest. These processes are resource-intensive and intricate. The current study attempts to decipher the ability to match different ADHs with their ideal substrates using machine learning algorithms. We explore the catalysis of 284 antibacterial ketone intermediates, against MDRs and SDRs to demonstrate a unique pattern of activity. To facilitate machine learning we curated a dataset comprising 33 features, encompassing 4 descriptors for each compound. Subsequently, an ensemble of machine learning techniques viz. Partial Least Squares (PLS) regression, k-Nearest Neighbors (kNN) regression, and Support Vector Machine (SVM) regression, was harnessed. Moreover, the assimilation of Principal Component Analysis (PCA) augmented precision and accuracy, thereby refining and demarcating diverse compound classes. As such, this classification is useful for discerning substrates amenable to diverse alcohol dehydrogenases, thereby mitigating the reliance on high-throughput screening or engineering in identifying the optimal enzyme for specific substrate. Show less

Summary
Background
Despite multifactorial evidence, the safe and effective elimination of free-floating microorganisms remain a significant scientific challenge. ZeBox Technology exploits microbial Zeta Potential, to extract and eliminate them from free-flowing air, using a non-ionizing electric field, in combination with a microbicidal surface.

Aim
Evaluation of ZeBox Technology against aerosolized SARS-CoV-2 and Mycobacterium tuberculosis under controlled… Show more

Summary
Background
Despite multifactorial evidence, the safe and effective elimination of free-floating microorganisms remain a significant scientific challenge. ZeBox Technology exploits microbial Zeta Potential, to extract and eliminate them from free-flowing air, using a non-ionizing electric field, in combination with a microbicidal surface.

Aim
Evaluation of ZeBox Technology against aerosolized SARS-CoV-2 and Mycobacterium tuberculosis under controlled conditions.

Methods
SARS-CoV2 and Mycobacterium tuberculosis: H37Ra were used in this study. Individual microorganisms were aerosolized using a Collison nebulizer inside an air-sealed test chamber. Air samples were collected from the chamber onto a Mixed Cellulose Ester membrane, at various time points, and used for enumeration. SARS-CoV2 was enumerated using qRT-PCR, while Mycobacterium tuberculosis H37Ra, was quantified using standard microbiological procedures.

Findings
We established a viable aerosolized microbial load of ∼10E9 and ∼10E6 for SARS-CoV-2 and Mycobacterium tuberculosis H37Ra respectively inside the test chamber. Under ideal conditions, the floating microbial load was at a steady-state level of 10E9 for SARS-CoV-2 and 10E6 for Mycobacterium tuberculosis. When the ZeBox Technology enabled device was operated, the microbial load reduced significantly. A reduction of ∼10E4.7 was observed for Mycobacterium tuberculosis, while ∼10E7 for SARS-CoV2 within a short duration. The reduction in airborne SARS-CoV-2 load was qualitatively and quantitatively measured using fluorescence analysis and qRT-PCR method respectively.

Conclusion
This validation testifies the efficacy of the developed technology against two of the deadliest microorganism, claiming millions of lives worldwide. In conjunction with the existing reports, the present validation proved the true broad-spectrum elimination capability of ZeBox technology. Show less

Flux balance analysis (FBA) and ordinary differential equation models have been instrumental in depicting the metabolic functioning of a cell. Nevertheless, they demonstrate a population’s average behavior (summation of individuals), thereby portraying homogeneity. However, living organisms such as Escherichia coli contain more biochemical reactions than engaging metabolites, making them an underdetermined and degenerate system. This results in a heterogeneous population with varying metabolic… Show more

Flux balance analysis (FBA) and ordinary differential equation models have been instrumental in depicting the metabolic functioning of a cell. Nevertheless, they demonstrate a population’s average behavior (summation of individuals), thereby portraying homogeneity. However, living organisms such as Escherichia coli contain more biochemical reactions than engaging metabolites, making them an underdetermined and degenerate system. This results in a heterogeneous population with varying metabolic patterns. We have formulated a population systems biology model that predicts this degeneracy by emulating a diverse metabolic makeup with unique biochemical signatures. The model mimics the universally accepted experimental view that a subpopulation of bacteria, even under normal growth conditions, renders a unique biochemical state, leading to the synthesis of metabolites and persister progenitors of antibiotic resistance and biofilms. We validate the platform’s predictions by producing commercially important heterologous (isobutanol) and homologous (shikimate) metabolites. The predicted fluxes are tested in vitro resulting in 32- and 42-fold increased product of isobutanol and shikimate, respectively. Moreover, we authenticate the platform by mimicking a bacterial population in the presence of glyphosate, a metabolic pathway inhibitor. Here, we observe a fraction of subsisting persisters despite inhibition, thus affirming the signature of a heterogeneous populace. The platform has multiple uses based on the disposition of the user. Show less

There are three prominent alcohol dehydrogenases superfamilies: Short-chain, Medium-chain, and Iron-containing alcohol dehydrogenases (FeADHs). Many members are valuable catalysts for producing industrially relevant products such as Active pharmaceutical Intermediates, Chiral synthons, Biopolymers, Biofuels and secondary metabolites. However, FeADHs are the least explored enzymes among the superfamilies for commercial tenacities. They portray a conserved structure having a ‘tunnel-like’… Show more

There are three prominent alcohol dehydrogenases superfamilies: Short-chain, Medium-chain, and Iron-containing alcohol dehydrogenases (FeADHs). Many members are valuable catalysts for producing industrially relevant products such as Active pharmaceutical Intermediates, Chiral synthons, Biopolymers, Biofuels and secondary metabolites. However, FeADHs are the least explored enzymes among the superfamilies for commercial tenacities. They portray a conserved structure having a ‘tunnel-like’ cofactor and substrate binding site with particular functions, despite representing high sequence diversity. Interestingly, phylogenetic analysis demarcates enzymes catalyzing distinct native substrates where closely related clades convert similar molecules. Further, homologs from various mesophilic and thermophilic microbes have been explored for designing a solvent and temperature resistant enzyme for industrial purposes. The review explores different Iron-containing alcohol dehydrogenases potential engineering of the enzymes and substrates helpful in manufacturing commercial products. Show less

Despite rigorous disinfection and fumigation, healthcare associated infection (HAI) remains a significant concern in health care settings. We have developed a novel airborne-microbicidal technology ”ZeBox” which clears over 99.999% of airborne microbial load under controlled lab conditions. In this study we evaluate the clinical performance of ZeBox in reducing airborne and surface microbial load. The study was conducted in single bed and multi bed ICU of two hospitals. Airborne and surface… Show more

Despite rigorous disinfection and fumigation, healthcare associated infection (HAI) remains a significant concern in health care settings. We have developed a novel airborne-microbicidal technology ”ZeBox” which clears over 99.999% of airborne microbial load under controlled lab conditions. In this study we evaluate the clinical performance of ZeBox in reducing airborne and surface microbial load. The study was conducted in single bed and multi bed ICU of two hospitals. Airborne and surface microbial loads were sampled pre- and post-deployment of ZeBox at pre-determined sites. Statistical significance of the reduction was determined using Mann-Whitney’s U test. ZeBox brought statistically significant reduction of both airborne and surface bacterial and fungal load. In both hospital ICUs, airborne and surface bacterial load decreased by 90% and 75% on average respectively, providing a low bioburden zone of ∼10-15 feet diameter around the unit. The reduced microbial level was maintained during ZeBox’s operation over several weeks. Most clinical bacterial isolates recovered from one of the hospitals were antibiotic resistant, highlighting ZeBox’s ability to eliminate antimicrobial-resistant bacteria among others. ZeBox significantly reduces airborne and surface microbial burden in clinical settings. It thereby serves an unmet need for reducing the incidence of HAI. Show less

Preventing nosocomial infection is a major unmet need of our times. Existing air decontamination technologies suffer from demerits such as toxicity of exposure, species specificity, noxious gas emission, environment-dependent performance and high power consumption. Here, we present a novel technology called “ZeBox” that transcends the conventional limitations and achieves high microbicidal efficiency. In ZeBox, a non-ionizing electric field extracts naturally charged microbes from flowing air… Show more

Preventing nosocomial infection is a major unmet need of our times. Existing air decontamination technologies suffer from demerits such as toxicity of exposure, species specificity, noxious gas emission, environment-dependent performance and high power consumption. Here, we present a novel technology called “ZeBox” that transcends the conventional limitations and achieves high microbicidal efficiency. In ZeBox, a non-ionizing electric field extracts naturally charged microbes from flowing air and deposits them on engineered microbicidal surfaces. The surface’s three dimensional topography traps the microbes long enough for them to be inactivated. The electric field and chemical surfaces synergistically achieve rapid inactivation of a broad spectrum of microbes. ZeBox achieved near complete kill of airborne microbes in challenge tests (5–9 log reduction) and >90% efficiency in a fully functional stem cell research facility in the presence of humans. Thus, ZeBox fulfills the dire need for a real-time, continuous, safe, trap-and-kill air decontamination technology. Show less

Black gram (Vigna mungo L. Hepper), is an extensively studied food crop which is affected by many abiotic and biotic factors, especially diseases. The yield potential of Black gram is shallow due to lack of genetic variability and biotic stress susceptibility. Core biotic stress factors include mung bean yellow mosaic virus (MYMV), urdbean leaf crinkle virus (UCLV), wilt (Fusarium oxysporum) and powdery mildew (Erysiphe polygoni DC). Although many studies determine resistant varieties to a… Show more

Black gram (Vigna mungo L. Hepper), is an extensively studied food crop which is affected by many abiotic and biotic factors, especially diseases. The yield potential of Black gram is shallow due to lack of genetic variability and biotic stress susceptibility. Core biotic stress factors include mung bean yellow mosaic virus (MYMV), urdbean leaf crinkle virus (UCLV), wilt (Fusarium oxysporum) and powdery mildew (Erysiphe polygoni DC). Although many studies determine resistant varieties to a particular disease, however, it is often complimented by low yield and susceptibility to other diseases. Hence, this study focuses on investigating the genetic relationships among three varieties and nine accessions of black gram having disease resistance to previously described diseases and susceptibility using random amplified polymorphic deoxyribonucleic acid (RAPD) markers. A total of 33 RAPD primers were used for diversity analysis and yielded 206 fragments. Number of amplified fragments ranged from two (OPN-1) to 13 (OPF-1). The highest similarity coefficient was observed between IC-145202 and IC-164118 (0.921), while lowest similarity was between PU-31 and IC-145202 (0.572). The genetic diversity obtained in this study along with disease analysis suggests PU31as a useful variety for the development of markers linked to MYMV, UCLV, wilt and powdery mildew resistance by marker-assisted back cross breeding and facilitates the production of crosses with multiple disease resistance. Show less

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and… Show more

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved Show less