Anju Chadha

Biocatalysis, one of the most ecologically friendly methods for synthesising chiral synthons, has emerged as a desirable process for manufacturing active pharmaceutical ingredients and agrochemicals, most of which contain one or more chiral amine moieties. Compared with the traditional biocatalytic processes for the synthesis of chiral amines involving lipases or transaminases, enzymatic imine reduction is a more promising approach. The single-step enzymatic reduction of prochiral imines to the corresponding amines can yield 100% of the required enantiomer without any by-products. Furthermore, the reduction of imines generated in situ through the condensation of amines and carbonyl compounds can be used to synthesise almost any primary, secondary, or tertiary amine. In the past decade, several imine-reducing enzyme families, such as Streptomyces imine reductases (IReds), native amine dehydrogenases and engineered leucine/phenylalanine dehydrogenases and opine dehydrogenases, have been explored. The ornithine cyclodeaminase/µ-crystallin (OCD/CRYM) superfamily consists of proteins capable of imine reduction, which have been relatively unexplored regarding the synthesis of chiral amines. The proteins in this family are ubiquitously distributed in all three domains of life and catalyse diverse and unique reactions. This review is aimed at summarising current knowledge on this superfamily and exploring its potential in biocatalysis. After a brief discussion of their discovery, the known members of the OCD/CRYM superfamily are broadly classified on the basis of the reactions that they catalyse, and their biochemical characteristics and biological roles are described in detail. This is followed by a discussion of the overall structure, active sites and proposed reaction mechanisms, with common themes drawn among members. Finally, the applications of these enzymes, particularly in the synthesis of various chiral synthons, are summarised.

Biocatalytic reduction of alkyl 2-oxopropanoates were carried out by utilizing the whole cells of Candida parapsilosis ATCC 7330 to form the optically enriched alkyl 2-hydroxypropanoates with good enantiomeric excess (ee) (≤91%) and isolated yields (≤68%). Enantiomerically enriched (S)-ethyl 3-bromo-2-hydroxypropanoate thus synthesized by biocatalytic reduction of ethyl 3-bromo-2-oxopropanoate is presented in this study for the first time in water under ambient reaction conditions in a reaction time of 4 h which is considerably less than earlier reported procedures.

Measurement in liquid media is a major challenge in real-time detection using resonant cantilevers. This is addressed in the present study by fabricating sub-micron thick cantilevers followed by functionalization for biomolecule detection. The fabricated cantilever resonator beams of thickness 165 nm were used for measurements in two systems: (i) human immunoglobulin (HIgG) as the antibody on the cantilever sensing mouse immunoglobulin (MIgG) as corresponding antigen, and (ii) detection of triglyceride (TG) based on the enzymatic hydrolysis with lipase, using tributyrin as a model. In both cases, the beams were functionalized for covalent bonding of the protein receptor. The label-free detection was carried out by measuring the shift in resonance frequency at higher modes, using a laser Doppler vibrometer in liquid and in air. The calibration showed a linear correlation between the bioanalyte concentration and change in the resonance frequency. Notably, detection of antigen mass as low as 434 ± 59fg and triglyceride concentration in the nM range with limit of detection as 7 nM in liquid interface was achieved, greatly improving the sensitivity of bioanalyte detection in liquid samples. Although frequency-based methods are highly sensitive, the issues with measurement liquid medium limit their application. In the present report, these issues were addressed by fabricating sub-micron thick cantilever beam, choosing an appropriate functionalization method without affecting the sensitivity, and measurement at higher modes. These have resulted in circumventing issues like damping and hydrodynamic loading thus improving its potential as real-time sensor.

Understanding the biosynthetic mechanism of gold nanoparticle formation is the key to controlling the size, dispersity, and morphology of the nanoparticles. Reduction of gold (III) to gold (0) in cell-free extracts of Candida parapsilosis ATCC 7330 is not only enzymatic, as confirmed by experiments with heat denatured extracts. In addition to proteins, cellular reducing equivalents also contribute to the formation of gold nanoparticles in a concentration-dependent manner. Characterization of the bio-synthesized gold nanoparticles using X-ray photoelectron spectroscopy and elemental analysis revealed that nanoparticles are stabilized by proteins. The importance of protein three-dimensional structure in producing stable gold nanoparticles is also addressed. Making free thiol groups (–SH) unavailable by derivatizing them in protein extracts resulted in monodisperse gold nanoparticles implying that free –SH increase aggregation and emphasize this as a possible strategy to produce monodisperse gold nanoparticles in biological extracts which is otherwise difficult.

In this study, a pH-induced self-assembly-based method has been developed to form silk fibroin nanoparticles (SFN-2) with a higher drug loading capacity (21.0 ± 2.1%) and cellular uptake than that of silk fibroin particles produced by a conventional desolvation method (SFN-1). Using the self-assembly method, rifampicin-encapsulated silk fibroin nanoparticles (R-SFN-2) were prepared with a size of 165 ± 38 nm at an optimum pH of 3.8. In silico analysis reveals that at acidic pH, the amino acid side chain charge neutralization of acidic residues, especially GLU64, promotes the formation of additional favorable interactions between the silk fibroin and the drug. The SFN-2 also possess a good aerosol property with a mass median aerodynamic diameter of 3.82 ± 0.71 μm and fine particle fraction of 64.0 ± 1.4%. These SFN-2 particles were selectively endocytosed by macrophages through clathrin- and caveolae-mediated endocytosis with a higher uptake efficiency (66.2 ± 2.1%) and were found to exhibit a sustained drug release in the presence of macrophage intracellular lysates. The cytokine and biomarker expression analyses revealed that SFN-2 could exhibit an immunomodulatory effect by polarizing the macrophages to an initial M1 phase and later M2 phase. Further, R-SFN-2 also exhibited an enhanced and sustained intracellular antibacterial activity against Mycobacterium smegmatis-infected macrophages than free rifampicin. Thus, the self-assembled silk fibroin particles with immunomodulatory action combined with a good aerosol and intracellular drug release property can be an attractive choice as a carrier for developing pulmonary drug delivery systems.

Nanoparticles of noble metals such as gold, silver, and platinum are useful for important applications in catalysis and biomedical science. Biological methods of nanofabrication include the use of easily available plants, microbes, and enzymes. This chapter presents an overview of microbial methods of preparing gold nanoparticles and the use of these gold nanoparticles in catalytic applications. Mainly, the catalytic performance of biosynthesized gold nanoparticles toward protecting the environment from aromatic toxic pollutants and textile dyes is discussed with relevant examples. The method of preparation of supported gold nanoparticles catalysts using conventional chemical and microbial methods for the hydrogenation/reduction of nitrobenzene to aniline is presented. Toward sustainable catalysis, the application of bio-supported nanoparticles (gold nanoparticles, palladium nanoparticles, and the combination), as heterogeneous catalysts for various chemical reactions like oxidation, reduction, and coupling, to yield value-added fine chemicals and intermediates under environmentally benign conditions is elaborated.

Biocatalytic reduction catalysed by alcohol dehydrogenases is a valuable tool for asymmetric synthesis of chiral alcohols. This study reports the broad substrate specificity of NADH dependent (S) - specific alcohol dehydrogenase (S-ADH) purified from Candida parapsilosis ATCC 7330. The substrates for this enzyme include aliphatic and aromatic ketones, cyclic and diketones, aldehydes, ketoesters, primary and secondary alcohols. The kinetic studies of different substrates indicate that ketones and secondary alcohols are the most preferred substrates of S-ADH with highest catalytic efficiencies reported for reduction of acetone (4153 s−1mM−1) and oxidation of 2-propanol (1358 s−1mM−1). The double reciprocal plots obtained for varied concentrations of acetophenone (0.2–16 mM) at a fixed concentration of NADH (0.05, 0.1 and 0.2 mM) and with varied concentrations of NADH (0.01–0.2 mM) at a fixed concentration of acetophenone (1, 4, 8 and 16 mM) showed intersecting lines indicating sequential kinetic mechanism. S-ADH follows Prelog's stereopreference in reducing prochiral carbonyl substrates to yield (S)-alcohols with >99% enantiomeric excess using a simple coupled substrate approach for cofactor recycling.

Docosahexaenoic acid (DHA) is an essential dietary supplement that is highly coveted due to its benefits for human health. Extensive research has been conducted for the sustainable commercial production of DHA by various strains in thraustochytrid family due to the accumulation of higher lipid content in the cells. The current study is focused on improving DHA production by investigating various key enzymes like glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME), and ATP-citrate lyase (ACL) involved in DHA production using Thraustochytrium sp. T01. The growth of this strain was compared in batch and fed-batch mode. The fed-batch yielded better Dry cell weight (40 g L−1), lipid (27.75 g L−1 or 693 mg g−1 of DCW), and DHA contents (11.10 g L−1 or 277 mg g−1 of DCW). G6PDH activity increased 4-fold during the glucose fed-batch, but ME and ACL did not increase significantly. Furthermore, a study was conducted to determine the effects of organic acids (pyruvate and malate) on key enzyme activities. The addition of pyruvate increased the lipid content by 1.35-fold, and ACL activity by 10-fold as compared with control (without added organic acids). Malate addition into the culture media increased DHA content 1.4-fold, and ME activity increased 14-fold compared with control.

Human alcohol dehydrogenases, specifically ADH1 oxidize primary alcohols to aldehydes in the skin. These aldehydes act as haptens, leading to sensitization. Inhibition of these ADH enzymes may help in combating skin allergies. In the present study, the recombinantly purified stereospecific enzyme from Candida parapsilosis ATCC 7330, Candida parapsilosis Carbonyl Reductase (CpCR) has 23.31 % sequence similarity with human alcohol dehydrogenase ADH1B1. These two enzymes have perfectly overlapping cofactor and zinc binding domains. CpCR was found to oxidize primary alcohols to their corresponding aldehydes. Oxidation of primary alcohols by CpCR was further optimized with cinnamyl alcohol as the model substrate that initially showed a conversion of 56 % ± 0.25, and upon optimization increased to 98.2 % ± 0.23. An increase of 20–50 % in the conversion rate has been observed for various primary alcohols under the optimized reaction conditions. A simple and efficient model was designed for the screening of compounds that inhibit CpCR with the possibility of mitigating the action of skin allergens by inhibiting ADH1. p-Nitrophenylglyoxal (PNG) was found to be a good inhibitor for CpCR which showed inhibitory activity at very low concentration (IC50,100 mM ± 1.27) as compared to the standard inhibitor 4-methyl pyrazole (4MP) (IC50, 400 mM ± 2.05).

Two representative substrates, a ketone 2-hydroxyacetophenone (2HAP) and an α-ketoester ethyl-2-oxo-4-phenylbutanoate (EOPB) are reduced by a purified (S)-specific carbonyl reductase (SRED) from Candida parapsilosis ATCC 7330 at different rates and kinetic behaviors (hyperbolic vs. sigmoidal), and the resulting alcohols show difference in optical purities. Further, an extensive study employing quantum mechanics/molecular mechanics methods to understand the bases for these differences reveals different reaction coordinates and energy profiles along the reduction paths of 2HAP and EOPB. These complement the experimental observations in which (a) the catalytic efficiency of SRED towards EOPB > 2HAP and (b) the optical purity of the (S)-alcohol is >99% for the reduction of 2HAP while it is ∼70% for EOPB. A plausible explanation for the different reduction mechanisms for 2HAP and EOPB at the atomic level is presented. This journal is