Evaluation of the efficiency of thermostable L-asparaginase from B. licheniformis UDS-5 for acrylamide mitigation during preparation of French fries.

A thermostable L-asparaginase was produced from Bacillus licheniformis UDS-5 (GenBank accession number, OP117154). The production conditions were optimized by the Plackett Burman method, followed by the Box Behnken method, where the enzyme production was enhanced up to fourfold. It secreted L-asparaginase optimally in the medium, pH 7, containing 0.5% (w/v) peptone, 1% (w/v) sodium chloride, 0.15% (w/v) beef extract, 0.15% (w/v) yeast extract, 3% (w/v) L-asparagine at 50 °C for 96 h. The enzyme, with a molecular weight of 85 kDa, was purified by ion exchange chromatography and size exclusion chromatography with better purification fold and percent yield. It displayed optimal catalysis at 70 °C in 20 mM Tris-Cl buffer, pH 8. The purified enzyme also exhibited significant salt tolerance too, making it a suitable candidate for the food application. The L-asparaginase was employed at different doses to evaluate its ability to mitigate acrylamide, while preparing French fries without any prior treatment. The salient attributes of B. licheniformis UDS-5 L-asparaginase, such as greater thermal stability, salt stability and acrylamide reduction in starchy foods, highlights its possible application in the food industry.

Distribution of bacterial community structures and spread of antibiotic resistome at industrially polluted sites of Mini River, Vadodara, Gujarat, India.

The influence of anthropogenic pollution on the distribution of bacterial diversity, antibiotic-resistant bacteria (ARBs), and antibiotic resistance genes (ARGs) was mapped at various geo-tagged sites of Mini River, Vadodara, Gujarat, India. The high-throughput 16S rRNA gene amplicon sequencing analysis revealed a higher relative abundance of Planctomycetota at the polluted sites, compared to the pristine site. Moreover, the relative abundance of Actinobacteriota increased, whereas Chloroflexi decreased in the water samples of polluted sites than the pristine site. The annotation of functional genes in the metagenome samples of Mini River sites indicated the presence of genes involved in the defence mechanisms against bacitracin, aminoglycosides, cephalosporins, chloramphenicol, streptogramin, streptomycin, methicillin, and colicin. The analysis of antibiotic resistome at the polluted sites of Mini River revealed the abundance of sulfonamide, beta-lactam, and aminoglycoside resistance. The presence of pathogens and ARB was significantly higher in water and sediment samples of polluted sites compared to the pristine site. The highest resistance of bacterial populations in the Mini River was recorded against sulfonamide (≥ 7.943 × 103 CFU/mL) and ampicillin (≥ 8.128 × 103 CFU/mL). The real-time PCR-based quantification of ARGs revealed the highest abundance of sulfonamide resistance genes sul1 and sul2 at the polluted sites of the Mini River. Additionally, the antimicrobial resistance genes aac(6')-Ib-Cr and blaTEM were also found abundantly at polluted sites of the Mini River. The findings provide insights into how anthropogenic pollution drives the ARG and ARB distribution in the riverine ecosystem, which may help with the development of antimicrobial resistance mitigation strategies.

Solvent tolerant enzymes in extremophiles: Adaptations and applications.

Non-aqueous enzymology has always drawn attention due to the wide range of unique possibilities in biocatalysis. In general, the enzymes do not or insignificantly catalyze substrate in the presence of solvents. This is due to the interfering interactions of the solvents between enzyme and water molecules at the interface. Therefore, information about solvent-stable enzymes is scarce. Yet, solvent-stable enzymes prove quite valuable in the present day biotechnology. The enzymatic hydrolysis of the substrates in solvents synthesizes commercially valuable products, such as peptides, esters, and other transesterification products. Extremophiles, the most valuable yet not extensively explored candidates, can be an excellent source to investigate this avenue. Due to inherent structural attributes, many extremozymes can catalyze and maintain stability in organic solvents. In the present review, we aim to consolidate information about the solvent-stable enzymes from various extremophilic microorganisms. Further, it would be interesting to learn about the mechanism adapted by these microorganisms to sustain solvent stress. Various approaches to protein engineering are used to enhance catalytic flexibility and stability and broaden biocatalysis's prospects under non-aqueous conditions. It also describes strategies to achieve optimal immobilization with minimum inhibition of the catalysis. The proposed review would significantly aid our understanding of non-aqueous enzymology.

Amylases from thermophilic bacteria: structure and function relationship.

Amylases hydrolyze starch to diverse products including dextrins and progressively smaller polymers of glucose units. Thermally stable amylases account for nearly 25% of the enzyme market. This review highlights the structural attributes of the α-amylases from thermophilic bacteria. Heterologous expression of amylases in suitable hosts is discussed in detail. Further, specific value maximization approaches, such as protein engineering and immobilization of the amylases are discussed in order to improve its suitability for varied applications on a commercial scale. The review also takes into account of the immobilization of the amylases on nanomaterials to increase the stability and reusability of the enzymes. The function-based metagenomics would provide opportunities for searching amylases with novel characteristics. The review is expected to explore novel amylases for future potential applications.

Inhibition of Phospholipase by Orlistat as an Alternate Therapy to Combat Opportunistic Mycosis Caused by C. albicans.

Candida albicans is one of the most important etiological agents causing an opportunistic mycosis, candidiasis. In the past, it was perceived to be associated with immunocompromised patients only. However, it has now been reported with several clinical complications with varying severity. Additionally, increasing incidences of multiple drug resistance associated with the infections have complicated its treatment as well. Therefore, an investigation of alternate therapy, for instance, inhibition of the virulence factors is desperately needed. In the present study, a multidrug-resistant Candida albicans SDL-4 was screened for secretion of the virulence factors: aspartyl proteases and phospholipases. The pathogen secreted phospholipases potentially compared to aspartyl proteases. Therefore, C. albicans SDL-4 phospholipase was purified to homogeneity, characterized, and its inhibition was studied subsequently. It catalysed the substrate, p-nitrophenyl palmitate, optimally in 0.1 M acetate buffer, pH 5, at 37 °C. In the present study, we also aimed to re-purpose orlistat, which is a commercially available anti-obesity drug. Orlistat, at the concentration of 360 µg/ml, could diminish the activity and stability of the candidal virulence factor. Its half-life was reduced in the presence of orlistat at 37 °C. As well, increase in Km and unaltered Vmax indicated that orlistat inhibited phospholipase competitively. The inhibition kinetics was supported by measuring alterations in the secondary structure of the candidal phospholipase upon treatment with orlistat by the circular dichroism spectroscopy and K2D3. Moreover, validation of the study at clinical level may establish orlistat as a supportive treatment to reduce invasiveness and related medical intricacies during candidiasis.

Immobilization of α-amylase on GO-magnetite nanoparticles for the production of high maltose containing syrup.

Robust amylases with stability and catalysis at multitude of extremities are the need of an hour. Enzyme immobilization may prove beneficial at commercial scale to achieve such attributes. In the present study, a commercially available amylase was immobilized on graphene oxide (GO) - magnetite (Fe3O4) nanoparticles through covalent bonding. The structural and morphological characterizations were conducted by XRD, SEM and TEM. Further, FTIR and TGA confirmed the interaction between amylase, GO and nanoparticles. The variables, such as concentrations of GO (1.3 mg), Fe3O4 (58 µg), and amylase (4.5 mg) were optimized by the response surface methodology using central composite design. High loading capacity of 77.58 µg amylase over 1 µg GO-magnetite nanoparticles was achieved under optimum conditions. Biochemically, the pH optimum remained unaltered, i.e., pH 7, whereas, the alkalitolerance was increased by ~20% in relative activities upon immobilization. The half-life of soluble amylase was 13 h, which enhanced to 20 h upon immobilization in 20 mM phosphate buffer, pH 7 at 50 °C. Besides, the thermodynamic parameters supported the stability trends. The immobilized amylase could be used for 11 subsequent cycles. The mentioned attributes and the dextrose equivalent values during the production of high maltose containing syrup highlighted its commercialization.

Biochemical, thermodynamic and structural characteristics of a biotechnologically compatible alkaline protease from a haloalkaliphilic, Nocardiopsis dassonvillei OK-18.

This report describes purification strategies, biochemical properties and thermodynamic analysis of an alkaline serine protease from a marine actinomycete, Nocardiopsis dassonvillei strain OK-18. The solvent tolerance, broad thermal-pH stability, favourable kinetics and thermodynamics suggest stability of the enzymatic reaction. The enzyme was active in the range of pH 7-12 and 37-90 °C, optimally at pH 9 and 70 °C. The deactivation rate constant (Kd), half-life (t½), enthalpy (ΔH*), entropy (ΔS*), activation energy (E) and change in free energy (ΔG*) suggested stability and spontaneity of the reaction. ß-Sheets as revealed by the Circular dichroism (CD) spectroscopy, were the major elements in the secondary structure of the enzyme, while Fourier-transform infrared spectroscopy (FTIR) indicated the presence of amide I and amide II. Based on the liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis, the amino acid sequence had only 38% similarity with other proteases of Nocardiopsis strains, suggesting its novelty. The Ramachandran Plot revealed the location of the amino acid residues in the most favored region. The blood de-staining, gelatin hydrolysis, silver recovery and deproteinization of crab shells established the biotechnological potential of the enzyme.