Laccase: Sustainable production strategies, heterologous expression and potential biotechnological applications.

Laccase is a multicopper oxidase enzyme that target different types of phenols and aromatic amines. The enzyme can be isolated and characterized from microbes, plants and insects. Its ubiquitous nature and delignification ability makes it a valuable tool for research and development. Sustainable production methods are being employed to develop low cost biomanufacturing of the enzyme while achieving high titers. Laccase have significant industrial application ranging from food industry where it can be used for wine stabilization, texture improvement and detection of phenolic compounds in food products, to cosmetics offering benefits such as skin brightening and hair colouring. Dye decolourization/degradation, removal of pharmaceutical products/emerging pollutants and hydrocarbons from wastewater, biobleaching of textile fabrics, biofuel production and delignification of biomass making laccase a promising green biocatalyst. Innovative methods such as using inducers, microbial co-culturing, recombinant DNA technology, protein engineering have pivotal role in developing laccase with tailored properties. Enzyme immobilization using new age compounds including nanoparticles, carbonaceous components, agro-industrial residues enhance activity, stability and reusability. Commercial formulations of laccase have been prepared and readily available for a variety of applications. Certain challenges including production cost, metabolic stress in response to heterologous expression, difficulty in purification needs to be addressed.

Production, purification, characterization, and applications of α-galactosidase from Bacillus flexus JS27 isolated from Manikaran hot springs.

α-Galactosidase hydrolyzes the α-1,6-linkage present at the non-reducing end of the sugars and results in the release of galactosyl residue from oligosaccharides like melibiose, raffinose, stachyose, etc. In the present study we report, α-galactosidase from Bacillus flexus isolated from Manikaran hot springs (India). Maximum enzyme production was obtained in guar gum and soybean meal after 72 h at 150 rpm. While, the temperature/pH of production was optimized at 50 °C and 7.0, respectively. Isoenzymes (α-gal I and II) were obtained and characterized based on temperature/pH optima along with their stability profile. JS27 α-Gal II was purified with a final purification fold of 11.54. Native and SDS-PAGE were used to determine the molecular weight of the enzyme as 86 and 41 kDa, respectively, indicating its homodimeric form. JS27 α-Gal II showed optimum enzyme activity at 55 °C and pH 7 (10 min). The enzyme displayed Km value of 2.3809 mM and Vmax of 2.0 × 104 µmol/min/ml with pNPG as substrate. JS27 α-Gal II demonstrated substrate hydrolysis and simultaneous formation of transgalactosylation products (α-GOS) with numerous substrates (sugar/sugar alcohols, oligosaccharides, and complex carbohydrates) which were verified by TLC and HPLC analysis. α-GOS are significant functional food ingredients and can be explored as prebiotics.

Insights on sustainable approaches for production and applications of value added products.

The increasing demand for the development of sustainable strategies to utilize and process agro-industrial residues paves new paths for exploring innovative approaches in this area. Biotechnology based microbial transformations provide efficient, low cost and sustainable approaches for the production of value added products. The use of organic rich residues opens new avenues for the production of enzymes, pigments, biofuels, bioactive compounds, biopolymers etc. with vast industrial and therapeutic applications. Innovative technologies like strain improvement, enzyme immobilization, genome editing, morphological engineering, ultrasound/supercritical fluid/pulse electric field extraction, etc. can be employed. These will be helpful in achieving significant improvement in qualitative and quantitative parameters of the finished products. The global trend for the valorisation of biowaste has boosted the commercialization of these products which has transformed the markets by providing new investment opportunities. The upstream processing of raw materials using microbes poses a limitation in terms of product development and recovery which can be overcome by modifying the bioreactor design, physiological parameters or employing alternate technologies which will be discussed in this review. The other problems related to the processes include product stability, industrial applicability and cost competitiveness which needs to be addressed. This review comprehensively discusses the recent progress, avenues and challenges in the approaches aimed at valorisation of agro-industrial wastes along with possible opportunities in the bioeconomy.

Microbial production and biotechnological applications of α-galactosidase.

α-Galactosidase, (E.C. 3.2.1.22) is an exoglycosidase that target galactooligosaccharides such as raffinose, melibiose, stachyose and branched polysaccharides like galactomannans and galacto-glucomannans by catalysing the hydrolysis of α-1,6 linked terminal galactose residues. The enzyme has been isolated and characterized from microbial, plant and animal sources. This ubiquitous enzyme possesses physiological significance and immense industrial potential. Optimization of the growth conditions and efficient purification strategies can lead to a significant increase in the enzyme production. To boost commercial productivity, cloning of novel α-galactosidase genes and their heterologous expression in suitable host has gained popularity. Enzyme immobilization leads to its greater reutilization, superior thermostability, pH tolerance and increased activity. The enzyme is well explored in food industry in the removal of raffinose family oligosaccharides (RFOs) in soymilk and sugar crystallization process. It also improves animal feed quality and biomass processing. Applications of the enzyme is in the area of biomedicine includes therapeutic advances in treatment of Fabry disease, blood group conversion and removal of α-gal type immunogenic epitopes in xenotransplantation. With considerable biotechnological applications, this enzyme has been vastly commercialized and holds greater future prospects.

Oral microbiome and health.

The oral microbiome is diverse in its composition due to continuous contact of oral cavity with the external environment. Temperatures, diet, pH, feeding habits are important factors that contribute in the establishment of oral microbiome. Both culture dependent and culture independent approaches have been employed in the analysis of oral microbiome. Gene-based methods like PCR amplification techniques, random amplicon cloning, PCR-RELP, T-RELP, DGGE and DNA microarray analysis have been applied to increase oral microbiome related knowledge. Studies revealed that microbes from the phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Fusobacteria, Neisseria, TM7 predominately inhabits the oral cavity. Culture-independent molecular techniques revealed the presence of genera Megasphaera, Parvimonas and Desulfobulbus in periodontal disease. Bacteria, fungi and protozoa colonize themselves on various surfaces in oral cavity. Microbial biofilms are formed on the buccal mucosa, dorsum of the tongue, tooth surfaces and gingival sulcus. Various studies demonstrate relationship between unbalanced microflora and development of diseases like tooth caries, periodontal diseases, type 2 diabetes, circulatory system related diseases etc. Transcriptome-based remodelling of microbial metabolism in health and disease associated states has been well reported. Human diets and habitat can trigger virus activation and influence phage members of oral microbiome. As it is said, "Mouth, is the gateway to the total body wellness, thus oral microbiome influences overall health of an individual".

Metagenomic analysis of bacterial and archaeal assemblages in the soil-mousse surrounding a geothermal spring.

The soil-mousse surrounding a geothermal spring was analyzed for bacterial and archaeal diversity using 16S rRNA gene amplicon metagenomic sequencing which revealed the presence of 18 bacterial phyla distributed across 109 families and 219 genera. Firmicutes, Actinobacteria, and the Deinococcus-Thermus group were the predominant bacterial assemblages with Crenarchaeota and Thaumarchaeota as the main archaeal assemblages in this largely understudied geothermal habitat. Several metagenome sequences remained taxonomically unassigned suggesting the presence of a repertoire of hitherto undescribed microbes in this geothermal soil-mousse econiche.

Metagenomic evaluation of bacterial and archaeal diversity in the geothermal hot springs of manikaran, India.

Bacterial and archaeal diversity in geothermal spring water were investigated using 16S rRNA gene amplicon metagenomic sequencing. This revealed the dominance of Firmicutes, Aquificae, and the Deinococcus-Thermus group in this thermophilic environment. A number of sequences remained taxonomically unresolved, indicating the presence of potentially novel microbes in this unique habitat.

Applications of ß-gal-III isozyme from Bacillus coagulans RCS3, in lactose hydrolysis.

Bacillus coagulans RCS3 isolated from hot water springs secreted five isozymes i.e. ß-gal I-V of ß-galactosidase. ß-gal III isozyme was purified using DEAE cellulose and Sephadex G 100 column chromatography. Its molecular weight characterization showed a single band at 315kD in Native PAGE, while two subunits of 50.1 and 53.7 kD in SDS PAGE. ß-Gal III had pH optima in the range of 6-7 and temperature optima at 65°C. It preferred nitro-aryl-ß-d-galactoside as substrate having K(m) of 4.16 mM with ONPG. More than 85% and 80% hydrolysis of lactose (1-5%, w/v) was recorded within 48 h of incubation at 55°C and 50°C respectively and pH range of 6-7. About 78-86% hydrolysis of lactose in various brands of standardized milk was recorded at incubation temperature of 50°C. These results marked the applications of ß-gal III in processing of milk/whey industry.

Competition between COX-2 inhibitors and some other drugs for binding sites on human serum albumin.

Competitive binding of six COX-2 inhibitors (celecoxib, valdecoxib, etoricoxib, parecoxib sodium, meloxicam and nimesulide) in the presence of three categories of drugs: an antidiabetic (gliclazide), antipsychotic (chlorpromazine) and antibiotic (ceftriaxone sodium) were studied by fluorescence spectroscopy. Data are expressed in terms of the percentage of drug bound in the absence and presence of competing drug and change in the percentage of free drug due to competitive binding. The results are discussed in terms of three cases: decrease in the binding of the parent drug; increase in binding; and no effect by the presence of the competing drug. The relative binding affinity of the parent drug and the displacing compound for human serum albumin (HSA) is not the only factor involved in the competitive mechanism. Binding behaviour of individual drugs was analysed, and explanations for different cases based on the competitive displacement, non-competitive interference, conformational changes in the HSA molecule, and independent binding are presented.

Complexation of cox-2 inhibitors with bovine serum albumin: interaction mechanism.

Mechanism of interaction of six cox-2 inhibitors--celecoxib, valdecoxib, etoricoxib, parecoxib sodium, meloxicam and nimesulide--with bovine serum albumin (BSA) was studied using fluorescence spectroscopic technique. Results were discussed in terms of the binding parameters, thermodynamics of the binding process, the nature of forces involved in the interaction and the fluorescence quenching mechanism involved. Association constants were of the order of 10(4)-10(5) for various drugs. Binding affinity varied with the nature of drug. Nature of forces involved in the interaction could be predicted from the thermodynamic parameters for the binding. Meloxicam and nimesulide shared common sites with hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS) on BSA molecule. Stern-Volmer analysis of the quenching data indicated that both tryptophan residues of BSA are fully accessible to the drugs and predominantly static quenching mechanism is involved in the interaction.

Quantitative correlation between theoretical molecular descriptors and drug-HSA binding affinities for various cox-2 inhibitors.

In this study, quantitative structure-protein binding relationships have been derived to predict the binding affinities of cox-2 inhibitor drugs to HSA from the structure of drug molecules. Drug-HSA binding affinities were determined using fluorescence spectroscopic technique. For the calculation of theoretical molecular descriptors and statistical treatment of data dragon, codessa and spss software were used. In the quantitative structure-protein binding model, one-parameter correlations were statistically not very sound. Quadratic equations gave better fit of the data in some cases. Two-parameter correlations could explain 94-98% of the variance in the data. Steric, geometric and topological descriptors outweighed all other parameters in the developed quantitative structure-protein binding relationships. There was some contribution from electrostatic descriptors as well, but the role of hydrophobic binding was completely ruled out in the protein binding of coxibs. The quantitative correlations derived in this paper are useful in predicting the protein binding of coxibs and may help in the design of cox-2 inhibitors with appropriate HSA binding properties.

Interaction of non-steroidal anti-inflammatory drugs, etoricoxib and parecoxib sodium, with human serum albumin studied by fluorescence spectroscopy.

The mechanism of interaction of the non-steroidal anti-inflammatory drugs, etoricoxib and parecoxib sodium, with human serum albumin (HSA) was studied using fluorescence spectroscopy. There was only one class of binding site with association constants of the order of 10(4). Thermodynamic parameters suggest that van der Waals and hydrogen bonding interactions in the case of etoricoxib, and electrostatic and hydrogen bonding interactions in the case of parecoxib sodium, are predominantly involved in the binding. Studies in the presence of the hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS), showed that hydrophobic interactions are not involved in the binding of these drugs to HSA. Displacement studies using the site-specific probe, dansylsarcosine piperidinium salt (DSS), showed that the drugs are bound at site II on the HSA molecule. However, etoricoxib and parecoxib sodium are bound at different regions within site II. Increase of pH and the presence of salt caused significant changes in the association constants and the concentration of free pharmacologically active drug. Stern-Volmer analysis of the binding data indicated that the tryptophan residues of albumin are not fully accessible to anionic parecoxib sodium and a predominantly static quenching mechanism is operative in each case.

Reversible binding of celecoxib and valdecoxib with human serum albumin using fluorescence spectroscopic technique.

Mechanism of interaction of non-steroidal anti-inflammatory drugs, celecoxib and valdecoxib with human serum albumin has been studied using fluorescence spectroscopic technique. There was only one high affinity site on serum albumin for both the drugs with association constants of the order of 10(4) in the case of celecoxib and 10(5) in the case of valdecoxib. Thermodynamic parameters for the binding indicated that hydrogen bonding interactions are predominantly involved in the binding of these drugs to human serum albumin. Binding studies in the presence of hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS) suggested that the mode of interaction of drugs and ANS with HSA is different and hydrophobic interactions are not primarily involved in the binding. Studies carried out in the presence of site-specific probe showed that drugs are bound at site II and phenolic oxygen of (411)Tyr is involved in binding. Stern-Volmer analysis of the quenching data indicated that predominantly static quenching mechanism is operative and the tryptophan residues of albumin are fully accessible to celecoxib and only partially accessible to valdecoxib. The presence of salt caused a decrease in the association constant and significant increase in the concentration of free drug.

Mechanism of interaction of the non-steroidal antiinflammatory drugs meloxicam and nimesulide with serum albumin.

The mechanism of interaction of the non-steroidal antiinflammatory drugs meloxicam and nimesulide with human and bovine serum albumin has been studied using fluorescence spectroscopy. There was only one high affinity site on serum albumin for both the drugs with association constants of the order of 10(5). Negative enthalpy (DeltaH(0)) and positive entropy (DeltaS(0)) values in the case of both meloxicam and nimesulide showed that both hydrogen bonding and hydrophobic interactions play a role in the binding of these drugs. Binding studies in the presence of the hydrophobic probe 1-anilinonaphthalene-8-sulfonate (ANS) showed that the binding of meloxicam and nimesulide to serum albumin involves predominantly hydrophobic interactions. Stern-Volmer analysis of the quenching data showed that quenching is highly efficient and that the tryptophan residues in hydrophobic regions of the proteins are fully exposed to the drugs. Thus these drugs are bound to albumin by hydrophobic interactions as well as hydrogen bonding at a site, which is close to the tryptophan residues. An increase of the pH and ionic strength caused an increase in the concentration of free drug, although the effect was not very significant.