An insight into microbial sources, classification, and industrial applications of xylanases: A rapid review.

Endo 1,4-ß-d-xylanases (EC3.2.1.8) are one of the key lignocellulose hydrolyzing enzymes. Xylan, which is present in copious amounts on earth, forms the primary substrate of endo-xylanases, which can unchain the constituent monosaccharides linked via ß-1,4-glycosidic bonds from the xylan backbone. Researchers have shown keen interest in the xylanases belonging to glycoside hydrolase families 10 and 11, whereas those placed in other glycoside hydrolase families are yet to be investigated. Various microbes such as bacteria and fungi harbor these enzymes for the metabolism of their lignocellulose fibers. These microbes can be used as miniature biofactories of xylanase enzymes for a plethora of environmentally benign applications in pulp and paper industry, biofuel production, and for improving the quality of food in bread baking and fruit juice industry. This review highlights the potential of microbes in production of xylanase for industrial biotechnology.

Pectinases as promising green biocatalysts having broad-spectrum applications: Recent trends, scope, and relevance.

Pectinases are a collection of multiple enzymes that have a common substrate, that is, pectin. They can act on different parts of pectin due to the structural heterogeneity of pectin. Therefore, they have been placed in different groups, such as protopectinases, polygalacturonases, polymethylesterases, pectin lyases, and pectate lyases. They are naturally present both in multicellular organisms such as higher plants and in unicellular organisms such as microbes. In past decade, it has been witnessed that chemical and mechanical methods employed in industrial processes have led to environmental hazards and serious health disorders, thus increasing the search for eco-friendly approaches with minimal health risks. Hence, microbial enzymes have been extensively used as safer alternative for these environmentally unsafe methods. Among these microbial enzymes, pectinases hold great significance and is one of the principal enzymes that have been used commercially. It is predominantly used as a green biocatalyst for fruit, fiber, oil, textile, beverage, pulp, and paper industry. Thus, this review focuses on the structure of pectin, microbial sources of pectin, and principle industrial applications of pectinases.

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.

GSTT1 and GSTM1 gene polymorphisms as major risk factors for asthma in a North Indian population.

BACKGROUND: According to the National Family Health Survey, asthma is one of the leading diseases in India. In order to understand the complexity of asthma, the susceptibility genes need to be targeted for their association. Glutathione S-transferases play a major role in the detoxification of metabolites of oxidative stress resulting in inflammation and asthma. In the present study, the hypothesis that GSTT1 and GSTM1 gene polymorphisms are associated with asthma was examined. METHODS: This is the first study to investigate the role of GSTT1 and GSTM1 gene polymorphisms in asthma pathogenesis in a North Indian population. A total of 824 subjects were recruited, of which 410 were asthma patients, including 323 patients suffering from allergic rhinitis. The other 414 recruits were healthy controls from regions of North India. Multiplex PCR was used for genotyping the GSTT1 and GSTM1 gene polymorphisms. RESULTS: The GSTT1 null allele was more prevalent in asthma patients (40 %) than in the control subjects (13.3 %), which yielded a nearly fourfold risk towards asthma with odds ratio (OR) (95 % CI) = 4.35 (3.04-6.24), χ(2) = 75.34, and p = 0.000. The GSTM1 polymorphism also revealed a greater prevalence of the GSTM1 null allele in asthma patients (46.6 %) than in controls (39.4 %). Statistical analysis yielded a marginal risk toward asthma with OR (95 % CI) = 1.34 (1.01-1.79), χ(2) = 4.37, and p = 0.036. CONCLUSIONS: Polymorphisms as a result of deletions in the GSTT1 and GSTM1 genes confer an increased risk towards asthma thereby suggesting the protective role of these functional genes in the development of the disease.

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".

PI3K/Akt/mTOR Intracellular Pathway and Breast Cancer: Factors, Mechanism and Regulation.

BACKGROUND: The most recurrent and considered second most frequent cause of cancer-related deaths worldwide in women is the breast cancer. The key to diagnosis is early prediction and a curable stage but still treatment remains a great clinical challenge. Origin of the Problem: A number of studies have been carried out for the treatment of breast cancer which includes the targeted therapies and increased survival rates in women. Essential PI3K/mTOR signaling pathway activation has been observed in most breast cancers. The cell growth and tumor development in such cases involve phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) complex intracellular pathway. HYPOTHESIS: Through preclinical and clinical trials, it has been observed that there are a number of other inhibitors of PI3K/Akt/mTOR pathway, which either alone or in combination with cytotoxic agents can be used for endocrine therapies. CONCLUSION: Structure and regulation/deregulation of mTOR provides a greater insight into the action mechanism. Also, through this review, one could easily scan first and second generation inhibitors for PI3K/Akt/mTOR pathway besides targeted therapies for breast cancer and the precise role of mTOR.

Multiple Pharmacological Properties of a Novel Parthenin Analog P16 as Evident by its Cytostatic and Antiangiogenic Potential Against Pancreatic Adenocarcinoma PANC -1 Cells.

Pancreatic ductal adenocarcinoma (PDA) remains one of the deadliest types of cancers. Median survival rate is very poor with the currently available chemotherapeutical regimens. Therefore, discovery of new antineoplastic agents against PDA is one of the focused areas of contemporary research. The present study was undertaken to explore the antitumour activity of a potent parthenin analog P16. Among PANC-1, Mia PaCa-2 and AsPC-1 pancreatic cancer cells, PANC-1 showed highest sensitivity to P16 with an IC50 value of 3.4 µM. Time dependent cell cycle studies revealed that P16 suppressed the growth of PANC-1 cells by arresting the progression through the cell cycle in G2/M phase via downregulation of cyclin B1 and cyclin A. However, P16 did not alter the expressions of CDK-1 and CDC25C in PANC-1 cells. The P16 induced cell cycle arrest, which consequently, led to induction of apoptosis, which was accompanied by activation of caspase-9 and -3. Interestingly, PANC-1 cells displayed increasing loss of mitochondrial potential, which seemed to be correlated to the activation of caspase-3. Additionally, P16 was also able to down-regulate the cell migration in PANC-1 cells. Furthermore, P16 treatment of hypoxic PANC-1 cells strongly suppressed the expression of proangiogenic factors VEGFR-2, HIF1α and HIF1ß. Antiangiogenic ability of P16 was also reflected in the human umbilical vascular endothelial cells (HUVECs), where it effectively suppressed the migration and inhibited the formation of the tube in a matrigel based assay. Therefore, cytostatic and antiangiogenic properties of P16 against pancreatic adenocarcinoma cells make it a suitable candidate for further investigation.

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.

Antiproliferative potential of a novel parthenin analog P16 as evident by apoptosis accompanied by down-regulation of PI3K/AKT and ERK pathways in human acute lymphoblastic leukemia MOLT-4 cells.

Leukemia is one of the deadliest types of cancer. Lack of effective treatment strategies has resulted in an extensive quest for new therapeutic molecules against it. This study explores the molecular mechanism of anticancer activity of P16, a semisynthetic analog of parthenin, against the human acute lymphoblastic leukemia MOLT-4 cells. P16 displayed antiproliferative activity in different cancer cell lines; however, MOLT-4 cells showed highest sensitivity for P16 with IC50 value of 0.6µM. Further studies revealed that P16 induced cell death by apoptosis. It caused mitochondrial stress, which was mediated by the translocation of Bax from cytosol to mitochondria and release of cytochrome c into the cytosol and consequent activation of caspase-9. However, P16 was also able to activate caspase-8, thus involving both extrinsic and intrinsic pathways of apoptosis. Further, activation of caspase-3 led to cleavage of its target proteins PARP-1 and ICAD, which resulted in apoptotic DNA damage. P16 induced apoptosis was accompanied by the down-regulation of important leukemic cell survival proteins like pAKT (S473), pAKT (T308), pP70S6K, pCRAF, and pERK1/2. However, inhibition of caspases by Z-VAD-FMK reversed the down-regulatory effect of P16 on pAKT (S473) and pP70S6K, as evident by the cell viability assay and flow cytometric analysis but this inhibition did not completely reverse the antiproliferative effect of P16, thereby indicating the role of additional factors apart from caspases in P16 induced apoptosis in MOLT-4 cells. Owing to its antiproliferative potential against leukemia cells, P16 can further be explored as an effective therapeutics against leukemia.

Association of the wild-type A/A genotype of MBL2 codon 54 with asthma in a North Indian population.

BACKGROUND: High serum MBL level as well as polymorphisms in the mannose-binding lectin 2 (MBL2) gene resulting in MBL deficiency are involved in the mechanism of a number of non-infectious diseases such as asthma, conferring either risk or protection in different population studies. MBL being the first reactant of the MBL pathway is also a major determinant of the fate of the anaphylatoxins such as C3a and C5a, which are also pro-inflammatory mediators. The MBL2 gene polymorphisms thus control the serum levels of MBL as well as C3a and C5a. OBJECTIVE: This is the first case-control study conducted in India, investigating the role of MBL2 codon 54 A/B polymorphism in asthma pathogenesis. METHODS: A case-control study was performed with a total of 992 adult subjects, including 410 adult asthmatics and 582 healthy controls from regions of North India. The MBL2 codon 54 A/B polymorphism was genotyped by PCR-RFLP. RESULTS: Statistical analysis for the codon 54 polymorphism revealed that the wild (A) allele was significantly associated with asthma with OR=1.9, 95% CI (1.4-2.4), and p< 0.001. CONCLUSION: The MBL2 codon 54 A/B polymorphism is significantly associated with asthma and its phenotypic traits as the wild (A/A) genotype confers a significant risk towards the disease in the studied North Indian population.

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.

No association of PTGDR -441C/T polymorphism with asthma in a North Indian population.

BACKGROUND: Asthma is the most prevalent disease in India according to the national survey conducted by NFHS 2 in 1998-399. Prostaglandin D2 (PGD2) is a bronchoconstriction inducing metabolite of arachidonic acid in the mast cells, which is produced on exposure to allergens and acts as a ligand for the Prostaglandin D2 Receptor (PTGDR). Polymorphisms in the PTGDR gene have been suggested to be involved in the mechanism of asthma. OBJECTIVE: This is the first study conducted in India, investigating the role of PTGDR -441C/T} promoter polymorphism in asthma pathogenesis. METHODS: A case-control study was performed with a total of 992 subjects, including 410 adult asthmatics and 582 healthy controls from regions of North India. The PTGDR -441C/T polymorphism was genotyped by Tetra-Primer Amplification Refractory Mutation System Polymerase Chain Reaction (Tetra-Primer ARMS PCR). RESULTS: Statistical analysis of the results between asthma cases and controls for the PTGDR −441C/T polymorphism showed Chi² (χ²) = 0.29, OR = 0.95, 95% CI (0.70-1.15) and p = 0.599. Neither the genotypic nor the allelic frequencies observed for the PTGDR −441C/T polymorphism, were significantly associated with asthma or asthma phenotypes. CONCLUSIONS: The PTGDR -441C/T polymorphism is not associated with asthma or its phenotypes in the studied North Indian population.

Metagenomics: Concept, methodology, ecological inference and recent advances.

Microorganisms constitute two third of the Earth's biological diversity. As many as 99% of the microorganisms present in certain environments cannot be cultured by standard techniques. Culture-independent methods are required to understand the genetic diversity, population structure and ecological roles of the majority of organisms. Metagenomics is the genomic analysis of microorganisms by direct extraction and cloning of DNA from their natural environment. Protocols have been developed to capture unexplored microbial diversity to overcome the existing barriers in estimation of diversity. New screening methods have been designed to select specific functional genes within metagenomic libraries to detect novel biocatalysts as well as bioactive molecules applicable to mankind. To study the complete gene or operon clusters, various vectors including cosmid, fosmid or bacterial artificial chromosomes are being developed. Bioinformatics tools and databases have added much to the study of microbial diversity. This review describes the various methodologies and tools developed to understand the biology of uncultured microbes including bacteria, archaea and viruses through metagenomic analysis.

Purification and characterisation of alkaline cellulase produced by a novel isolate, Bacillus sphaericus JS1.

A novel strain of Bacillus sphaericus JS1 producing thermostable alkaline carboxymethyl cellulase (CMCase; endo-1,4-beta-glucanase, E.C. 3.2.1.4) was isolated from soil using Horikoshi medium at pH 9.5. CMCase was purified 192-fold by (NH(4))(2)SO(4) precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 23%. The CMCase is a multimeric protein with a molecular weight estimated by native-PAGE of 183 kDa. Using SDS-PAGE a single band is found at 42 kDa. This suggests presence of four homogeneous polypeptides, which would differentiate this enzyme from other known alkaline cellulases. The activity of the enzyme was significantly inhibited by bivalent cations (Fe(3+) and Hg(2+), 1.0 mM each) and activated by Co(2+), K(+) and Na(+). The purified enzyme revealed the products of carboxymethyl cellulose (CMC) hydrolysis to be CM glucose, cellobiose and cellotriose. Thermostability, pH stability, good hydrolytic capability, and stability in the presence of detergents, surfactants, chelators and commercial proteases make this enzyme potentially useful in laundry detergents.

Production and characterization of a thermostable beta-galactosidase from Bacillus coagulans RCS3.

A strain of Bacillus coagulans RCS3 isolated from a hot-water spring produced significant beta-galactosidase activity at 10 days of growth in a flask. While enzyme production was maximum at 50 degrees C, the highest activity was at 65 degrees C, where the half-life was 2 h. A 2 degrees C decrease in temperature increased the half-life to 15 h without significantly changing the activity, suggesting that 63 degrees C is the temperature of preference compared with 65 degrees C for a combination of good activity and stability. The beta-galactosidase was also stable over pH 5-8, with peak activity at pH 6-7. It was strongly and competitively inhibited by the hydrolysis product galactose. Bivalent cations (Cu(2+), Ni(2+) and Hg(2+)) in the concentration range of 0.5-2.0 mM also inhibited enzyme activity. Both lactose solution and whey could be hydrolysed substantially within 36 h at 50 degrees C. The thermostability and pH-stability and good hydrolytic capability make this enzyme potentially useful in the dairy industry.