Shiwani Guleria

Purification and characterization of detergent stable alkaline protease from Bacillus amyloliquefaciens SP1 isolated from apple rhizosphere

A thermostable extracellular alkaline protease producing Bacillus amyloliquefaciens SP1 was isolated from apple rhizosphere having multifarious plant growth promoting activities. Strain SP1 was purified to 6.48‐fold using four‐step purification protocol and characterized in detail for its robustness and ecofriendly application in leather and detergent industries. Structural analysis revealed that the protease was monomeric and had a molecular weight of 43 kDa. It exhibited optimum activity at 60°C in alkaline environment (pH 8.0) and stable in the presence of surfactants and oxidizing agents. Enzyme was thermostable at 50°C and retained more than 70% activity after 30 min incubation. It has shown stain removal property and dehairing of goat skin without chemical assistance and hydrolyzing fibrous proteins. This protease showed Km of 0.125 mg ml−1 and Vmax of 12820 μg ml−1 indicating its excellent affinity and catalytic role. Thermal inactivation of the pure enzyme followed first‐order kinetics. The half life of the pure enzyme at 50, 60, and 65°C was 77, 19.80, and 13.33 min, respectively. The activation energy was 37.19 KJ mol−1. The results suggest that the B. amyloliquefaciens SP1 has a potential application in different industries.

Impact of Fungicide Mancozeb at Different Application Rates on Soil Microbial Populations, Soil Biological Processes, and Enzyme Activities in Soil

The use of fungicides is the continuous exercise particularly in orchard crops where fungal diseases, such as white root rot, have the potential to destroy horticultural crops rendering them unsaleable. In view of above problem, the present study examines the effect of different concentrations of mancozeb (0–2000 ppm) at different incubation periods for their harmful side effects on various microbiological processes, soil microflora, and soil enzymes in alluvial soil (pH 6.8) collected from apple orchards of Shimla in Himachal Pradesh (India). Low concentrations of mancozeb were found to be deleterious towards fungal and actinomycetes population while higher concentrations (1000 and 2000 ppm) were found to be detrimental to soil bacteria. Mancozeb impaired the process of ammonification and nitrification. Similar results were observed for nitrifying and ammonifying bacteria. Phosphorus solubilization was increased by higher concentration of mancozeb, that is, 250 ppm and above. In unamended soil, microbial biomass carbon and carbon mineralization were adversely affected by mancozeb. Soil enzymes, that is, amylase, invertase, and phosphatase showed adverse and disruptive effect when mancozeb used was above 10 ppm in unamended soil. These results conclude that, to lessen the harmful effects in soil biological processes caused by this fungicide, addition of higher amount of nitrogen based fertilizers is required.

Modification in the properties of paper by using cellulase-free xylanase produced from alkalophilic Cellulosimicrobium cellulans CKMX1 in biobleaching of wheat straw pulp

Alkalophilic Cellulosimicrobium cellulans CKMX1 isolated from mushroom compost is an actinomycete that produces industrially important and environmentally safer thermostable cellulase-free xylanase, which is used in the pulp and paper industry as an alternative to the use of toxic chlorinated compounds. Strain CKMX1 was previously characterized by metabolic fingerprinting, whole-cell fatty acids methyl ester analysis, and 16S rDNA and was found to be C. cellulans CKMX1. Crude enzyme (1027.65 U/g DBP) produced by C. cellulans CKMX1, having pH and temperature optima of 8.0 and 60 °C, respectively, in solid state fermentation of apple pomace, was used in the production of bleached wheat straw pulp. Pretreatment with xylanase at a dose of 5 U/g after pulping decreased pulp kappa points by 1.4 as compared with the control. Prebleaching with a xylanase dose of 5 U/g pulp reduced the chlorine charge by 12.5%, increased the final brightness points by approximately 1.42% ISO, and improved the pulp strength properties. Xylanase could be substituted for alkali extraction in C-Ep-D sequence and used for treating chemically bleached pulp, resulting in bleached pulp with higher strength properties. Modification of bleached pulp with 5 U of enzyme/g increased pulp whiteness and breaking length by 1.03% and 60 m, respectively; decreased tear factor of pulp by 7.29%; increased bulk weight by 3.99%, as compared with the original pulp. Reducing sugars and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. Cellulosimicrobium cellulans CKMX1 has a potential application in the pulp and paper industries.

Molecular Cloning and Sequencing of AlkalophilicCellulosimicrobium cellulans CKMX1 Xylanase Gene Isolated from Mushroom Compost and Characterization of the Gene Product

A xylanolytic bacterium was isolated from mushroom compost by using enrichment technique. Results from the metabolic fingerprinting, whole-cell fatty acids methyl ester analysis and 16S rDNA sequencing suggested the bacterium to be Cellulosimicrobium cellulans CKMX1. Due to the xylanolytic activity of this bacterium, isolation and characterization of the xylanase gene were attempted. A distinct fragment of about 1671 bp was successfully amplified using PCR and cloned into Escherichia coli DH5α. A BLAST search confirmed that the DNA sequence from the amplified fragment was endo-1, 4-beta-xylanase, which was a member of glycoside hydrolase family 11. It showed 98% homology withCellulosimicrobium sp. xylanase gene (Accession no. FJ859907.1) reported from the gut of Eisenia fetida in Korea. In silicophysico-chemical characterization of amino acid sequence of xylanase showed an open reading frame encoding a 556 amino acid sequence with a molecular weight of 58 kDa and theoretical isolectric point (pI) of 4.46 was computed using Expasys ProtParam server. Secondary and homology based 3D structure of xylanase was analysed using SOPMA and Swiss-Prot software.

Tricalcium phosphate solubilization and nitrogen fixation by newly isolated Aneurinibacillus aneurinilyticus CKMV1 from rhizosphere of Valeriana jatamansi and its growth promotional effect

Aneurinibacillus aneurinilyticus strain CKMV1 was isolated from rhizosphere of Valeriana jatamansi and possessed multiple plant growth promoting traits like production of phosphate solubilization (260 mg/L), nitrogen fixation (202.91 nmol ethylene mL−1 h−1), indole-3-acetic acid (IAA) (8.1 μg/mL), siderophores (61.60%), HCN (hydrogen cyanide) production and antifungal activity. We investigated the ability of isolate CKMV1 to solubilize insoluble P via mechanism of organic acid production. High-performance liquid chromatography (HPLC) study showed that isolate CKMV1 produced mainly gluconic (1.34%) and oxalic acids. However, genetic evidences for nitrogen fixation and phosphate solubilization by organic acid production have been reported first time for A. aneurinilyticus strain CKMV1. A unique combination of glucose dehydrogenase (gdh) gene and pyrroloquinoline quinone synthase (pqq) gene, a cofactor of gdh involved in phosphate solubilization has been elucidated. Nitrogenase (nif H) gene for nitrogen fixation was reported from A. aneurinilyticus. It was notable that isolate CKMV1 exhibited highest antifungal against Sclerotium rolfsii (93.58%) followed by Fusarium oxysporum (64.3%), Dematophora necatrix (52.71%), Rhizoctonia solani (91.58%), Alternaria sp. (71.08%) and Phytophthora sp. (71.37%). Remarkable increase was observed in seed germination (27.07%), shoot length (42.33%), root length (52.6%), shoot dry weight (62.01%) and root dry weight (45.7%) along with NPK (0.74, 0.36, 1.82%) content of tomato under net house condition. Isolate CKMV1 possessed traits related to plant growth promotion, therefore, could be a potential candidate for the development of biofertiliser or biocontrol agent and this is the first study to include the Aneurinibacillus as PGPR.

Endophytic Bacteria: Role in Phosphate Solubilization

The worldwide need to increase agricultural and horticultural production from a consistently diminishing and degraded land resource has set remarkable strain in light of agro biological systems. The current methodology is to keep up and enhance agricultural and horticultural productivity only by means of the utilization of chemical fertilizers and pesticides. Despite the fact that the utilization of chemical fertilizers is credited with almost fifty percent of increase in agricultural production yet they are closely associated with environmental contamination and health problems in human beings and animals. Microbial assorted qualities in the soil are viewed as critical for keeping up for the manageability of agriculture and horticulture systems. Nonetheless, the connections between microbial differences and environmental processes are not surely known. Rhizosphere soil strongly affects a range of procedures influencing crop yield. Rhizobacteria that are present inside plant roots, framing more close associations, are known as endophytes. These endophytes are likewise called intracellular plant growth-promoting rhizobacteria (PGPR) microorganisms dwelling inside plant cells, producing nodules and being present inside these specific structures. These incorporate an extensive variety of soil microorganisms framing less formal relationship than the rhizobia-legume advantageous interaction called symbiosis, endophytes may empower plant development, directly or indirectly and incorporate the rhizobia. In this review, we essentially concentrate on the plant development by Phosphate solubilization furthermore by different means. Phosphorus is normally lacking in most characteristic soils since it is settled as insoluble iron and aluminum phosphates in acidic soils or calcium phosphates in soluble soils. Phosphate-solubilizing bacteria (PSB) as inoculants have the ability to convert insoluble forms of phosphorus to an usable form for high plant yields. This chapter mainly focuses on endophytic P-solubilizing bacteria, mechanism of P-solubilization, genetic diversity of P-solubilizers, and mass production of inoculants inoculant production and response of the crop to P-solubilizers bioinoculants.

Diacylglycerol acyl transferase: A pathogenicity related gene in Colletotrichum gloeosporioides.

To gain more insight into the molecular mechanisms of Colletotrichum gloeosporioides pathogenesis, restriction enzyme‐mediated integration (REMI) mutagenesis identified the mutants of C. gloeosporioides impaired in pathogenicity. Transformants screened for defects in pathogenicity using detached leaves and fruits. Of the 20 REMI transformants tested, two mutants (H4 and H7) showed reduced pathogenicity on leaves of apple, kiwi, mango, peach, and fruits of guava, apple, and capsicum. One tagged gene from the genome sequence of mutant H4 was recovered by inverse PCR. Sequence analysis of the tagged site in mutant H4 revealed insertion in diacylglycerol acyltransferase gene which encodes diacylglycerol acyltransferase enzyme, catalyzing the steps involved in the biosynthesis of triacylglycerol, an important component of biological membranes and source of energy. Therefore, tagging of diacylglycerol acyltransferase gene in mutant H4 resulted in reduced pathogenicity, indicating possible role of this gene in pathogenicity of C. gloeosporioides.

Mycovirus associated hypovirulence, a potential method for biological control of Fusarium species

Fusarium is a large genus of filamentous fungi belongs to the division Ascomycota and was first described as Fusisporium. Innumerable members of this genus act as pathogens, endophytes and saprophytes and can be recovered from plants and soils worldwide. Many of these members are known to be phytopathogens. It is among the most diverse and widely dispersed phyto-pathogenic fungi which cause economically important blights, rots, wilts and cankers of many ornamental, field, horticultural and forest crops both in agricultural commodities and natural ecosystems. Some species, e.g. F. graminearum and F. verticillioides have a narrow host range and mainly infect the cereals, whereas F. oxysporum has effects on both monocotyledonous and dicotyledonous plants. Attempts have been made to control the diseases caused by Fusarium sp. and to minimize crop yield losses. Till date, effective and eco-friendly methods have not been devised for the control of this devastating pathogen. A new potential of using mycovirus associated hypovirulence as biocontrol method against Fusarium species has been proposed. The present review taking into account of worldwide researches to provide possible insights for Fusarium-mycovirus coevolution.

Metagenomics of Fermented Foods: Implications on Probiotic Development

Fermented foods act as delivery vehicles of probiotic cells in human body. These food products boost human health through enhanced nutrition content, digestibility, microbial stability, and detoxification. The importance of fermented foods as probiotics is increasing continuously as they play significant roles in regulating and balancing intestinal microflora. Therefore, efforts are needed toward mining and characterization of microbial communities of fermented foods. The application of metagenomics techniques provides a right way to explore and characterize the unexplored beneficial microbial flora. Furthermore, for commercial development of a probiotic from fermented food, there are some prerequisite, which need to be fulfilled. Here we present the benefits, pitfall, and development of fermented food as probiotic.

Genetic Engineering: A Possible Strategy for Protein–Energy Malnutrition Regulation

Protein–energy malnutrition (PEM) has adversely affected the generations of developing countries. It is a syndrome that in severity causes death. PEM generally affects infants of 1–5 age group. This manifestation is maintained till adulthood in the form of poor brain and body development. The developing nations are continuously making an effort to curb PEM. However, it is still a prime concern as it was in its early years of occurrence. Transgenic crops with high protein and enhanced nutrient content have been successfully developed. Present article reviews the studies documenting genetic engineering-mediated improvement in the pulses, cereals, legumes, fruits and other crop plants in terms of nutritional value, stress tolerance, longevity and productivity. Such genetically engineered crops can be used as a possible remedial tool to eradicate PEM.