Thermostable and alkalistable exopolygalacturonase of Bacillus pumilus dcsr1: Characteristics and applicability.

The bioactive form of thermostable and alkali stable pectinase of Bacillus pumilus dcsr1 is a homodimer of the molecular mass of 60 kDa with a pI of 4.6. The enzyme is optimally active at 50 °C and pH 10.5, and its Michaelis constant (Km), maximum rate of reaction (Vmax), activation energy (Ea), and temperature quotient (Q10) values (for citrus pectin) are 0.29 mg mL-1, 116 µmole mg-1 min-1, 74.73 KJmol-1 and 1.57, respectively. The enzyme has a shelf life of one and a half years at room temperature as well as 4 °C. The activity of the enzyme is stimulated by Mn2+ and Ca2+ and inhibited by Hg+, Cd2+, Co2+, Zn2+, Fe2+, Pb2+, EDTA and urea to a varied extent. The conformational studies of the enzyme revealed a high ß-sheet content in the bioactive dimer, and high α-helix in the inactive monomer. The Circular Dichroism (CD) spectra of the dimer in the presence of inhibitors suggested a marked decrease in ß-sheet, and a significant increase in α-helix, suggesting a key role of ß-sheets in the enzyme catalysis. Based on the end product analysis, the enzyme is an exopolygalacturonase with a unique ability of transglycosylation. When ramie fibers were treated with the enzyme, removal of gummy material (pectin) was visible, confirming its applicability in the degumming process.

Recombinant exochitinase of the thermophilic mould Myceliopthora thermophila BJA: Characteristics and utility in generating N-acetyl glucosamine and in biocontrol of phytopathogenic fungi.

Chitinase from the thermophilic mould Myceliopthora thermophila BJA (MtChit) is an acid tolerant, thermostable and organic solvent stable biocatalyst which does not require any metal ions for its activity. To produce high enzyme titres, reduce fermentation time and overcome the need for induction, this enzyme has been heterologously expressed under GAP promoter in the GRAS yeast, Pichia pastoris. The production medium supplemented with the permeabilizing agent Tween-20 supported two-fold higher rMtChit production (5.5 × 103 U L-1 ). The consensus sequences S(132)xG(133)G(134) and D(168)xxD(171)xD(173)xE(175) in the enzyme have been found to represent the substrate binding and catalytic sites, respectively. The rMtChit, purified to homogeneity by a two-step purification strategy, is a monomeric glycoprotein of ∼48 kDa, which is optimally active at 55°C and pH 5.0. The enzyme is thermostable with t1/2 values of 113 and 48 min at 65 and 75°C, respectively. Kinetic parameters Km , Vmax , kcat , and kcat /Km of the enzyme are 4.655 mg mL-1 , 34.246 nmol mg-1  s-1 , 3.425 × 106 min-1 , and 1.36 × 10-6 mg mL-1  min-1 , respectively. rMtChit is an unique exochitinase, since its action on chitin liberates N-acetylglucosamine NAG. The enzyme inhibits the growth of phytopathogenic fungi like Fusarium oxysporum and Curvularia lunata, therefore, this finds application as biofungicide at high temperatures during summer in tropics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:70-80, 2017.

Fungal phytases: characteristics and amelioration of nutritional quality and growth of non-ruminants.

Fungal phytases are histidine acid phosphatases, a subclass of acid phosphatases, which catalyse the hydrolysis of phytic acid resulting in the release of phosphate moieties and thus mitigate its antinutritional properties. The supplementation of feed with phytases increases the bioavailability of phosphorus and minerals in non-ruminant animals and reduces the phosphorus pollution due to phosphorus excretion in the areas of intensive livestock production. Although phytases are reported in plants, animals and micro-organisms, fungal sources are used extensively for the production of phytases on a commercial scale. Phytases have been produced by fungi in both solid-state fermentation (SSF) and submerged fermentation (SmF). The fungal phytases are high molecular weight proteins ranging from 35 to 500 kDa. They are optimally active within pH and temperature ranges between 4.5 and 6.0, and 45 and 70 °C respectively. Phytate degradation leads to amelioration in the nutritional status of foods and feeds by improving the availability of minerals, phosphorus and proteins in non-ruminant animals and human beings and thus mitigates the environmental phosphorus pollution. Our article focuses on the role of fungal phytases in improving nutritional value of foods and feeds with concomitant increase in growth of non-ruminant animals and mitigating environmental phosphorus pollution.

Biotechnological Potential of Agro Residues for Economical Production of Thermoalkali-Stable Pectinase by Bacillus pumilus dcsr1 by Solid-State Fermentation and Its Efficacy in the Treatment of Ramie Fibres.

The production of a thermostable and highly alkaline pectinase by Bacillus pumilus dcsr1 was optimized in solid-state fermentation (SSF) and the impact of various treatments (chemical, enzymatic, and in combination) on the quality of ramie fibres was investigated. Maximum enzyme titer (348.0 ± 11.8 Ug(-1) DBB) in SSF was attained, when a mixture of agro-residues (sesame oilseed cake, wheat bran, and citrus pectin, 1 : 1 : 0.01) was moistened with mineral salt solution (a(w) 0.92, pH 9.0) at a substrate-to-moistening agent ratio of 1 : 2.5 and inoculated with 25% of 24 h old inoculum, in 144 h at 40°C. Parametric optimization in SSF resulted in 1.7-fold enhancement in the enzyme production as compared to that recorded in unoptimized conditions. A 14.2-fold higher enzyme production was attained in SSF as compared to that in submerged fermentation (SmF). The treatment with the enzyme significantly improved tensile strength and Young's modulus, reduction in brittleness, redness and yellowness, and increase in the strength and brightness of ramie fibres.

Production of acid-stable and high-maltose-forming α-amylase of Bacillus acidicola by solid-state fermentation and immobilized cells and its applicability in baking.

Among matrices used for immobilizing Bacillus acidicola cells [calcium alginate, chitosan + alginate, scotch brite, and polyurethane foam (PUF)], α-amylase production was highest by PUF-immobilized cells (9.1 U ml(-1)), which is higher than free cells (7.2 U ml(-1)). The PUF-immobilized cells could be reused over seven cycles with sustained α-amylase production. When three variables (moisture, starch, and ammonium sulfate), which significantly affected enzyme production in solid-state fermentation (SSF), were optimized using response surface methodology, 5.6-fold enhancement in enzyme production was attained. The enzyme production in SSF is 3.8-fold higher than that in submerged fermentation. The bread made by supplementing dough with α-amylase of B. acidicola scored better than those with the xylanase of Bacillus halodurans and thermostable α-amylase of Geobacillus thermoleovorans.

Production, characteristics and applications of the cell-bound phytase of Pichia anomala.

Among several yeasts isolated from dried flowers of Woodfordia fruticosa, Pichia anomala produced a high titre of cell-bound phytase. The optimization of fermentation variables led to formulation of media and selection of cultural variables that supported enhanced phytase production. The enzyme productivity was very high in fed batch fermentation in air-lift fermentor as compared to that in stirred tank fermentor. Amelioration in the cell-bound phytase activity was observed when yeast cells were permeabilized with Triton-X-100. The enzyme is thermostable and acid stable with broad substrate specificity, the characteristics that are desirable for enzymes to be used in the animal feed industry. The phytase-encoding gene was cloned and sequenced. The 3D structure of the enzyme was proposed by comparative modeling using phytase of Debaryomyces occidentalis (50% sequence identity) as template. When broiler chicks, and fresh water and marine fishes were fed with the feed supplemented with yeast biomass containing phytase, improvement in growth and phosphorus retention, and decrease in the excretion of phosphorus in the faeces were recorded. The cell-bound phytase of P. anomala could effectively dephytinize wheat flour and soymilk.

High maltose-forming, Ca2+-independent and acid stable α-amylase from a novel acidophilic bacterium, Bacillus acidicola.

Bacillus acidicola TSAS1 produced a novel acid-stable, thermostable, Ca(2+)-independent and high maltose-forming α-amylase with optimum activity at pH 4.0 and 60°C, and T(1/2) of 27 min at 90°C. The enzyme saccharified raw as well as soluble starches, and ameliorated bread quality when the dough was supplemented with the enzyme.

Plant growth promotion by an extracellular HAP-phytase of a thermophilic mold Sporotrichum thermophile.

Phytase of the thermophilic mold Sporotrichum thermophile Apinis hydrolyzed and liberated inorganic phosphate from Ca(+2), Mg(+2), and Co(+2) phytates more efficiently than those of Al(3+), Fe(2+), Fe(3+), and Zn(2+). The hydrolysis rate was higher at 60 degrees C as compared to 26 degrees Celsius. Among all the organic acids tested, citrate was more effective in enhancing solubilization of insoluble phytate salts by phytase than others. The dry weight and inorganic phosphate contents of the wheat plants were high when supplemented with phytase or fungal spores. The plants provided with 5 mg phytate per plant exhibited enhanced growth and inorganic phosphate. With increase in the dosage of phytase, there was increase in growth and inorganic phosphate of plants, the highest being at 20 U per plant. The compost made employing the combined native microflora of the wheat straw and S. thermophile promoted growth of the plants. The plant-growth-promoting effect was also higher with the compost made using S. thermophile than that from only the native microflora.

Phytase production by a thermophilic mould Sporotrichum thermophile in solid state fermentation and its potential applications.

Phytase production by a thermophilic mould Sporotrichum thermophile Apinis was investigated in solid state fermentation (SSF) using sesame oil cake as the substrate. Scanning electron microscopy of the fermented sesame oil cake revealed a dense growth of the mould with abundant conidia. Glucose, ammonium sulphate and incubation period were identified as the most significant factors by Plackett-Burman design. The optimum values of the critical components determined by central composite design of response surface methodology for the maximum phytase production were glucose 3%, ammonium sulphate 0.5% and incubation period 120 h. An overall 2.6-fold improvement in phytase production was achieved due to optimization. Highest enzyme production (348.76 U/g DMR) was attained at a substrate bed depth of 1.5 cm in enamel coated metallic trays. The enzyme liberated inorganic phosphate from wheat flour and soymilk with concomitant dephytinization and liberation of soluble inorganic phosphate.

Economical glucoamylase production by alginate-immobilized Thermomucor indicae-seudaticae in cane molasses medium.

AIMS: The present investigation is aimed at assessing the suitability of cane molasses as a cheaper carbon and energy source for glucoamylase production using alginate-immobilized Thermomucor indicae-seudaticae. METHODS AND RESULTS: The culture variables for glucoamylase production were optimized by 'one-variable-at-a-time' strategy and response surface methodology (RSM). A high glucoamylase titre was attained when 40 alginate beads (c. 5x10(6) immobilized spores) were used to inoculate 50 ml of cane molasses (8%) medium in 250-ml Erlenmeyer flasks. Response surface optimization of fermentation parameters (cane molasses 7%, inoculum level 44 alginate beads per 50 ml of medium and ammonium nitrate 0.25%) resulted in 1.8-fold higher glucoamylase production (27 U ml(-1)) than that in the unoptimized medium (15 U ml(-1)). Enzyme production was also sustainable in 22 l of laboratory air-lift bioreactor. CONCLUSIONS: Cane molasses served as an excellent carbon and energy source for the economical production of glucoamylase, which was almost comparable with that in sucrose yeast-extract broth. The statistical model developed using RSM allowed determination of optimum levels of the variables for improving glucoamylase production. SIGNIFICANCE AND IMPACT OF THE STUDY: The cost of glucoamylase produced in cane molasses supplemented with ammonium nitrate was considerably lower (euro1.43 per million U) than in synthetic medium containing sucrose and yeast-extract (euro35.66 per million U). The reduction in fermentation time in air-lift bioreactor with sustainable glucoamylase titres suggested the feasibility of scale up of the process.

Improving production of hyperthermostable and high maltose-forming alpha-amylase by an extreme thermophile Geobacillus thermoleovorans using response surface methodology and its applications.

By cultivating Geobacillus thermoleovorans in shake flasks containing cane molasses medium at 70 degrees C, the fermentation variables were optimized by 'one variable at a time' approach followed by response surface methodology (RSM). The statistical model was obtained by central composite design (CCD) using three variables (cane-molasses, urea and inoculum density). An overall 1.6- and 2.1-fold increase in enzyme production was achieved in the optimized medium in shake flasks and fermenter, respectively. The alpha-amylase titre increased significantly in cane-molasses medium (60 U ml(-1)) as compared to that in the synthetic medium (26 U ml(-1)). Thus the cost of enzyme produced in cane molasses medium (0.823 euros per million U) was much lower than that produced in the synthetic starch-yeast extract-tryptone medium (18.52 euros per million U). The shelf life of bread was improved by supplementing dough with alpha-amylase, and thus, the enzyme was found to be useful in preventing the staling of bread. Reducing sugars liberated from 20% and 30% raw pearl millet starch were fermented to ethanol; ethanol production levels attained were 35.40 and 28.0 g l(-1), respectively.

A marked enhancement in phytase production by a thermophilic mould Sporotrichum thermophile using statistical designs in a cost-effective cane molasses medium.

AIMS: Statistical optimization of phytase production by a thermophilic mould Sporotrichum thermophile in a cost-effective cane molasses medium. METHODS AND RESULTS: Sporotrichum thermophile secreted phytase in cane molasses medium at 45 degrees C and 250 rev min(-1) after 5 days. The important factors identified by Plackett-Burman design (magnesium sulfate, Tween 80, ammonium sulfate and incubation period) were further optimized by response surface methodology (RSM). An overall 107% improvement in phytase production was achieved due to optimization. Supplementation of the medium with inorganic phosphate repressed the enzyme synthesis. When inorganic phosphate was reduced from the cane molasses medium by treatment with calcium chloride, the enzyme production increased. The phytase activity was not affected by the enzyme treatment with trypsin and pepsin. CONCLUSIONS: A twofold increase in phytase production was achieved due to optimization using statistical designs in a cost-effective cane molasses medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytase production was doubled due to optimization. The enzyme, being resistant to trypsin and pepsin, thermostable and acid stable, can find application in animal feed industry for improving nutritional status of the feed and combating environmental phosphorus pollution.

Phytase production by thermophilic mold Sporotrichum thermophile in solid-state fermentation and its application in dephytinization of sesame oil cake.

The phytase production by Sporotrichum thermophile TLR50 was recorded on all the commonly used animal feed ingredients tested to varying degrees in solid-state fermentation. Enzyme production increased to 180 U/g of dry moldy residue (DMR) in sesame oil cake at 120 h and 45 degrees C at the initial substrate-to-moisture ratio of 1:2.5 and aw of 0.95. Supplementation of sesame oil cake with glucose and ammonium sulfate further enhanced phytase titer (282 U/g of DMR). An overall 76% enhancement in phytase production was achieved owing to optimization. The mold secreted acid phosphatase, amylase, xylanase, and lipase along with phytase. By the action of phytase, inorganic phosphate was liberated efficiently, leading to dephytinization of sesame oil cake.

Evidence that whitefly-transmitted cowpea mild mottle virus belongs to the genus Carlavirus.

Two strains of whitefly-transmitted cowpea mild mottle virus (CPMMV) causing severe (CPMMV-S) and mild (CPMMV-M) disease symptoms in peanuts were collected from two distinct agro-ecological zones in India. The host-range of these strains was restricted to Leguminosae and Chenopodiaceae, and each could be distinguished on the basis of symptoms incited in different hosts. The 3'-terminal 2500 nucleotide sequence of the genomic RNA of both the strains was 70% identical and contains five open reading frames (ORFs). The first three (P25, P12 and P7) overlap to form a triple gene block of proteins, P32 encodes the coat protein, followed by P12 protein located at the 3' end of the genome. Genome organization and pair-wise comparisons of amino acid sequences of proteins encoded by these ORFs with corresponding proteins of known carlaviruses and potexviruses suggest that CPMMV-S and CPMMV-M are closely related to viruses in the genus Carlavirus. Based on the data, it is concluded that CPMMV is a distinct species in the genus Carlavirus.

Comparative pharmacognosy of medicinally important Indian vitex species.

Vitex genera is reputed for their medicinal properties. Of the 12 species reported to be present in Indian only 8 species are medicinally useful. Of these six species were colled and their pharmacognostic characters were studied and described.