Cyclic extraction of phosphate from soybean meal using immobilized Aspergillus oryzae SBS50 phytase.

Phytase enzyme found in plants, animals, and microorganisms is mainly involved in catalyzing the systematic removal of a phosphate group from phytic acid. Enzyme immobilization is one of the cost-effective methods for the wide usage of enzymes in the industrial sector. This paper reports the covalent immobilization of phytase on glutaraldehyde-activated aluminum oxide beads. The immobilization yield, efficiency, and activation energy were found to be 47.8%, 71.5%, and 15.78 J/mol, respectively. The bound enzyme displayed a shift in pH optima from 5.5 to 4.5, which is more beneficial to increase digestibility in comparison with the free enzyme. Immobilized phytase retained 42.60% of its activity after 1.0 h incubation at 80 °C, whereas free enzyme retained only 4.20% of its activity. Thermodynami increase in half-lives, D-values, enthalpy and free energy change after covalent immobilization could be credited to the enhanced stability. Immobilized phytase could be reused for five consecutive cycles retaining 51% of its initial activity with sodium phytate. The immobilized phytase was also found effective to hydrolyze the soybean meal, thus increasing the digestibility of poultry feed. The hydrolyzing reaction of soybean meal was carried out for six consecutive cycles and immobilized phytase retained nearly 50% of activity till the fifth cycle. The amount of phosphorus released after treatment with immobilized phytase was far higher than that from free phytase. Immobilization on this support is significant, as this support can sustain high mechanical resistance at high pH and temperature. This considerable stability and reusability of the bound enzyme may be advantageous for its industrial application.

Production, Characterization and Prebiotic Potential of Xylooligosaccharides Produced from Wheat Bran using Enterobacter hormaechei KS1 Xylanase.

In the present investigation, xylooligosaccharides were produced from wheat bran and wheat bran extracted xylan through enzymatic hydrolysis using xylanase from novel Enterobacter hormaechei KS1. Xylooligosaccharides/reducing sugars production from wheat bran was found maximum (374 mg/g) when 4.0% of wheat bran was treated with 375 units (IU/mL) of Enterobacter hormaechei KS1 xylanase at pH 6.0 and incubated at 50 °C for 24 h of incubation. In case of wheat bran extracted xylan 419 mg/g of xylooligosaccharides were produced when 3% of extracted xylan was incubate for 8 h. Analysis of the enzymatic hydrolysate through high performance liquid chromatography equipped with refractive index detector showed the presence of xylose, xylopentose and xylohexose. The decrease in pH with 1.0% dose of xylooligosacchaides produced from extracted xylan hydrolysis using E. hormaechei KS1 xylanase showed more decrease with L. rhamnosus (6.72 to 5.94) followed by L. brevis (6.71 to 6.15) and L. plantarum (6.71 to 6.41). In case of increase in optical density both wheat bran and wheat bran extracted xylan generated xylooligosaccharides exhibited similar pattern i.e., L. rhamnosus > L. plantarum > L. brevis.

Biochemical assessment of oat genotypes revealed variability in grain quality with nutrition and crop improvement implications.

Oat is a potent source of nutrients and bioactive compounds offering potential health benefits and role in combating micronutrient malnutrition problems. To exploit nutritional and quality traits of oats, a biochemical assessment of 112 oat genotypes was conducted. The high range of variability for total phenol (1.7-31.3 mg/g), ß-glucan (1.0-8.0 mg/g), calcium (1.91-4.34 mg/g), zinc (3.80-6.50 mg/100 g), iron (0.66-4.89 mg/100 g) and manganese (2.88-8.0 mg/100 g) was revealed among genotypes. A higher amount of iron and zinc was found in genotypes OS-6, HFO-638, HFO-915 & HFO-918, whereas, elevated levels of manganese and zinc were recorded in genotypes OS-403 & OL-1804. The results revealed groups of low phytic acid oat genotypes containing high crude protein (HFO-52, HFO-270, HFO-330), ß-glucan (HFO-62, HFO-588, HFO-926). A significant positive correlation was obtained between copper with iron, manganese, and calcium content. These findings could be useful for developing value-added oat food products and novel oat varieties.

Immobilization of xylanase purified from Bacillus pumilus VLK-1 and its application in enrichment of orange and grape juices.

This study was conducted to evaluate the efficacy of purified free and immobilized xylanase in enrichment of fruit juices. Extracellular xylanase produced from Bacillus pumilus VLK-1 was purified to apparent homogeneity by 15.4-fold with 88.3 % recovery in a single step using CM-Sephadex C-50. Purified xylanase showed a single band on SDS-polyacrylamide gel with a molecular mass of 22.0 kDa. The purified enzyme was immobilized on glutaraldehyde-activated aluminum oxide pellets and the immobilization process parameters were optimized statistically through response surface methodology. The bound enzyme displayed an increase in optimum temperature from 60 to 65 ºC and pH from 8.0 to 9.0. The pH and temperature stability of the enzyme was also enhanced after immobilization. It could be reused for 10 consecutive cycles with 58 % residual enzyme activity. The potential of purified xylanase (free and immobilized) in juice enrichment from grape (Vitis amurensis) and orange (Citrus sinensis) pulps has been investigated. The optimization of this process using free xylanase revealed maximum juice yield, clarity and reducing sugar on treatment with 20 IU/g fruit pulp for 30 min at 50 ºC. Treatment of both the fruit pulps with xylanase under optimized conditions resulted in an increase in juice yield, clarity, reducing sugars, titratable acidity, and filterability but a decline in turbidity and viscosity. Immobilized enzyme was more effective in improving juice quality as compared to its soluble counterpart. The results showed B. pumilus VLK-1 xylanase, in both free and immobilized form, as a potential candidate for use in fruit juice enrichment.

Modulation of xylanase production from alkaliphilic Bacillus pumilus VLK-1 through process optimization and temperature shift operation.

This study was aimed at enhancing the production of xylanase from an alkaliphilic Bacillus pumilus VLK-1 in submerged fermentation using wheat bran, a cheap and abundantly available agro-residue, through process optimization and to monitor the effect of temperature shift operation on it. The potential of xylanase in saccharification of wheat straw was also investigated. The results showed that optimization of the fermentation process by one variable approach increased the enzyme yield from 402 to 4,986 IU/ml. Subsequently, optimization of nitrogen and carbon sources through response surface methodology led to high level xylanase production (7,295 IU/ml) which was 1.46-fold greater than one variable approach after 56 h of cultivation at 30 °C. Temperature shift operation during fermentation resulted in maximum xylanase production in lesser duration (48 h instead of 56 h). Enzymatic hydrolysis of the alkali pre-treated wheat straw with 500 IU xylanase alone released 173 ± 8 mg sugars/g whereas in combination with cellulase and ß-glucosidase released 553 ± 12 mg sugars/g dry substrate in 6 h, indicating its potential in saccharification of the lignocellulosic substrate. Temperature shift operation is likely to be attractive for large scale industrial fermentation due to significant reduction in the operating cost. To our knowledge, this is the first report which showed the effect of temperature shift operation on xylanase production from bacteria. The xylanase production from Bacillus sp. in the present study is close to the highest titre reported in the literature. An enhanced xylanase production using wheat bran, a cheap and abundantly available agro-residue, will apparently reduce the enzyme cost, which would be beneficial for industry.

Covalent immobilization of xylanase produced from Bacillus pumilus SV-85S on electrospun polymethyl methacrylate nanofiber membrane.

Polymethyl methacrylate (PMMA) nanofiber membrane (NFM) was synthesized by an electrospinning technique. These membranes were utilized as a support for immobilization of xylanase enzyme to study its pH stability, thermal stability, and reusability. The morphology of aligned NFM was studied by optical microscopy and scanning electron microscopy. The PMMA NFM was functionalized with phenylenediamine and activated with glutaraldehyde to yield an aldehyde group on its surface for covalent immobilization of xylanase. The Fourier transform infrared analysis of the covalently immobilized xylanase confirmed that the enzyme was immobilized on PMMA NFM via amide linkages. The immobilization efficiency of covalently bound xylanase was found experimentally to be 90%. A forward shift in pH optima from 6.0-7.0 (soluble enzyme) to 7.0-9.0 (immobilized enzyme) was observed after xylanase immobilization. The pH and temperature stability of xylanase were enhanced upon its covalent immobilization. The immobilized enzyme was active on repeated use and retained ∼80% of its initial activity after 11 reaction cycles. The improved thermal and operational stability of the covalently immobilized enzyme on PMMA NFM might be advantageous for industrial applications.

Biobleaching application of cellulase poor and alkali stable xylanase from Bacillus pumilus SV-85S.

The potential of extracellular alkali stable and thermo tolerant xylanase produced by Bacilluspumilus SV-85S through solid state fermentation was investigated in pulp bleaching in association with conventional bleaching using chlorine and chlorine dioxide. The biobleaching of kraft pulp with xylanase was the most effective at an enzyme dose of 10 IU/g oven dried pulp, pH 9.0 and 120 min incubation at 55 °C. Under the optimized conditions, xylanase pretreatment reduced Kappa number by 1.6 points and increased brightness by 1.9 points. Subsequently, chlorine dioxide and alkaline bleaching sequences (CDE1D1D2) finally resulted in brightness gain of 2.7 points as compared with the control. The pretreatment of pulp with xylanase resulted in 29.16 % reduction in chlorine consumption by maintaining the same brightness as in control. An improvement in pulp strength properties was also observed after bleaching of xylanase pretreated pulp. Scanning electron microscopy revealed loosening and swelling of pulp fibers after enzyme treatment. These results clearly demonstrated that the B. pumilus SV-85S xylanase was effective as a pulp biobleaching agent. The decrease in chlorine consumption by pretreatment of pulp with xylanase apparently made the biobleaching process not only economical but also eco-friendly.

Production of alkali tolerant cellulase free xylanase in high levels by Bacillus pumilus SV-205.

The fermentation conditions were optimized for hyper production of xylanase from Bacillus pumilus SV-205. The bacterium secretes high levels (7382.7±1200 IU/mL) of cellulase-free xylanase using wheat bran led to 21.63 fold increase in activity. A combination of yeast extract and peptone stimulated highest xylanase production (2448.0 IU/mL) as compared to other combinations. The most important characteristic of the enzyme is its high pH stability (100%) over a broad pH range of 6-11 for 24h. Thermostability studies revealed that enzyme retained 65% activity after an incubation of 2h at 60°C. The level of production is remarkable as compared to earlier reports.

Hyper production of alkali stable xylanase in lesser duration by Bacillus pumilus SV-85S using wheat bran under solid state fermentation.

High level production of an extracellular cellulase-poor alkali stable xylanase has been conceded from newly isolated Bacillus pumilus SV-85S under solid state fermentation using wheat bran as a substrate. Optimization of the fermentation conditions enhanced the enzyme production to 73,000 ± 1,000 IU/g dry substrate, which was 13.8-fold higher than unoptimized conditions (5,300 IU/g). The enzyme titre was highest after 48 h of incubation at 30°C with 1:3 ratios of substrate to moistening agent using wheat bran as a carbon source. The enzyme could be produced in significant levels by using either tap water or distilled water alone as a moistening agent. An elevated production of xylanase by B. pumilus SV-85S in the presence of wheat bran, a cheap and easily available agro-residue, in shorter duration would apparently reduce the enzyme cost substantially. The enzyme was completely stable over a broad pH (5-11) range and retained 52% of its activity at a temperature of 70°C for 30 min. The desired characteristics of this enzyme together with economic production would be important for its application in paper and pulp industry.

Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation.

This paper reports the production of a cellulase-free and alkali-stable xylanase in high titre from a newly isolated Bacillus pumilus SV-85S using cheap and easily available agro-residue wheat bran. Optimization of fermentation conditions enhanced the enzyme production to 2995.20 +/- 200.00 IU/ml, which was 9.91-fold higher than the activity under unoptimized basal medium (302.2 IU/ml). Statistical optimization using response-surface methodology was employed to obtain a cumulative effect of peptone, yeast extract, and potassium nitrate (KNO(3)) on enzyme production. A 2(3) central composite design best optimized the nitrogen source at the 0 level for peptone and yeast extract and at the -alpha level for KNO(3), along with 5.38-fold increase in xylanase activity. Addition of 0.1% tween 80 to the medium increased production by 1.5-fold. Optimum pH for xylanase was 6.0. The enzyme was 100% stable over the pH range from 5 to 11 for 1 h at 37 degrees C and it lost no activity, even after 3 h of incubation at pH 7, 8, and 9. Optimum temperature for the enzyme was 50 degrees C, but the enzyme displayed 78% residual activity even at 65 degrees C. The enzyme retained 50% activity after an incubation of 1 h at 60 degrees C. Characteristics of B. pumilus SV-85S xylanase, including its cellulase-free nature, stability in alkali over a long duration, along with high-level production, are particularly suited to the paper and pulp industry.