Afsheen Aman

Degradation of complex carbohydrate: immobilization of pectinase from Bacillus licheniformis KIBGE-IB21 using calcium alginate as a support.

Pectinases are heterogeneous group of enzymes that catalyse the hydrolysis of pectin substances which is responsible for the turbidity and undesirable cloudiness in fruits juices. In current study, partially purified pectinase from Bacillus licheniformis KIBGE-IB21 was immobilized in calcium alginate beads. The effect of sodium alginate and calcium chloride concentration on immobilization was studied and it was found that the optimal sodium alginate and calcium chloride concentration was 3.0% and 0.2 M, respectively. It was found that immobilization increases the optimal reaction time for pectin degradation from 5 to 10 min and temperature from 45 to 55°C, whereas, the optimal pH remained same with reference to free enzyme. Thermal stability of enzyme increased after immobilization and immobilized pectinase retained more than 80% of its initial activity after 5 days at 30°C as compared with free enzyme which showed only 30% of residual activity. The immobilized enzyme also exhibited good operational stability and 65% of its initial activity was observed during third cycle. In term of pectinase immobilization efficiency and stability, this calcium alginate beads approach seemed to permit good results and can be used to make a bioreactor for various applications in food industries.

Immobilization of pectin degrading enzyme from Bacillus licheniformis KIBGE IB-21 using agar-agar as a support

Pectinase from Bacillus licheniformis KIBGE IB-21 was immobilized in agar-agar matrix using entrapment technique. Effect of different concentrations of agar-agar on pectinase immobilization was investigated and it was found that maximum immobilization was achieved at 3.0% agar-agar with 80% enzyme activity. After immobilization, the optimum temperature of enzyme increased from 45 to 50 °C and reaction time from 5 to 10 minutes as compared to free enzyme. Due to the limited diffusion of high molecular weight substrate, K(m) of immobilized enzyme slightly increased from 1.017 to 1.055 mg ml(-1), while Vmax decreased from 23,800 to 19,392 μM min(-1) as compared to free enzyme. After 120 h entrapped pectinase retained their activity up to 82% and 71% at 30 °C and 40 °C, respectively. The entrapped pectinase showed activity until 10th cycle and maintain 69.21% activity even after third cycle.

Polygalacturonase: production of pectin depolymerising enzyme from Bacillus licheniformis KIBGE IB-21.

Polygalacturonase is an enzyme that hydrolyzes external and internal α (1-4) glycosidic bonds of pectin to decrease the viscosity of fruits juices and vegetable purees. Several bacterial strains were isolated from soil and rotten vegetables and screened for polygalacturonase production. The strain which produced maximum polygalacturonase was identified Bacillus licheniformis on the basis of taxonomic studies and 16S rDNA analysis. The isolated bacterial strain produced maximum polygalacturonase at 37 °C after 48 h of fermentation. Among various carbon sources apple pectin (1.0%) showed maximum enzyme production. Different agro industrial wastes were also used as substrate in batch fermentation and it was found that wheat bran is capable of producing high yield of enzyme. Maximum polygalacturonase production was obtained by using yeast extract (0.3%) as a nitrogen source. It was observed that B. licheniformis KIBGE IB-21 is capable of producing 1015 U/mg of polygalacturonase at neutral pH.

Structural analysis and characterization of dextran produced by wild and mutant strains of Leuconostoc mesenteroides.

An exopolysaccharide known as dextran was produced by Leuconostoc mesenteroides KIBGE-IB22 (wild) and L. mesenteroides KIBGE-IB22M20 (mutant). The structure was characterized using FTIR, (1)H NMR, (13)C NMR and 2D NMR spectroscopic techniques, whereas surface morphology was analyzed using SEM. A clear difference in the spectral chemical shift patterns was observed in both samples. All the spectral data indicated that the exopolysaccharide produced by KIBGE-IB22 is a mixture of two biopolymers. One was dextran in α-(1 → 6) configuration with a small proportion of α-(1 → 3) branching and the other was levan containing β-(2 → 6) fructan fructofuranosyl linkages. However, remarkably the mutant only produced dextran without any concomitant production of levan. Study suggested that the property of KIBGE-IB22M20, regarding improved production of high molecular weight dextran in a shorter period of fermentation time without any contamination of other exopolysaccharide, could be employed to make the downstream process more feasible and cost effective on large scale.

Agar–agar entrapment increases the stability of endo-β-1,4-xylanase for repeated biodegradation of xylan

Microbial xylanases, specially endo-β-1,4-xylanase catalyzes the hydrolysis of xylan, is considered one of the most significant hydrolases. It has numerous applications but most extensively is utilized in paper and pulp industry as a bio-bleaching agent. Immobilization technique is comprehensively studied with the expectation of modifying and improving enzyme stability and characteristics for commercial purposes. Currently, matrix entrapment technique is applied to immobilize endo-β-1,4-xylanase within agar-agar gel beads produced by Geobacillus stearothermophilus KIBGE-IB29. Maximal enzyme immobilization yield was achieved at 2.5% of agar-agar concentration. Optimized conditions demonstrated an increase in the optimal reaction time from 05 min to 30 min and incubation temperature from 50 °C to 60 °C with reference to free enzyme whereas; no effect was observed for optimum pH. Entrapment technique uniquely changed the kinetic parameters of immobilized endo-β-1,4-xylanase (Km: 0.5074 mg min(-1) to 0.5230 mg min(-1) and Vmax: 4773 U min(-1) to 968 U min(-1)) as compared to free enzyme. However, immobilized enzyme displayed broad thermal stability and retained 79.0% of its initial activity at 80 °C up to 30 min whereas; free enzyme completely lost its activity at this temperature. With respect to economic feasibility, the immobilized enzyme showed impressive recycling efficiency up to six reaction cycles.

Hyper production of cellulose degrading endo (1,4) β-d-glucanase from Bacillus licheniformis KIBGE-IB2

Abstract Cellulase hydrolyzes β (1,4) glycosidic linkages of cellulose polymer to soluble sugar. An extracellular enzyme production by Bacillus licheniformis KIBGE-IB2 (GenBank accession No. http://ncbi-n:GU216259) was studied under various environmental conditions. Maximum enzyme production was measured in the liquid fermentation medium after 48 h, containing (gL−1), CMC, 5.0; peptone, 15.0; yeast extract, 15.0; CaCl2·2H2O, 0.001; FeSO4·7H2O, 0.001; K2HPO4, 5.0; NaH2PO4, 5.0 and MgSO4·7H2O, 1.0. The optimal pH and temperature for enzyme production was found to be 6.0 and 37°C, respectively. It was also found that beside soluble sugars, a significant amount of enzyme production was obtained when biomass (wheat bran and orange peel) were examined as a sole carbon source. The current findings indicate that endo (1,4) β-d-glucanase from B. licheniformis KIBGE-IB2 can be beneficial for commercial purpose.

Production of xylan degrading endo-1, 4-β-xylanase from thermophilic Geobacillus stearothermophilus KIBGE-IB29

Abstract Xylan degrading bacterial strain was isolated from soil and identified as Geobacillus stearothermophilus KIBGE-IB29 on the basis of morphological, biochemical and 16S rDNA sequence analysis. Optimization of medium and culture conditions in submerged fermentation was investigated for maximum endo-1, 4-β-xylanase production. High yield of xylan degrading endo-1, 4-β-xylanase was achieved at 60°C and pH-6.0 with 24h of fermentation. Maximum enzyme was produced using 0.5% xylan as a carbon source, 0.5% peptone, 0.2% yeast extract and 0.1% meat extract as nitrogen sources. Di-potassium hydrogen phosphate (0.25%), calcium chloride (0.01%), potassium hydrogen phosphate (0.05%) and ammonium sulfate (0.05%) were also incorporated in the fermentation medium to enhance the enzyme production.

High production of cellulose degrading endo-1,4-β-d-glucanase using bagasse as a substrate from Bacillus subtilis KIBGE HAS

Sugarcane bagasse is a cheap carbon source for endo-1,4-β-D-glucanase production as it is easily available as by-product from sugar industries. Fermentation conditions for endo-1,4-β-D-glucanase production by Bacillus subtilis KIBGE HAS were optimized by using un-treated sugarcane bagasse for induction of endo-1,4-β-D-glucanase and it was found that 2.0 g% bagasse in fermentation medium induced maximum endo-1,4-β-D-glucanase production. It was also found that when sugarcane bagasse was supplemented with different carbon sources, the results showed that lactose, xylose, maltose and sucrose favored endo-1,4-β-D-glucanase production, whereas cellobiose and fructose inhibit enzyme production. Maximum endo-1,4-β-D-glucanase production was obtained at 40 °C keeping the initial pH of the medium at 7.0 before sterilization. Maximum endo-1,4-β-D-glucanase production was obtained after 48 h incubation. Among different nitrogen sources, ammonium nitrate enhanced endo-1,4-β-D-glucanase production. The optimal temperature and pH for enzyme activity were 60 °C and 7.0, respectively.

Immobilization of pectin depolymerising polygalacturonase using different polymers.

Polygalacturonase catalyses the hydrolysis of pectin substances and widely has been used in food and textile industries. In current study, different polymers such as calcium alginate beads, polyacrylamide gel and agar-agar matrix were screened for the immobilization of polygalacturonase through entrapment technique. Polyacrylamide gel was found to be most promising one and gave maximum (89%) immobilization yield as compared to agar-agar (80%) and calcium alginate beads (46%). The polymers increased the reaction time of polygalacturonase and polymers entrapped polygalacturonases showed maximum pectinolytic activity after 10 min of reaction as compared to free polygalacturonase which performed maximum activity after 5.0 min of reaction time. The temperature of polygalacturonase for maximum enzymatic activity was increased from 45°C to 50°C and 55°C when it was immobilized within agar-agar and calcium alginate beads, respectively. The optimum pH (pH 10) of polygalacturonase was remained same when it was immobilized within polyacrylamide gel and calcium alginate beads, but changed from pH 10 to pH 9.0 after entrapment within agar-agar. Thermal stability of polygalacturonase was improved after immobilization and immobilized polygalacturonases showed higher tolerance against different temperatures as compared to free enzyme. Polymers entrapped polygalacturonases showed good reusability and retained more than 80% of their initial activity during 2nd cycles.

Dextranase: Hyper production of dextran degrading enzyme from newly isolated strain of Bacillus licheniformis

Dextranase, 6-alpha-D-glucan 6-glucanohydrolase catalyzes the degradation of dextran (polymer of D-glucose) in to low molecular weight fractions. Dextranolytic bacterial strains were isolated from various natural sources and plate assay methods were developed for screening of highest extracellular dextranase producing isolate. Bacillus licheniformis, identified on the basis of taxonomic characterization was subjected to UV radiation and highest enzyme producing mutant obtained led to 7 times more dextranase production than wild. Optimization of major physico-chemical parameters affecting enzyme production; including medium composition, pH, cultivation time and temperature revealed that maximum enzyme production was obtained in a self designed medium (pH 6.0) containing 1% Dextran 5000 Da, after 24 h culture incubation at 37 °C. Dextranase reported in this study is of great commercial importance as it is strictly inducible in nature and B. licheniformis being non-pathogenic removes the safety concerns associated with production of dextran fractions for clinical and pharmaceutical usage.