Development of Pectinase Based Nanocatalyst by Immobilization of Pectinase on Magnetic Iron Oxide Nanoparticles Using Glutaraldehyde as Crosslinking Agent.

To increase its operational stability and ongoing reusability, B. subtilis pectinase was immobilized on iron oxide nanocarrier. Through co-precipitation, magnetic iron oxide nanoparticles were synthesized. Scanning electron microscopy (SEM) and energy dispersive electron microscopy (EDEX) were used to analyze the nanoparticles. Pectinase was immobilized using glutaraldehyde as a crosslinking agent on iron oxide nanocarrier. In comparison to free pectinase, immobilized pectinase demonstrated higher enzymatic activity at a variety of temperatures and pH levels. Immobilization also boosted pectinase's catalytic stability. After 120 h of pre-incubation at 50 °C, immobilized pectinase maintained more than 90% of its initial activity due to the iron oxide nanocarrier, which improved the thermal stability of pectinase at various temperatures. Following 15 repetitions of enzymatic reactions, immobilized pectinase still exhibited 90% of its initial activity. According to the results, pectinase's catalytic capabilities were enhanced by its immobilization on iron oxide nanocarrier, making it economically suitable for industrial use.

Single step immobilization of CMCase within agarose gel matrix: Kinetics and thermodynamic studies.

In the current study, CMCase from Bacillus licheniformis KIBGE-IB2 was immobilized within the matrix of agarose gel through entrapment technique. Maximum immobilization yield (%) of the enzyme was obtained when 2.0 % agarose was used. The activation energy (Ea) of the enzyme increased from 16.38 to 44.08 kJ mol-1 after immobilization. Thermodynamic parameters such as activation energy of deactivation (ΔGd), enthalpy (ΔHd) and entropy (ΔSd) of deactivation, deactivation rate constant (Kd), half-life (t1/2), D-value and z-value were calculated for native/free and immobilized CMCase. The maximum reaction rate (Vmax) of the native enzyme was found to be 8319.47 U ml-1 min-1, which reduced to 7218.1 U ml-1 min-1after immobilization process. However, the Michaelis-Menten constant (Km) value of the enzyme increased from 1.236 to 2.769 mg ml-1 min-1 after immobilization. Immobilized enzyme within agarose gel matrix support can be reuse up to eight reaction cycles. Broad stability profile and improved catalytic properties of the immobilized CMCase indicated that this enzyme can be a plausible candidate to be used in various industrial processes.

Production of commercially important enzymes from Bacillus licheniformis KIBGE-IB3 using date fruit wastes as substrate.

BACKGROUND: Pakistan is one of the top five date fruit-producing countries and produced more than 30% wastes in picking, packing, storage, and commercialization stages. The date fruit wastes are usually considered inedible for humans and only used for livestock feed. In current research, Bacillus licheniformis KIBGE-IB3 was screened for pectinase, xylanase, cellulase, and amylase production using date fruit wastes as substrate through solid state fermentation. RESULTS: The B. licheniformis KIBGE-IB3 produced higher concentration of pectinase using date fruit wastes as substrate as compared to amylase, cellulase, and xylanase. B. licheniformis KIBGE-IB3 produced maximum pectinase using 5.0 g/dl date fruit wastes and 0.5 g/dl yeast extract. B. licheniformis KIBGE-IB3 required pH 7.0, 37 °C incubation temperature, and 72 h incubation period for maximum production of pectinase. CONCLUSION: It has been concluded that date fruit waste is a good source of biomass and can be utilized for the commercial production of pectinase.

Utilization of different polymers for the improvement of catalytic properties and recycling efficiency of bacterial maltase.

Current study deals with the comparative study related to immobilization of maltase using synthetic (polyacrylamide) and non-synthetic (calcium alginate, agar-agar and agarose) polymers via entrapment technique. Polyacrylamide beads were formed by cross-linking of monomers, agar-agar and agarose through solidification while alginate beads were prepared by simple gelation. Results showed that the efficiency of enzyme significantly improved after immobilization and among all tested supports agar-agar was found to be the most promising and biocompatible for maltase in terms of immobilization yield (82.77%). The catalytic behavior of maltase was slightly shifted in terms of reaction time (free enzyme, agarose and polyacrylamide: 5.0 min; agar-agar and alginate: 10.0 min), pH (free enzyme, alginate and polyacrylamide: 6.5; agar-agar, agarose: 7.0) and temperature (free enzyme: 45 °C; alginate: 50 °C; polyacrylamide: 55 °C; agarose: 60 °C; agar-agar: 65 °C). Stability profile of immobilized maltase also revealed that all the supports utilized have significantly enhanced the activity of maltase at higher temperatures then its free counterpart. However, recycling data showed that agar-agar entrapped maltase retained 20.0% of its initial activity even after 10 cycles followed by agarose (10.0%) while polyacrylamide and alginate showed no activity after 8 and 6 cycles respectively.

Production of α-1,4-glucosidase from Bacillus licheniformis KIBGE-IB4 by utilizing sweet potato peel.

In the current study, sweet potato peel (Ipomoea batatas) was observed as the most favorable substrate for the maximum synthesis of α-1,4-glucosidase among various agro-industrial residues. Bacillus licheniformis KIBGE-IB4 produced 6533.0 U ml-1 of α-1,4-glucosidase when growth medium was supplemented with 1% dried and crushed sweet potato peel. It was evident from the results that bacterial isolate secreted 6539.0 U ml-1 of α-1,4-glucosidase in the presence of 0.4% peptone and meat extract with 0.1% yeast extract. B. licheniformis KIBGE-IB4 released 6739.0 and 7190.0 U ml-1 of enzyme at 40 °C and pH 7.0, respectively. An improved and cost-effective growth medium design resulted 8590.0 U ml-1 of α-1,4-glucosidase with 1.3-fold increase as compared to initial amount from B. licheniformis KIBGE-IB4. This enzyme can be used to fulfill the accelerating demand of food and pharmaceutical industries. Further purification and immobilization of this enzyme can also enhance its utility for various commercial applications. Graphical abstract Pictorial representation of maltase production from sweet potato peel.

Polyacrylamide Gel-Entrapped Maltase: An Excellent Design of Using Maltase in Continuous Industrial Processes.

Bacterial maltase catalyzes the hydrolysis of maltose and is known as one of the most significant hydrolases. It has several applications in different industrial processes but widely used in food fermentation technology and alcohol production. In the current study, entrapment technique was comprehensively examined using polyacrylamide gel as a matrix support to improve the stability and catalytic efficiency of maltase for continuous use. Maximum entrapment yield of maltase was achieved at 10 % polyacrylamide concentration with 3.0-mm bead size. Optimized conditions indicated an increase in the reaction temperature from 45 to 55 °C after maltase entrapment while no change was observed in the reaction time and pH. An increase in the K m value of entrapped maltase was attained whereas V max value decreased from 8411.0 to 6813.0 U ml(-1) min(-1) with reference to its free counterpart. Entrapped maltase showed remarkable thermal stability and retained 16 % activity at 70 °C even after 120.0 min. Entrapped maltase also exhibited excellent recycling efficiency up to eight consecutive reaction cycles. With respect to economic feasibility, entrapped maltase indicates its high potential to be used in various biotechnological applications.

Continuous degradation of maltose by enzyme entrapment technology using calcium alginate beads as a matrix.

Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme-substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in Vmax of immobilized enzyme from 8411.0 to 4919.0 U ml-1 min-1 whereas, Km apparently increased from 1.71 to 3.17 mM ml-1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.

Morphological and molecular based identification of pectinase producing Bacillus licheniformis from rotten vegetable.

Pectinase catalyzed the degradation of pectin substances and has been used in various biotechnological industries. In the current study, 23 bacterial strains were isolated from rotten vegetables, soil and air. The isolated bacterial strains were qualitatively screened for pectinase production on pectin agar medium and only three strains HR 4, HR 21 and HR 23 were observed to produce extracellular pectinase. These strains were further screened quantitatively for pectinase production through submerged fermentation technology in pectin containing fermentation medium. Strain HR 4 from rotten brinjal (Solanum melongena) was found to produce higher pectinase as compared to others. The maximum pectinase producing bacterial strain was identified as Bacillus licheniformis on the basis of morphological, physiological and biochemical characteristics. For further confirmation of identification, 16S rDNA sequence analysis was performed. The 16S rDNA sequences were aligned and the phylogenetic tree was constructed. The phylogenetic tree confirmed that the strain was belonging to B. licheniformis. The 16S rDNA sequences of this new strain were submitted to GenBank and designated as B. licheniformis KIBGE-IB21 with the GenBank accession number JQ 411812. The newly isolated pectinase producing B. licheniformis used apple pectin as carbon and yeast extract as nitrogen source for maximum pectinase production.

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.

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.

Influence of phytic acid and its metal complexes on the activity of pectin degrading polygalacturonase.

Polygalacturonase is one of the important requirements of different microorganism to cause pathogenicity and spoilage of fruits and vegetables that involved in degradation of pectin during plant tissue infections. In current study, 20 mM phytic acid inhibited 70% activity of polygalacturonase. The effect of different concentration of metal ions such as Cu(+2), Al(+3) and V(+4) were studied separately and it was found that the 20 mM of these metal ions inhibited 37.2%, 79%, and 53% activity of polygalacturonase, respectively. Finally, the complexes of phytic acid and these metals ions were prepared and 1:1 ratio of phytic acid and metal ions complexes showed maximum inhibitory activity of enzyme as compared to complexes having 1:2 and 1:3 ratio except phytate-copper complexes which showed no inhibitory effect on the activity of polygalacturonase.

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.