Synthesis and analysis of silica nanocarriers for pectinase immobilization: Enhancing enzymatic stability for continuous industrial applications.

Pectinolytic enzymes are among the important group of industrial enzymes that have wide applications in different food industries. In this study, pectinase-based silica nanocarriers were synthesized using co-precipitation and cross-linking techniques. The resulting silica nanoparticles were investigated using scanning electron microscopy (SEM), energy-dispersive electron microscopy (EDEX), and X-ray diffraction (XRD) for determination of its morphology, elemental composition, and crystalline pattern. Under the optimal immobilization conditions like 1.5 % glutaraldehyde, 3000 IU/mg pectinase concentration, 90 min immobilization time and 40 °C immobilization temperature, pectinase showed maximum immobilization yield. The immobilization of pectinase onto the silica nanocarriers led to enhanced catalytic characteristics, displaying higher enzymatic activity across various temperature and pH levels compared to soluble pectinase. Moreover, the immobilization substantially improved the temperature stability of pectinase, exhibiting 100 % of its initial activity even after 120 h of pre-incubation at 50 °C. Additionally, the silica nanocarrier pectinase retained 100 % of its original activity even after being reused 10 times in a single batch of reactions. These findings indicate that the immobilization of silica nanocarriers effectively enhances pectinase's industrial capabilities, making it economically feasible for industrial use and an efficient system for various biotechnological applications.

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.

Exploration of a three-dimensional matrix as micro-reactor in the form of reactive polyaminosaccharide hydrogel beads using multipoint covalent interaction approach.

OBJECTIVES: Diversity in backbone polymer composition makes hydrogel-based resources open to broad spectrum of applications. Biomacromolecules which have reactive functional groups in their structural frame and can also exhibit hydrogel properties could be utilized in biomedical, pharmaceutical and drug delivery applications after some chemical modifications. RESULTS: Present study aims towards development of chitosan-based hydrogel system crosslinked together with glucosyltransferase. Hydrogel structure worked as an immobilization matrix and as a microreactor system to catalyze the cleavage of a disaccharide. Uniform chitosan hydrogel beads were prepared and dextransucrase was attached using multipoint covalent interaction approach. Strong interaction was developed by linking polymeric hydrogel with the biocatalyst utilizing glutaraldehyde as spacer arms. This bifunctional crosslinking agent performed two important tasks that includes functionalization of hydrogel beads and crosslinking of this activated matrix system with enzyme fragments. Hydrogel beads required 18.0 h crosslinking time with enzyme (6.5 mg ml-1, 189.9 DSU) under specific environment (4 °C, 100 rpm) to saturate all available ends. Enzyme fragments were observed bound with hydrogel beads when screened for surface topology indicating successful crosslinking. Steady state kinetics of crosslinked dextransucrase was studied in detail and it was revealed that it can catalyse sucrose in 30.0 min at 35 °C (pH 5.5) with an energy of activation around 15.23 kJ mol-1 with increased Vmax (785 DSU ml-1) and Km (256 mM) values as compared to soluble enzyme version. Thermal stability of the crosslinked dextransucrase also particularly improved 2.5 fold at 45 °C in comparison with soluble enzyme. Improved catalytic performance suggests that multipoint covalent immobilization protocol adapted using hydrogel system could be tailored as microreactor for catalysis of profitable macromolecules.

Preparation, characterization and stability studies of cross-linked α-amylase aggregates (CLAAs) for continuous liquefaction of starch.

In current study, α-amylase of fungal origin was immobilized using cross-linking strategy. The influence of precipitant (ammonium sulphate) and cross-linker (glutaraldehyde) concentration revealed that 60% (w/v) precipitant and 1.5% (v/v) cross-linker saturation was required to attain optimum activity. Cross-linked amylase aggregates (CLAAs) were characterized and 10-degree shift in optimum temperature (soluble enzyme: 50 °C; cross-linked: 60 °C) and 1-unit shift in pH (soluble enzyme: pH -6; cross-linked: pH -7) was observed after immobilization. The Vmax for soluble α-amylase and its cross-linked form was 1225 U ml-1 and 3629 U ml-1, respectively. The CLAAs was more thermostable than its soluble form and retained its 30% activity even after 60 min of incubation at 70 °C. Moreover, cross-linked amylase retained its activity after two months while its soluble counterpart lost its complete activity after 10 and 20 days at 30 °C and 4 °C storage, respectively. Reusability test showed that cross-linked amylase could retain 13% of its residual activity after 10 repeated cycles. Therefore, 10 times more glucose was produced after cross-linking than soluble amylase when it was utilized multiple times. This study indicates that amylase aggregates are highly effective for continuous liquefaction of starch, hence have strong potential to be used for different industrial processes.

Inhibitory mechanism of BAC-IB17 against ß-lactamase mediated resistance in methicillin-resistant Staphylococcus aureus and application as an oncolytic agent.

Cancer remains a foremost cause of deaths worldwide, despite several advances in the medical science. The conventional chemotherapeutic methods are not only harmful for normal body cells but also become inactive due to the development of resistance by cancer cells. Therefore, the demand of safe anticancer agents is increasing and enforced the bottomless research on the bacteriocins. Several studies have reported the selective anticancer property of bacteriocins. Current research is the contribution to explore the exact mechanism of action and in vitro application of bacteriocin (BAC-IB17) as an oncolytic agent. In this study, ß-lactamase mediated resistance of methicillin resistant Staphylococcus aureus (MRSA) was studied and inhibitory mechanism of MRSA by BAC-IB17 was investigated. Cytotoxic studies were conducted to analyze the anticancerous potential of BAC-IB17. Results revealed that BAC-IB17 inhibited the ß-lactamase and produced profound effect on the membrane integrity of MRSA confirmed by scanning electron microscope (SEM). FTIR spectroscopic analysis revealed the changes in the functional groups of bacterial cells before and after treatment with BAC-IB17. BAC-IB17 also found anticancer in nature as it kills HeLa cell lines with the IC50 value of 12.5 µg mL-1 with no cytotoxic effect on normal cells at this concentration. This specific anticancer property of BAC-IB17 will make it a promising candidate for the treatment of cancer after further clinical trials. Moreover, BAC-IB17 may control MDR bacteria responsible for the secondary complications in cancer patients.

Encapsulation of pectinase within polyacrylamide gel: characterization of its catalytic properties for continuous industrial uses.

Pectinase as a biocatalyst play a significant role in food and textile industries. In this study, the pectinase was immobilized by encapsulation within polyacrylamide gel to enhance its catalytic properties and ensure the reusability for continuous industrial processes. 9.5% acrylamide and 0.5% N, N'- methylenebisacrylamide concentration gave high percentage of pectinase immobilization yield within gel. The catalytic properties of immobilized pectinase was determined with comparison of soluble pectinase. The immobilization of pectinase within polyacrylamide gel didn't effect catalytic properties of pectinase and both the free and immobilized pectinase showed maximum pectinolytic activity at 45 °C and pH 10. The Michaelis-Menten kinetic behavior of pectinase was slightly changed after immobilization and immobilized pectinase showed somewhat higher Km and lower Vmax value as compared to soluble pectinase. Polyacrylamide gel encapsulation enhanced the thermal stability of pectinase and encapsulated pectinase showed higher thermal stability against various temperature ranging from ranging from 30 °C to 50 °C as compared free pectinase. Furthermore, the surface topography of polyacrylamide gel was analyzed using scanning electron microscopy and it was observed that the surface topography of polyacrylamide gel was changed after encapsulation. The encapsulation of pectinase within polyacrylamide gel enhanced the possibility of reutilization of pectinase in various industries and pectinase retained more than 50% of its initial activity even after seven batch of reactions.

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.

Isolation and characterization of bacteriophage to control multidrug-resistant Pseudomonas aeruginosa planktonic cells and biofilm.

Pseudomonas aeruginosa is Gram-negative bacterium, one of the leading cause of drug-resistant nosocomial infections in developing countries. This bacterium possesses chromosomally encoded efflux pumps, poor permeability of outer-membrane and high tendency for biofilm formation which are tools to confer resistance. Bacteriophages are regarded as feasible treatment option for control of resistant P. aeruginosa. The aim of the current study was isolate and characterized a bacteriophage against P. aeruginosa with MDR and biofilm ability. A bacteriophage MA-1 with moderate host range was isolated from waste water. The phage was considerable heat and pH stable. Electron microscopy revealed that phage MA-1 belongs to Myoviridae family. Its genome was dsDNA (≈50 kb), coding for eighteen different proteins (ranging from 12 to 250 KDa). P. aeruginosa-2949 log growth phase was significantly reduced by phage MA-1 (2.5 × 103 CFU/ml) as compared to control (without phage). Phage MA-1 also showed significant reductions of 2.0, 2.5 and 3.2 folds in 24, 48, and 74 h old biofilms after 6 h treatment with phage respectively as compared to control. It was concluded from this study that phage MA-1 has capability of killing P. aeruginosa planktonic cells and biofilm, but for complete eradication cocktail will more effective to avoid resistance.

Maltose deterioration approach: Catalytic behavior optimization and stability profile of maltase from Bacillus licheniformis KIBGE-IB4.

Maltase is an economically valuable enzyme that is used to catalyze the hydrolytic process of maltose and yields d-glucose as a product. In this study, the catalytic behavior of maltase was optimized under various physicochemical condition. Results indicated that bacterial maltase exhibited maximum catalytic activity at 45 °C and pH-6.5 after 5.0 min. It presented greater stability within 0.1 M K2HPO4 buffer having pH-6.5 and showed 100 % activity even after 1.0 h. It retained 83.6 % and 45.0 % activity at 40 °C after 1.0 and 3.0 h, respectively. The enzyme retained 90.0 % activity at -20 °C even after 60 days. The molecular weight of enzyme was deduced to be 157.2 kDa as calculated using polyacrylamide gel electrophoresis (PAGE) and zymography. It was concluded that the characterized maltase has notable stability profile with reference to temperature, pH and other reaction conditions which anticipates its utilization in various starch and maltose hydrolyzing processes for the synthesis of glucose.

Improvement of catalytic properties of starch hydrolyzing fungal amyloglucosidase: Utilization of agar-agar as an organic matrix for immobilization.

In this study, amyloglucosidase was immobilized within agar-agar through entrapment technique for the hydrolysis of soluble starch. Enzymatic activities of soluble and entrapped amyloglucosidase were compared using soluble starch as a substrate. Partially purified enzyme was immobilized and maximum immobilization yield (80%) was attained at 40 gL-1 of agar-agar. Enzyme catalysis reaction time shifted from 5.0 min to 10 min after immobilization. Similarly, a five-degree shift in temperature (60 °C-65 °C) and a 0.5 unit increase in pH (pH-5.0 to pH-5.5) were also observed. Substrate saturation kinetics revealed that Km of entrapped amyloglucosidase increased from 1.41 mg ml-1 (soluble enzyme) to 3.39 mg ml-1 (immobilized enzyme) whereas, Vmax decreased from 947 kU mg-1 (soluble enzyme) to 698 kU mg-1 (immobilized enzyme). Entrapped amyloglucosidase also exhibited significant catalytic performance during thermal and storage stability when compared with soluble enzyme. Reusability of entrapped amyloglucosidase for hydrolysis of soluble starch demonstrated its recycling efficiency up to six cycles which is an exceptional characteristic for continuous bioprocessing of soluble starch into glucose.

Characterization of cross-linked amyloglucosidase aggregates from Aspergillus fumigatus KIBGE-IB33 for continuous production of glucose.

Current research deals with immobilization of amyloglucosidase through carrier-free approach using cross-linking strategy. Cross-linked amyloglucosidase aggregates (CLAAs) with aggregation yield of 94% were prepared in 04 h by incorporating 40% ammonium sulfate and 1.5% glutaraldehyde in enzyme solution. CLAAs were characterized by optimizing various conditions including reaction time, pH, temperature and substrate concentration. It was noticed that after cross-linking no change in optimum reaction time and substrate concentration was observed however, a 5-degree shift in optimum temperature from 60 °C to 65 °C was obtained as compared to soluble amyloglucosidase. Activation energy (Ea) of amyloglucosidase as calculated from Arrhenius plot was 5.5 kcal mol-1 and 5.2 kcal mol-1 for soluble and cross-linked aggregates, respectively. Stability studies revealed that CLAAs can be used at higher temperatures for longer time period than soluble amyloglucosidase. Furthermore, data of recycling studies showed that CLAAs can be efficiently reused for 20 cycles with the retention of 63% of its initial activity. Due to the continuous reusability of CLAAs, the product formation is also increased 8 times from 5.71 mg ml-1 (soluble enzyme) to 46.548 mg ml-1 (CLAAs). Findings of this research show that carrier-free strategy is more effective for continuous hydrolysis of starch and production of glucose.

Characterization and interplay of bacteriocin and exopolysaccharide-mediated silver nanoparticles as an antibacterial agent.

Metallic nanoparticles have a substantial scientific interest because of their distinctive physicochemical and antimicrobial properties and the emergence of multidrug resistant pathogens could unlock the potential of nanoparticles to combat infectious diseases. The aim of the current study is to enhance the antibacterial potential of purified bacteriocin by combining bacteriocin and antibacterial silver nanoparticles (AgNPs). Hence, the interaction of natural antimicrobial compounds and antibacterial nanoparticles can be used as a potential tool for combating infectious diseases. In this study, a green, simple and effective approach is used to synthesize antibacterial AgNPs using fungal exopolysaccharide as both a reducing and stabilizing agent. The AgNPs were characterized by spectroscopic analysis, Scanning Electron Microscopy (SEM), Energy Dispersive X-ray spectroscopy (EDX) and Dynamic Light Scattering (DLS). Furthermore, the synergistic effect of bacteriocin-AgNPs was determined against pathogenic strains. The histogram of AgNPs indicated well-dispersed, stabilized and negatively charged particles with variable size distribution. The combination of bacteriocin with nanoparticles found to be more effective due to broad antibacterial potential with possibly lower doses. The current study is imperative to provide an alternative for the chemical synthesis of silver nanoparticles. It showed environmental friendly and cost effective green synthesis of antibacterial nanoparticles.

Role of two polysaccharide matrices on activity, stability and recycling efficiency of immobilized fungal amyloglucosidase of GH15 family.

Current study deals with immobilization of amyloglucosidase using two different strategies (entrapment and covalent binding). Chitosan beads were prepared using neutralization method while alginate beads were synthesized by simple gelation. Results of this study showed that percent recovery of amyloglucosidase after covalent binding was 85% however in case of entrapment it was 66%. Immobilization was optimized by standardizing various conditions including concentrations of polysaccharide (alginate: 4%; chitosan: 3%), divalent ions (0.2M) and glutaraldehyde (5%). Slight shift in catalytic efficiency of soluble amyloglucosidase in terms of reaction time, pH and temperature was also noticed after immobilization. Activation energy decreased after immobilization due to which stability of amyloglucosidase increased for longer time period as compared to soluble enzyme. Results of recycling studies showed that covalently bound amyloglucosidase retained more enzymatic activity even after 15 cycles as compared to the entrapped enzyme that lost its activity within 10 cycles.

Influence of different metals on the activation and inhibition of α-amylase from thermophilic Bacillus firmus KIBGE-IB28.

Thermophilic Bacillus firmus KIBGE-IB28 produced extracellular α-amylase at temperature 70°C. Enzyme was partially purified by ammonium sulfate precipitation with 42.80 fold purification and specific activity of 1889.6 U/mg. Effects of various metals on enzyme activity were determined and it was found that enzyme activity boosted significantly in presence of Ca(2+), K(2+), Ba(2+), Co(2+) and Ni(2+) whereas Zn(2+), Mg(2+), Na(2+) and Cu(2+) were found inhibitory at concentration 10mM.

Reversal of haloperidol induced motor deficits in rats exposed to repeated immobilization stress.

Stress is defined as a non specific response of body to any physiological and psychological demand. Preclinical studies have shown that an uncontrollable stress condition produces neurochemical and behavioral deficits. The present study was conducted to test the hypothesis that a decrease in the responsiveness of somatodendritic 5-hydroxytryptamine (5-HT)-1A receptors following adaptation to stress could attenuate haloperidol induced acute parkinsonian like effect. Results showed that single exposure (2h) to immobilization stress markedly decreased food intake, growth rate and locomotor activity but these stress-induced behavioral deficits were not observed following repeated (2h/day for 5 days) exposure of immobilization stress suggesting behavioral tolerance occurs to similar stress. An important finding of present study is a reversal of haloperidol-induced motor deficits in animals exposed to repeated immobilization stress than respective control animals. It is suggested that stress induced possible desensitization of somatodendritic 5-HT-1A as well as 5-HT-2C receptors could release dopamine system from the inhibitory influence of serotonin. On the other hand, an increase in the effectiveness of postsynaptic 5-HT-1A receptors elicits a direct stimulatory influence on the activity of dopaminergic neuron and is possibly involved in the reversal of haloperidol-induced parkinsonian like symptoms in repeatedly immobilized rats.

Saccharification and liquefaction of cassava starch: an alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process.

BACKGROUND: Cassava starch is considered as a potential source for the commercial production of bioethanol because of its availability and low market price. It can be used as a basic source to support large-scale biological production of bioethanol using microbial amylases. With the progression and advancement in enzymology, starch liquefying and saccharifying enzymes are preferred for the conversion of complex starch polymer into various valuable metabolites. These hydrolytic enzymes can selectively cleave the internal linkages of starch molecule to produce free glucose which can be utilized to produce bioethanol by microbial fermentation. RESULTS: In the present study, several filamentous fungi were screened for production of amylases and among them Aspergillus fumigatus KIBGE-IB33 was selected based on maximum enzyme yield. Maximum α-amylase, amyloglucosidase and glucose formation was achieved after 03 days of fermentation using cassava starch. After salt precipitation, fold purification of α-amylase and amyloglucosidase increased up to 4.1 and 4.2 times with specific activity of 9.2 kUmg⁻¹ and 393 kUmg⁻¹, respectively. Concentrated amylolytic enzyme mixture was incorporated in cassava starch slurry to give maximum glucose formation (40.0 gL⁻¹), which was further fermented using Saccharomyces cerevisiae into bioethanol with 84.0% yield. The distillate originated after recovery of bioethanol gave 53.0% yield. CONCLUSION: An improved and effective dual enzymatic starch degradation method is designed for the production of bioethanol using cassava starch. The technique developed is more profitable due to its fast liquefaction and saccharification approach that was employed for the formation of glucose and ultimately resulted in higher yields of alcohol production.