Development of the magnetic beads for dye ligand affinity chromatography and application to magnetically stabilized fluidized bed system

Magnetic poly(2-hydroxyethylmethacrylate) [mPHEMA] beads were prepared by suspension polymerization of HEMA in the presence of Fe₃O₄ nano-powder. Cibacron Blue F3GA was covalently immobilized to the mPHEMA beads via nucleophilic substitution reaction between chloride of its triazine ring and hydroxyl groups of HEMA under alkaline conditions. The mPHEMA/Cibacron Blue F3GA beads (100–140 μm in diameter) carrying 68.3 μmol Cibacron Blue F3GA per gram polymer were used for β-casein adsorption studies. Adsorption studies were performed under different conditions in a batch system (i.e., pH, β-casein initial concentration, temperature, and ionic strength) and then in a magnetically stabilized fluidized bed (MSFB) system. The swelling ratio of the mPHEMA was 62.1%. The maximum adsorption capacity for batch system was 20.2% lower as compared to the value obtained in MSFB. The mPHEMA/Cibacron Blue F3GA beads could be repeatedly applied for β-casein adsorption without significant losses in the adsorption capacity.     

Preparation and characterization of mixed-mode magnetic adsorbent with p-amino-benzamidine ligand: Operated in a magnetically stabilized fluidized bed reactor for purification of trypsin from bovine pancreas

The magnetic beads were synthesized using glycidylmethacrylate (GMA) and methylmethacrylate (MMA) monomers. A multimodal ligand (i.e., p-amino-benzamidine) was covalently immobilized onto magnetic beads after glutaraldehyde activation, and consequently used for purification of the trypsin from bovine pancreas. The p-amino-benzamidine ligand immobilized magnetic beads were characterized by FTIR, VSM, SEM, and analytical methods. Trypsin adsorption experiments were investigated under different experimental conditions (i.e., medium pH, initial trypsin concentration, temperature, and ionic strength) in a batch system. Maximum trypsin adsorption capacity was found to be 75.9 ± 2.6 mg/g beads. Adsorbed trypsin was eluted by using (0.1 M acetate buffer, pH 3.0) with a 97% recovery. The purification factor of trypsin from crude pancreas extract was 8.7 folds. The purity of the eluted trypsin from p-amino-benzamidine functionalized magnetic beads was determined as 86% by HPLC. The method developed in this report was successfully applied for purification of the trypsin from crude pancreas extract in a magnetically stabilized fluidized bed reactor.     

Genetic algorithm-based medium optimization for enhanced production of fluorescent pseudomonad R81 and siderophore

Fluorescent pseudomonad R81, a root-colonizing bacterium, is a potential bio-inoculant due to its plant growth promoting characteristics. It produces hydroxamate-type siderophore which is involved in disease suppression in plants. Genetic algorithm (GA) methodology was applied for the optimization of siderophore and cell mass production simultaneously in shake flask experiments. A total of 10 medium components were optimized within 80 experiments. A high siderophore concentration of 1.9 g/L and cell mass concentration of 2.8 g/L was achieved in the optimized medium. The application of GA was well suited for determination of optimum concentration levels of the medium constituents for a bi-objective function. GA was able to increase the siderophore concentration by 2.8-fold when compared to RSM-based optimization. Further, the batch fermentation of the GA-optimized medium in 14 L bioreactor without pH control produced 2.2 g/L siderophore in 36 h, the highest reported so far. GA was also successfully used to estimate the kinetic parameters of the mathematical models of the batch fermentation.     

Cold-active extracellular α-amylase production from novel bacteria Microbacterium foliorum GA2 and Bacillus cereus GA6 isolated from Gangotri glacier,Western Himalaya

Microbial cold-active α-amylases offer various economical and ecological benefits through energy savings by overcoming the heating requirements and also provide large biotechnological potentials. The objective of present study was to isolate new cold-adapted bacterial strains for production of cold-active α-amylases and their production optimization. Out of 30 cold-active α-amylase producing bacteria, isolated from soil of Gangotri glacier, Western Himalaya, India, two potential isolates, designated as GA2 and GA6, were selected for enzyme production. The α-amylase production was found maximum at 20 °C and pH 9 after 120 h incubation for GA2; and 20 °C and pH 10 after 96 h incubation for GA6. Among the carbon sources, lactose and glycerol was most suitable for GA2 and GA6, respectively. However, yeast extract and ammonium acetate was found best as nitrogen source by GA2 and GA6, respectively. Out of two potential isolates, maximum enzyme production (5870 units) was achieved with GA2 followed by GA6 (4746 units). GA2 was resistant to penicillin (10 μg) among tested antibiotics and as per plasmid curing results, amylase production was a plasmid mediated characteristic. The phylogenetic analysis revealed that GA2 and GA6 have highest homology with Microbacterium foliorum (99%) and Bacillus cereus (98%), respectively. This was the first report on cold-active α-amylase production by M. foliorum strain GA2 and B. cereus strain GA6, also their 16S rRNA sequences assigned an accession number HQ832574 and HQ832575, respectively from NCBI.     

Treatment of high-strength ammonium wastewater by polyvinyl alcohol–sodium alginate immobilization of activated sludge

We employed microorganism embedding immobilization technology to treat high-strength ammonium(NH₄⁺-N) wastewater. Experiments were conducted in batch reactors with different initial ammonium concentrations (50–400 mg/L), 10% particle dosage rates, 7.5–8.5 pH, and 495-min operation cycle. Stable treatment efficiency was reached in the 28th, 40th, 55th, 58th, and 58th cycles with average ammonium removal rates of 100, 100, 80.9, 64.6, and 48.0%, respectively. The ammonium removal reaction followed zero-order reaction kinetics. Brunauer-Emmett-Teller (BET) and Scanning Electron Microscopy (SEM) demonstrated that the specific surface area and pore size of beads in stable phase were larger than corresponding values for the unused embedding beads, and microorganisms were found in the interior and external surface of beads. High-throughput sequencing illustrated that the microbial community composition significantly differed between the interior and external surface of embedding beads. And the existence of heterotrophic nitrifying and aerobic denitrifying bacteria may provide additional pathways for biological nitrogen removal in the reactors.     

Biotechnological potential of Azolla filiculoides for biosorption of Cs and Sr: Application of micro-PIXE for measurement of biosorption

The presence of Cs and Sr in culture medium of Azolla filiculoides caused about 27.4% and 46.3% inhibition of biomass growth, respectively, in comparison to A. filiculoides control weight which had not metals. Biosorption batch experiments were conducted to determine the Cs and Sr binding ability of native biomass and chemically modified biosorbents derived from Azolla namely ferrocyanide Azolla sorbents type 1 and type 2 (FAS1 and FAS2) and hydrogen peroxide Azolla sorbent (HAS). The best Cs and Sr removal results were obtained when A. filiculoides was treated by 2 M MgCl₂ and 30 ml H₂O₂ 8 mM at pH 7 for 12 h and it was then washed by NaOH solution at pH 10.5 for 6 h. Pretreatment of Azolla have been suggested to modify the surface characteristics which could improve biosorption process. The binding of Cs and Sr on the cell wall of Azolla was studied with micro-PIXE and FT-IR.     

Immobilization of the cross-linked para-nitrobenzyl esterase of Bacillus subtilis aggregates onto magnetic beads

The para-nitrobenzyl esterase (PNBE), which was encoded by pnbA gene from Bacillus subtilis, was immobilized on amino-functionalized magnetic supports as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE-CLEA immobilized on the magnetic beads using glutaraldehyde as a coupling agent was 31.4 mg/g of beads with a 78% activity recovery after the immobilization. The performance of immobilized PNBE-CLEA was evaluated under various conditions. As compared to its free form, the optimal pH and temperature of PNBE-CLEA were 1 unit (pH 8.0) and 5 °C higher (45 °C), respectively. Under different temperature settings, the residual enzyme activity was highest for the PNBE-CLEA, followed by covalently fixed PNBE without further cross-linking and the free PNBE. During 40 days of storage pried, the PNBE-CLEA maintained more than 90% of its initial activity while the free PNBE maintained about 60% under the same condition. PNBE-CLEA also retained more than 80% activity after 30 reuses with 30 min of each reaction time, indicating stable reusability under aqueous medium.     

A flow-through enzymatic microreactor immobilizing lipase based on layer-by-layer method for biosynthetic process: Catalyzing the transesterification of soybean oil for fatty acid methyl ester production

A simple and convenient method was proposed in this paper to develop a flow-through enzymatic micro-reactor made from polytetrafluoroethylene (PTFE). It consisted of the polydopamine layer (functioned as a primer) and layer by layer (LBL) coatings composed of polyethylenimine (PEI) and lipase. The multiple deposition of PEI and lipase was the key factor of increasing the enzyme loading on microreactor. After 8 PEI/lipase layers, enzyme loading on the inner surface of 5-m microchannel reached a maximum (350 μg to 400 μg), compared with approximately 20 μg in single layer. Microreactor with higher enzyme loading was successfully applied on transesterification of soybean oil for effective fatty acid methyl ester (FAME, biodiesel) production. A 95.2% conversion rate of biodiesel can be achieved in 53 min under optimized conditions, instead of a couple of hours in the traditional batch reaction.     

Gallic acid interference on polyphenolic amperometric biosensing using Trametes versicolor laccase

The present work reports the gallic acid (GA) interference on polyphenolic amperometric biosensing using Trametes versicolor laccase (TvLac). GA′ inhibitory effect on TvLac activity was investigated on the oxidation of caffeic acid (CA) by free TvLac and its immobilised form on modified polyethersulfone membrane (PES/TvLac), using spectrophotometric and amperometric biosensor detection methods. The results have indicated that GA presents inhibitory behaviour on TvLac activity in a concentration-dependent manner. The GA concentration leading to 50% activity lost, IC₅₀, was determined to be 19.15 ± 0.11 μM and 5.11 ± 0.19 μM for free and immobilised enzyme, respectively. The results have also shown that GA exhibited a competitive and a mixed inhibition types on the TvLac activity for spectrophotometric and amperometric biosensor methods, respectively. Further GA′ and CA′ cyclic voltammetry studies have demonstrated that GA′ oxidation products interfered with CA′ redox reaction products. In fact, a decrease of the reduction current was observed at cyclic voltammograms of CA, when mixed with GA. Therefore, the GA′ interference on polyphenolic amperometric biosensing is the result of the combination of two factors: on one hand, we have the inhibitory enzymatic effect, and on the other, the reaction of GA′ oxidation products with the o-quinones obtained by the enzymatic oxidation of CA. Both gave rise to the amperometric signal decreasing effect.     

Improved yield and stability of amylase by multipoint covalent binding on polyglutaraldehyde activated chitosan beads: Activation of denatured enzyme molecules by calcium ions

In this study raw starch digesting amylase (RSDA) from Aspergillus carbonarius (Bainier) Thom IMI 366159 was stabilized by covalent binding on polyglutaraldehyde (PG), glutaraldehyde (G) activated chitosan beads or post immobilization cross linking of enzyme adsorbed on chitosan. Presence of Ca²⁺ ions (0.5–1.5 mM) activated the PG and G derivatives but repressed the crosslinked enzyme. Optimum pH for cross linked derivative increased by 2 units but was unaltered for PG and G derivatives. Immobilized amylase exhibited improved thermal and storage stability. Immobilized derivatives had no loss of activity after 1 month storage and retained above 90% activity after 10 batch reactions of 60 min each. Immobilization successfully stabilized RSDA and immobilized enzyme from A. carbonarius can be applied in numerous industries for cheap, cost effective and environmentally friendly starch hydrolytic processes to simple sugars.     

Development of Novel Glucose oxidase Immobilization on Graphene/Gold nanoparticles/Poly Neutral red modified electrode

Glucose oxidase (GOx) was immobilized onto glassy carbon electrode (GCE) that modified by reduced graphene oxide-gold nanoparticles- poly neutral red (RGO/AuNPs/PNR) nanocomposite. The composite was analyzed by scanning electron microscope (SEM), energy dispersive x-ray (EDX) spectroscopy, atomic force microscopy (AFM), attenuated total reflectance (ATR), cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS). SEM/EDX analysis showed the morphological of the nanocomposite. AFM results showed the morphology and structure of the RGO/AuNPs and RGO surfaces. The covalent bonding between glucose oxidase and composite was confirmed by ATR technique. The electrochemical experiments were done in 100 mM phosphate buffer at pH 7 and temperature of 25 °C with three electrodes including Ag/AgCl, platinum wire and the modified GCE as the reference electrode, the auxiliary electrode and working electrode respectively. The electrochemical results confirmed the activity and direct electron transfer of immobilized enzyme. The immobilized electroactive GOx concentration was estimated 3.06 × 10⁻¹¹ mol cm⁻². The results showed the immobilized enzyme had a good stability and maintained 90% of its performance after two weeks. The nanocomposite bioanode in an air-birthing biofuel cell and 100 mM glucose concentration showed 176 μWcm⁻² Power density. This strategy could be used for GOx-based biofuel cells.     

Surfactant-mediated permeabilization of Pseudomonas putida KT2440 and use of the immobilized permeabilized cells in biotransformation

Surfactants were used to permeabilize cells of Pseudomonas putida KT2440 so as to maximize retention of the arginine deiminase (ADI) activity within the treated cells. The surfactants cetyltrimethylammoniumbromide (CTAB), sodium dodecyl sulfate (SDS) and Triton X100 were tested separately. Statistical models were developed for the effects on the ADI activity of the following factors: the concentration of the surfactant, the length of the treatment period and the concentration of the cells. For all surfactants, the concentration of cells was the most significant factor in influencing permeabilization. All permeabilization treatments used mild conditions (pH 7, 37 °C). The permeabilized cells were immobilized in alginate beads for the biotransformation of arginine to citrulline. The optimal conditions for immobilization and biotransformation were as follows: 2% (w/v, g/100 mL) sodium alginate, 100 g/L of treated cells, 40 mM arginine, pH 6.0, a temperature of 35 °C and an agitation speed of 150 rpm. The immobilized biocatalyst retained nearly 90% of its initial activity after nine cycles of repeated use in batch operations. In contrast, the freely suspended cells were barely active after the second use cycle.     

Poly(glycidylmethacrylate) brushes generated on poly(VBC) beads by SI-ATRP technique: Hydrazine and amino groups functionalized for invertase adsorption and purification

Crosslinked-poly(vinylbenzylchloride), poly(VBC), beads were prepared by suspension polymerization and poly(glycidylmethacrylate) was grafted by surface-initiated-atom radical polymerization (SI-ATRP) technique. Epoxy groups of the grafted poly(GMA) were reacted with hydrazine and ammonia to create an affinity binding sites. The hydrazine and amine functionalized poly(VBC-g-GMA) beads were used as an affinity support for adsorption of invertase from solution and yeast crude extract. The influence of pH, equilibrium time, ionic strength and initial invertase concentration on the adsorption capacities of both hydrazine and amine functionalized beads has been investigated. Maximum invertase adsorptions onto hydrazine and amine functionalized beads, were 86.7 and 30.4 mg/g at pH 4.0 and 5.5, respectively. The experimental equilibrium data fitted well to the Temkin isotherm model. Finally, the hydrazine functionalized poly(VBC-g-GMA) beads were used for the purification of invertase from crude yeast extract in a batch system and the purity of the eluted invertase from the hydrazine functionalized beads was determined as 92% by HPLC from single step purification protocol.     

Halogenol- versus alkanol-induced structural transitions of acid-denatured glucoamylase: Characterization of alcohol-induced states

Different probes such as far- and near-UV CD spectral signals, ANS binding, Trp fluorescence and three-dimensional fluorescence were used to study halogenol- versus alkanol-induced conformational transitions in the acid-denatured state (pH 1.0) of Aspergillus niger glucoamylase (GA). These alcohols showed significant retrieval of the protein structure, inducing both secondary and tertiary structural changes, as evident from the increase in the α-helix and decrease in ANS binding, respectively. However, halogenols were found more competent than alkanols, requiring lesser alcohol concentration to induce similar spectral change. The effectiveness of these alcohols showed the order: HFIP > TFE > 2-chloroethanol for halogenols while tert-butanol > 1-propanol > 2-propanol > ethanol > methanol for alkanols. Both Trp fluorescence and near-UV CD spectra showed anomalous pattern, though the order of effectiveness remained the same as found with far-UV CD spectra and ANS fluorescence. Three-dimensional fluorescence results of the acid-denatured state (pH 1.0) of GA in the presence of 5.5 M tert-butanol agreed well with the data obtained from far-UV CD and Trp fluorescence. All these results suggested the formation of partially folded states of GA obtained in the presence of these alcohols, being more effective with halogenols than alkanols.     

Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH₄Cl, and l-tryptophan at a constant feeding rate of 16 mL h⁻¹, the final concentration of violacein reached 4.13 g L⁻¹, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L⁻¹ and 82.6 mg L⁻¹ h⁻¹, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.     

Immobilization of urease on vapour phase stain etched porous silicon

Porous silicon layers fabricated by the reaction-induced vapor phase stain etch method were coated with 5% polyethylenimine. Urease from Canavalia brasiliensis beans was immobilized on this support through covalent linking with 2.5% glutaraldehyde. The pH and temperature profile of the immobilized and free urease exhibited higher activity at pH 6.5 and 37 °C. After being stored for 30 days at 4 °C, the immobilized enzyme had 75% of the initial activity. The maximum apparent Michaelis constant for free urease (K_m) was 94.33 mM whereas for immobilized urease was 53.04 mM. The maximum reaction velocity (V_max) for free urease was 3.51 mmol/min and for immobilized urease was 1.57 mmol/min.     

Decolorization of azo dye by immobilized Pseudomonas luteola entrapped in alginate–silicate sol–gel beads

Pseudomonas luteola was immobilized by entrapment in alginate–silicate sol–gel beads for decolorization of the azo dye, Reactive Red 22. The influences of biomass loading and operating conditions on specific decolorization rate and dye removal efficiency were studied in details. The immobilized cells were found to be less sensitive to changes in agitation rates (dissolved oxygen levels) and pH values. Michaelis–Menten kinetics could be used to describe the decolorization kinetics with the kinetic parameters being 36.5 mg g⁻¹ h⁻¹, 300.1 mg l⁻¹ and 18.2 mg g⁻¹ h⁻¹, 449.8 mg l⁻¹ for free and immobilized cells, respectively. After five repeated batch cycles, the decolorization rate of the free cells decreased by nearly 54%, while immobilized cells still retained 82% of their original activity. The immobilized cells exhibited better thermal stability during storage and reaction when compared with free cells. From SEM observation, a dense silicate gel layer was found to surround the macroporous alginate–silicate core, which resulted in much improved mechanical stability over that of alginate beads when tested under shaking conditions. Alginate–silicate matrices appeared to be the best matrix for immobilization of P. luteola in decolorization of Reactive Red 22 when compared with previous results using synthetic or natural polymer matrices.     

Cyclodextrin glucanotransferase synthesis by semicontinuous cultivation of magnetic biocatalysts from cells of Bacillus circulans ATCC 21783

Cells of the alkalotolerant producer of cyclodextrin glucanotransferase (CGTase) Bacillus circulans ATCC 21783 were used as a model for preparing of magnetic biocatalysts applied for CGTase synthesis in batch and semicontinuous processes. The cell immobilization was carried out with four types of magnetic nano- and microparticles: magnetite microparticles (1–5 μm), entrapped in agar gel beads with bacterial cells (AM-biocatalyst); silanized magnetite (20–40 nm) covalently bound on the cell surface (SM-biocatalyst); and alkaline and citrate ferrofluids (10–20 nm), attached on the cell wall by an ionic interaction (FF-alkaline and FF-citrate biocatalyst). The highest CGTase production was achieved after 96 h of semicontinuous process using SM-biocatalysts (particularly, these composed of 80 mg silanized magnetite and 140 mg bacterial cells) when the specific enzyme activity was 8.4-fold higher compared to that of free cells. Cells modified with magnetic alkaline and citrate ferrofluids exhibited 2.19- and 1.55-fold increase of the specific CGTase activities. Magnetic nanoparticles linked on the cell walls by ionic interactions were partially released during the cultivation, while the covalent bond between the activated magnetite and the cells was very stable. The data obtained demonstrate convincingly the effect of the magnetic technologies for an effective enzyme production.     

Lipase immobilized microstructured fiber based flow-through microreactor for facile lipid transformations

A facile continuous flow-through Candida antarctica lipase B immobilized silica microstructured optical fiber (SMOF) microreactor for application in lipid transformations has been demonstrated herewith. The lipase was immobilized on the amino activated silica fiber using glutaraldehyde as a bifunctional reagent. The immobilized lipase activity in the SMOF was tested calorimetrically by determination of p-nitrophenyl butyrate hydrolysis products. The specific activity of the immobilized lipase was calculated to be 0.91 U/mg. The SMOF microreactor performance was evaluated by using it as a platform for synthesis of butyl laurate from lauric acid and n-butanol in n-hexane and n-heptane at 50 °C, with products identified by gas chromatography–mass spectrometry (GC–MS). Different substrate mole ratios were evaluated, with 1:3, lauric acid:n-butanol showing best performance. Remarkably, percentage yields of up to 99% were realized with less than ∼38 s microreactor residence time. In addition, the SMOF microreactor could be reused many times (at least 7 runs) with minimal reduction in the activity of the enzyme. The enzyme stability did not change even with storage of the microreactor in ambient conditions over one month.     

Treatment of dye-rich wastewater by an immobilized thermophilic cyanobacterial strain: Phormidium sp.

The removal of Remazol Blue and Reactive Black B by the immobilized thermophilic cyanobacterial strain Phormidium sp. was investigated under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye removal. In the experiments, performed at pH 8.5, with different initial dye concentrations between 9.1 mg l⁻¹ and 82.1 mg l⁻¹ and at 45 °C, calcium alginate immobilized Phormidium sp. showed high dye decolorization, with maximum uptake yields ranging from 50% to 88% at all dye concentrations tested. When the effects of high dye concentrations on dye removal were investigated, the highest uptake yield in the beads was 50.3% for 82.1 mg l⁻¹ Remazol Blue and 60.0% for 79.5 mg l⁻¹ Reactive Black B. The highest color removal was detected at 45 °C and 50 °C incubation temperatures for all dye concentrations. As the temperature decreased, the removal yield of immobilized Phormidium sp. also decreased. At about 75 mg l⁻¹ initial dye concentrations, the highest specific dye uptake measured was 41.29–41.17 mg g⁻¹ for Remazol Blue and 47.69–43.82 mg g⁻¹ for Reactive Black B at 45 °C and 50 °C incubation temperatures, respectively, after 8 days incubation.