Immobilization of chitosan-modified invertase on alginate-coated chitin support via polyelectrolyte complex formation

Saccharomyces cerevisiae invertase was chemically modified with chitosan and further immobilized on sodium alginate-coated chitin support. The yield of immobilized protein was determined as 85% and the enzyme retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 9 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was four-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 80% of the original catalytic activity after 50 h under continuous operational regime in a packed bed reactor.     

Polyelectrolyte complex formation mediated immobilization of chitosan-invertase neoglycoconjugate on pectin-coated chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on pectin-coated chitin support via polyelectrolyte complex formation. The yield of immobilized enzyme protein was determined as 85% and the immobilized biocatalyst retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 10 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 4-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 96 and 95% of the original catalytic activity after ten cycles of reuse and 74 h of continuous operational regime in a packed bed reactor, respectively.     

Immobilization of the recombinant invertase INVB from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on Nylon-6

The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 °C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 °C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 °C. The values for kinetic parameters were determined as: 984 and 98 mM for of immobilized and free re-INVB, respectively. values for immobilized and free enzymes were 6.1 × 10² and 1.2 × 10⁴ s⁻¹, respectively, and immobilized re-INVB showed of 158.73 μmol h min⁻¹ mg⁻¹. Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 °C and 70% at 40 °C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme.     

Immobilization of chitosan-invertase neoglycoconjugate on carboxymethylcellulose-modified chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on a carboxymethylcellulose-coated chitin support via polyelectrolyte complex formation. The yield of immobilized protein was determined to be 72% and the enzyme retained 68% of the initial invertase activity. The optimum temperature for invertase was increased by 5 degrees C and its thermostability was enhanced by about 9 degrees C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 12.6-fold more resistant to thermal treatment at 65 degrees C than the native counterpart. The prepared biocatalyst retained 98% and 100% of the original catalytic activity after 10 cycles of reuse and 70 h of continuous operational regime in a packed bed reactor, respectively. The immobilized enzyme retained 95% of its activity after 50 days of storage at 37 degrees C.     

Continuous removal of Cr(VI) from aqueous solution catalysed by palladised biomass of Desulfovibrio vulgaris

Growth-decoupled cells of Desulfovibrio vulgaris NCIMB 8303 can be used to reduce Pd(II) to cell-bound Pd(0) (Bio-Pd⁰), a bioinorganic catalyst capable of reducing hexavalent chromium to less toxic Cr(III), using formate as the electron donor. Magnetic resonance imaging showed that Bio-Pd⁰, immobilized in chitosan and agar beads, is distinguishable from the surrounding gel and is evenly dispersed within the immobilization matrix. Agar-immobilized Bio-Pd⁰ and `chemical Pd⁰' were packed into continuous-flow reactors, and challenged with a solution containing 100 m Cr(VI) (pH 7) at a flow rate of 2.4 ml h^–1. Agar-immobilized chemical Pd⁰ columns lost Cr(VI) reducing ability by 160 h, whereas columns containing immobilized Bio-Pd⁰ maintained 90% reduction until 680 h, after which reduction efficiency was gradually lost.     

Development of an Amperometric-Based Glucose Biosensor to Measure the Glucose Content of Fruit

An amperometric enzyme-electrode was introduced where glucose oxidase (GOD) was immobilized on chitosan membrane via crosslinking, and then fastened on a platinum working electrode. The immobilized enzyme showed relatively high retention activity. The activity of the immobilized enzyme was influenced by its loading, being suppressed when more than 0.6 mg enzyme was used in the immobilization. The biosensor showing the highest response to glucose utilized 0.21 ml/cm² thick chitosan membrane. The optimum experimental conditions for the biosensors in analysing glucose dissolved in 0.1 M phosphate buffer (pH 6.0) were found to be 35°C and 0.6 V applied potential. The introduced biosensor reached a steady-state current at 60 s. The apparent Michaelis-Menten constant (KMapp) of the biosensor was 14.2350 mM, and its detection limit was 0.05 mM at s/n > 3, determined experimentally. The RSD of repeatability and reproducibility of the biosensor were 2.30% and 3.70%, respectively. The biosensor was showed good stability; it retained ~36% of initial activity after two months of investigation. The performance of the biosensors was evaluated by determining the glucose content in fruit homogenates. Their accuracy was compared to that of a commercial glucose assay kit. There was no significance different between two methods, indicating the introduced biosensor is reliable.     

Immobilized, thermostable S- and F-forms of the extracellular invertase from Candida utilis can hydrolyse sucrose up to 100 °C

When grown on a sucrose-containing medium, Candida utilis synthesizes and secretes two invertases: one of molecular size of 280 kDa (the S-form – Slow-migrating) and a new form of Mr of 62 kDa (the F-form – Fast-migrating). Prior to immobilization, purification of S- and F-forms of invertase increased the immobilization yield to 89–100%, in comparison with that of crude invertase preparation (52%). The immobilized purified S- and F-form of invertase remained partially active after 15 min at 100 °C; the F-form retained almost 30% of its maximum activity. The immobilized S-form or F-form of invertase almost completely inverted (95% hydrolysis) 60% (w/v) sucrose over 5 h continuous reaction at 80 °C. Moreover, at 90 °C the immobilized F-form hydrolysed 70% of 60% (w/v) sucrose over 5 h, while the capability of the immobilized S-form of inverting sucrose over 5 h reaction decreased from 80% to 45%.     

Characterization of <Emphasis Type="Italic">Mucor miehei</Emphasis> lipase immobilized on polysiloxane-polyvinyl alcohol magnetic particles

Mucor miehei lipase was immobilized on magnetic polysiloxane-polyvinyl alcohol particles by covalent binding. The resulting immobilized biocatalyst was recycled by seven assays, with a retained activity around 10% of its initial activity. K_m and V_max were respectively 228.3 M and 36.1 U mg of protein^–1 for immobilized enzyme. Whereas the optimum temperature remained the same for both soluble and immobilized lipase (45 °C), there was a shift in pH profiles after immobilization. Optimum pH for the immobilized lipase was 8.0. Immobilized enzyme showed to be more resistant than soluble lipase when assays were performed out of the optimum temperature or pH.     

Immobilization of invertase on a new type of macroporous glycidyl methacrylate

Invertase was immobilized via its carbohydrate moiety. The immobilized enzyme has a specific activity of 5500 IU g^–1, with 45% activity yield on immobilization. In a packed bed reactor, 90% 2.5 M sucrose was converted at a flow rate of 4 bed volumes h^–1. The obtained specific productivity at 40 °C of 3 kg l^–1 h^–1 is the best one so far. Long-term stability was 290 days in 2.5 M sucrose at 40 °C and at a flow rate of 3 bed volumes h^–1.     

Stimulation of acid invertase activity by indol-3yl-acetic acid in tissues undergoing cell expansion

The soluble acid invertase activity of young, excised P. vulgaris internodal segments fell when they were incubated in water, and their elongation ceased within 6–7 h. IAA (10 M) promoted segment elongation and stimulated an increase in the specific activity of acid invertase to a level greater than that originally present. The rate of segment elongation in the presence of IAA was closely and positively correlated with the specific activity of the enzyme. Optimum concentration of IAA for both elongation and stimulation of invertase activity was 10 M. Concurrent protein synthesis was necessary for these responses to IAA. Segments cut from mature, fully-elongated internodes did not responsd to IAA.Inclusion of Ca²⁺, vanadate or mannitol in the incubation medium abolished IAA-induced segment elongation but did not inhibit the stimulation of acid invertase activity by IAA. Auxin-induced elongation and acid invertase activity were both substantially increased in the presence of up to 25 mM D-glucose or up to 50 mM sucrose. Inclusion of either sugar in the medium considerably increased tissue hexose concentrations. Under some circumstances cell growth and invertase synthesis may compete for available hexose substrate.It is concluded that IAA-induced promotion of acid invertase in P. vulgaris internodal segments is not simply an indirect consequence of removal of end-product (hexose) during IAA-induced cell growth and that a more direct action of IAA on enzyme turnover is involved.     

Production of cyclodextrin glycosyltransferase by immobilized <Emphasis Type="Italic">Bacillus</Emphasis> sp. on chitosan matrix

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol–sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml^?1 (36 h), 47.50 U ml^?1 (36 h) and 68.36 U ml^?1 (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml^?1 (18 h) on cassava, 79.17 U ml^?1 (12 h) on potato and 55.37 U ml^?1 (in 6 h and max 77.75 U ml^?1 in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.     

Immobilization of Bacillus licheniformis L-Arabinose Isomerase for Semi-Continuous L-Ribulose Production

Bacillus licheniformis L-arabinose isomerase (BLAI) with a broad pH range, high substrate specificity, and high catalytic efficiency for L-arabinose was immobilized on various supports. Eupergit C, activated-carboxymethylcellulose, CNBr-activated agarose, chitosan, and alginate were tested as supports, and Eupergit C was selected as the most effective. After determination of the optimum enzyme concentration, the effects of pH and temperature were investigated using a response surface methodology. The immobilized BLAI enzyme retained 86.4% of the activity of the free enzyme. The optimal pH for the immobilized BLAI was 8.0, and immobilization improved the optimal temperature from 50 °C (free enzyme) to a range between 55 and 65 °C. The half life improved from 2 at 50 °C to 212 h at 55 °C following immobilization. The immobilized BLAI was used for semi-continuous production of L-ribulose. After 8 batch cycles, 95.1% of the BLAI activity was retained. This simple immobilization procedure and the high stability of the final immobilized BLAI on Eupergit C provide a promising solution for large-scale production of L-ribulose from an inexpensive L-arabinose precursor.     

Chemically surface modified gel (CSMG): an excellent enzyme-immobilization matrix for industrial processes

Invertase from S. cerevisiae has been immobilized on porous silica matrix, formed using sol-gel chemistry, with surface area of approximately 650 m(2)/g. The co-condensation of silica sol with 3-aminopropyl(triethoxy)silane produced an amino-chemically surface modified silica gel (N-CSMG) with a very high ligand loading of 3.6 mmol/g SiO(2); significantly higher than commercially available matrices. Surface amine groups were activated with glutaraldehyde to produce GA-N-CSMG, and invertase covalently attached by the aldehyde. Invertase was used as a model enzyme to measure the immobilizing character of the GA-N-CSMG material. Using an optimized immobilization protocol, a very high loading of 723 mg invertase per gram GA-N-CSMG is obtained; 3-200-fold higher than values published in literature. The reproducible, immobilized activity of 246,000 U/g GA-N-CSMG is also greater than any other in literature. Immobilized invertase showed almost 99% retention of free enzyme activity and no loss in catalytic efficiency. The apparent kinetic parameters K(M) and V(M) were determined using the Michealis-Menten kinetic model. K(M) of the free invertase was 1.5 times greater than that of the immobilized invertase--indicating a higher substrate affinity of the immobilized invertase. These findings show considerable promise for this material as an immobilization matrix in industrial processes.     

Immobilization and stabilization of invertase on Cajanus cajan lectin support

Use of lectins as ligands for the immobilization and stabilization of glycoenzymes has immense application in enzyme research and industry. But their widespread use could be limited by the high cost of their production. In the present study preparation of a novel and inexpensive lectin support for use in the immobilization of glycoenzymes containing mannose or glucose residues in their carbohydrate moiety has been described. Cajanus cajan lectin (CCL) coupled covalently to cyanogen bromide activated Seralose 4B could readily bind enzymes such as invertase, glucoamylase and glucose oxidase. The immobilized and glutaraldehyde crosslinked preparations of invertase exhibited high resistance to inactivation upon exposure to enhanced temperature, pH, denaturants and proteolysis. Binding of invertase to CCL-Seralose was however found to be readily reversible in the presence of 1.0 M methyl alpha-D mannopyranoside. In a laboratory scale column reactor the CCL-Seralose bound invertase was stable for a month and retained more than 80% of its initial activity even after 60 days of storage at 4 degrees C. CCL-Seralose bound invertase exhibited marked stability towards temperature, pH changes and denaturants suggesting its potential to be used as an excellent support for the immobilization of other glycoenzymes as well.     

Sucrose hydrolysis by thermostable immobilized inulinases from aspergillus ficuum

The possibility of using thermostable inulinases from Aspergillus ficuum in place of invertase for sucrose hydrolysis was explored. The commercial inulinases preparation was immobilized onto porous glass beads by covalent coupling using activation by a silane reagent and glutaraldehyde before adding the enzyme. The immobilization steps were optimized resulting in a support with 5,440 IU/g of support (sucrose hydrolysis) that is 77% of the activity of the free enzyme. Enzymatic properties of the immobilized inulinases were similar to those of the free enzymes with optimum pH near pH 5.0. However, temperature where the activity was maximal was shifted of 10 degrees C due to better thermal stability after immobilization with similar activation energies. The curve of the effect of sucrose concentration on activity was bi-phasic. The first part, for sucrose concentrations lower than 0.3 M, followed Michaelis-Menten kinetics with apparent K(M) and Vm only slightly affected by immobilization. Substrate inhibition was observed at values from 0.3 to 2 M sucrose. Complete sucrose hydrolysis was obtained for batch reactors with 0.3 and 1 M sucrose solutions. In continuous packed-bed reactor 100% (for 0.3 M sucrose), 90% (1 M sucrose) or 80% sucrose conversion were observed at space velocities of 0.06-0.25 h(-1). The operational half-life of the immobilized inulinases at 50 degrees C with 2 M sucrose was 350 days.     

Immobilization of a keratinolytic protease from Purpureocillium lilacinum on genipin activated-chitosan beads

Keratinase from Purpureocillium lilacinum LPSC # 876 was immobilized on chitosan beads using two different cross-linking agents: glutaraldehyde and genipin. For its immobilization certain parameters were optimized such as cross-linker concentration, activation time and activation temperature. Under optimum conditions, enzyme immobilization resulted to be 96 and 92.8% for glutaraldehyde and genipin, respectively, with an activity recovery reaching up to 81% when genipin was used. The immobilized keratinase showed better thermal and pH stabilities compared to the soluble form, retaining more than 85% of its activity at pH 11 and 74% at 50 °C after 1 h of incubation. The residual activity of immobilized keratinase remained more than 60% of its initial value after five hydrolytic cycles. The results in this study support that glutaraldehyde could be replaced by genipin as an alternative cross-linking eco-friendly agent for enzyme immobilization.     

Immobilization of lactoperoxidase on graphene oxide nanosheets with improved activity and stability

Objectives

The purpose of this study was to develop a facile and efficient method to enhance the stability and activity of lactoperoxidase (LPO) by using its immobilization on graphene oxide nanosheets (GO-NS).

Methods

Following the LPO purification from bovine whey, it was immobilized onto functionalized GO-NS using glutaraldehyde as cross-linker. Kinetic properties and stability of free and immobilized LPO were investigated.

Results

LPO was purified 59.13 fold with a specific activity of 5.78 U/mg protein. The successful immobilization of LPO on functionalized GO-NS was confirmed by using dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR). The overall results showed that the stability of the immobilized LPO was considerably improved compared to free LPO. Apparent K_m and V_max of LPO also indicated that the immobilized enzyme had greater affinity to the substrate than the native enzyme.

Conclusions

Graphene oxide nanosheets are effective means for immobilization of LPO.

     

Immobilization of yeast cells in hydroxyethylmethacrylate gels

Summary Whole cells of Saccharomyces cerevisiae were entrapped in polymers of 2-hydroxyethylmetha-crylate and sucrose hydrolysis catalysed by its invertase was investigated.Analysis of the experimental results confirmed that diffusional resistance to mass transfer of reactant and product was not induced by immobilization.For the yeast cells in the hydrogel, invertase activity obeyed a Michaelis-Menten kinetic and the value of Km (40 mM) was the same as that for yeast cells in bulk phase.The recovery of biocatalyst activity ranged between 17% and 23%, depending on immobilization temperature; the optimum pH range was found to be slightly wider.Storage stability at refrigerator temperature was quite satisfactory; invertase half-life was 267 days. Operational stability of immobilized cells at 45°C (half-life 110 days) was almost twice that of free cells.Finally, cell distribution in the polymer, observed with a scanning electron microscope, was found to be uniform.Symbols C Active cell concentration, g/mg - Ea Activation energy, cal/mol - Kd kinetic constant of the enzyme deactivation reaction, h - Km Michaelis constant, mM - Nc Active cell amount, mg - r Enzymatic reaction rate, mol/min - S Substrate concentration, mM - t Reaction time, h or days - T Reaction temperature, °C or °K - Tp Polymerization temperature, °C - V _max Kinetic constant of enzymatic reaction, mol/min     

Phytate dephosphorylation by free and immobilized cells ofSaccharomyces cerevisiae

Summary Saccharomyces cerevisiae in the form of baker's yeast, cells cultivated on a yeast extract-peptone-glucose medium, as well as cells immobilized in 18% (w/v) polyacrylamide gel showed the ability to hydrolyze 1.727 mM sodium phytate solution at 45°C, pH 4.6, in a stirred tank reactor. Seventy percent yield of dephosphorylation was observed after 2 h using a baker's yeast concentration of 5.8 g dry matter per 100 ml. Hydrolytic activity at 1.8–2.0 M Pi min^–1 was observed between 1st and 3rd h of the reaction in cells cultured 24 or 48 h. No inhibition by the substrate was found at sodium phytate concentrations of 0.587–1.727 mM. After 1.5 h of hydrolysis a single, well distinguished peak ofmyo-inositol-triphosphate was the main product found. By means of immobilization the stability of the biocatalyst was enhanced 3.3-fold and reached its half-life at 64 ninety-minute runs.     

Properties of carbohydrate-metabolizing enzymes immobilized in sol-gel beads: stabilization of invertase and β;-glucosidase by Blue Dextran**

When immobilized in sol-gels, invertase (;-fructofuranosidase) from Candida utilis and ;-glucosidase from Pyrococcus furiosus had activity recovery values of 30 and 28%, respectively. However, if Blue Dextran (0.04%) was included in the immobilization-reaction mixture, the respective recovery values increased to 63 and 52%. Glucose dehydrogenase from Thermoplasma acidophilum immobilized by the same method lost most of its activity and Blue Dextran had no effect on the recovery of activity during the immobilization procedure. The immobilized enzymes required treatment with glutaraldehyde in order to maintain their activity within the sol-gel matrix during continuous reaction with their respective substrates.