Immobilization of Invertase in DAD Type Polymers: Combination of Benzothiadiazole Acceptor Unit and 3,4-Ethylenedioxythiophene and Thiophene Donor Units

Conductive polymers with donor-acceptor-donor (DAD) type units; benzothiadiazole acceptor unit and 3,4-ethylenedioxythiophene (EDOT) and thiophene (Th) donor units, were investigated for immobilization of invertase. The polymers were prepared potentiodynamically from their monomers, M₁ (Th-benzothiadiazole-Th), M₂ (EDOT-benzothiadiazole-EDOT), M₃ (Th-benzothiadiazole-EDOT) and a copolymer, which is a homolog to homopolymer of M₃, was prepared from M₁ and M₂. Invertase was trapped between a two layer-composite; DAD polymer and polypyrrole. The characterization of immobilized invertase was performed using a spectrophotometric method at 540 nm. Kinetic parameters, V _max, maximum reaction rate and K_m , Michaelis-Menten constant values of immobilized invertase in DAD type homopolymer (P₃), 0.95 μmol min^?1 and 22.7 mM, respectively, are found between those of homopolymers P₁ and P₂ and are better than the copolymer. Polymers and copolymers exhibited a broad optimal temperature profile between 30 and 50°C compared to the free enzyme. Optimum pH (5.0) is the same as for free invertase. The electrodes were found to be stable with 100% activity during one day for 40 consecutive measurements. As to the self-life, 25% of the initial activity was lost in the first ten days, then the electrodes were stable with 75% activity for a 40 day storage at 4°C.     

Immobilization of Invertase in a Novel Proton Conducting Poly(vinylphosphonic acid) – poly(1-vinylimidazole) Network

A novel proton conducting polymer blend was prepared by mixing poly(vinylphosphonic acid) (PVPA) with poly(1-vinylimidazole) (PVI) at various stoichiometric ratios via changing molar ratio of monomer repeating unit to achieve the highest protonation. The polymer network having the most suitable stoichiometric ratio for substantial proton conductivity was prepared and characterized by FT-IR spectroscopy and proton conductivity measurements. The network was used for immobilization of invertase and some important kinetic parameters such as the maximum reaction rate (V_max) and Michaelis-Menten constant (K_m) were investigated for the immobilized invertase. Additionally, optimum temperature and pH were determined to acquire the best conditions for the highest enzyme activity. Operational stability of the entrapped enzyme was also examined. The results reveal that the most stable and highly proton conducting polymer network may play a pioneer role in the biosensors applications as given by FT-IR, elemental analysis, impedance spectroscopy and storage stability experiments.     

Entrapment of plant invertase within novel composite of agarose-guar gum biopolymer membrane

Invertase or β-d-Fructofuranosidase (E.C.3.2.1.26) was extracted from Cucumis melo. L. fruit (Family-Cucurbitaceae). Soluble, plant invertase enzyme was immobilized in novel composite of agarose-guar gum biopolymer matrix in the form of hydrophilic, porous membranes. The immobilized invertase was characterized for sucrose hydrolytic activity and leakage from the matrix support. The efficiency of immobilization was found to be 91% with negligible leaching. The kinetic parameters K_m and V_max for free and immobilized invertase were also determined. Immobilized invertase was optimally active in the wide pH range of 4.5-6.5. The immobilization process also enhanced the thermal stability of enzyme up to 65 °C. Immobilized invertase membranes showed excellent storage stability with shelf life of 110 days. Entrapped invertase showed better operational stability and reusability up to 12 cycles. The fluorescence spectra of the composite membranes were studied and compared with that of soluble enzyme. All these characteristics of the immobilized invertase membranes make them suitable for the fabrication of biosensors.     

Screening of Dowex® anion-exchange resins for invertase immobilization

Commercial yeast invertase (Bioinvert^®) was immobilized by adsorption on anion-exchange resins, collectively named Dowex^® (1×8:50–400, 1×4:50–400, and 1×2:100–400). Optimal binding was obtained at pH 5.5 and 32°C. Among different polystyrene beads, the complex Dowex-1×4–200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/invertase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The K _m and V _max for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL.     

Man-tailored cellulose-based carriers for invertase immobilization

Cellulose-based carriers Granocel were specially prepared and optimised for covalent immobilization of enzymes. The effects of carrier characteristics such as pore size, chemistry of anchor groups and their density on invertase immobilization efficiency were evaluated. It was found that the preferential adsorption/binding of the enzyme to a carrier during coupling and its activity after immobilization depended on microenvironmental effects created by hydrophilic surface of the carrier, functional groups and their activators. The best preparations (activity approx. 300 U/mL, high storage stability) were obtained for NH₂-Granocel activated with glutaraldehyde. It is probably due to Granocel modification with pentaethylenehexamine that gave a 19-atom spacer arm. The enzyme concentration in coupling mixture was optimised as well. The kinetic parameters of sucrose hydrolysis for native and immobilized invertase were evaluated. Compared to the native invertase, K _m value of immobilized enzyme was only twice higher with about three times lower substrate inhibition. Reaction runs in a well mixed batch reactors with native and immobilized invertase showed slightly slower reaction rate in the case of the enzyme covalently bound to Granocel. Very good stability of cellulose-based carrier was proved experimentally by 20 successive reaction runs in a batch reactor.     

Immobilization of invertase in conducting copolymers of 3-methylthienyl methacrylate

Immobilization of invertase in conducting copolymer matrices of 3-methylthienyl methacrylate with pyrrole and thiophene was achieved by constant potential electrolysis using sodium dodecyl sulfate (SDS) as the supporting electrolyte. Polythiophene (PTh) was also used in entrapment process for comparison. Kinetic parameters, Michaelis-Menten constant, K(m), and the maximum reaction rate, V(max), were investigated. Operational stability and temperature optimization of the enzyme electrodes were also examined.     

Preparation and properties of sclerotium rolfsii invertase immobilized on indion 48-R

Crude extracellular invertase fromSclerotium rolfsii, when coupled to glutaraldehyde activated Indion 48-R, retained 70–80% activity of the soluble enzyme. Immobilization resulted in a decrease in the pH and temperature optima but it increased the temperature stability. K_m and V_max also increased as a result of immobilization. Both soluble and immobilized invertase showed inhibition at high substrate concentrations. The bound enzyme showed excellent stability to repeated use and retained approx 90% of its initial activity after 8 cycles of use.     

Characterization and Potential Applications of Immobilized Glucose Oxidase and Polyphenol Oxidase

Pyrrole functionalized polystyrene (PStPy) was copolymerized with pyrrole to obtain a conducting copolymer, P(PStPy‐co‐Py) which is used as the immobilization matrix. Glucose oxidase and polyphenol oxidase enzymes were immobilized via the entrapment method by electrochemical polymerization. Enzyme electrodes were prepared by electrolysis at a constant potential using sodium dodecyl sulfate (SDS) as the supporting electrolyte during the copolymerization of PStPy with pyrrole. Maximum reaction rates (V_max) and enzyme affinities (Michaelis‐Menten constants, K_m) were determined for the enzyme entrapped both in polypyrrole (PPy) and P(PStPy‐co‐Py) matrices. Optimizations of enzyme electrodes were done by examining the effects of temperature and pH on enzymes' activities along with the shelf life and operational stability investigations. Glucose oxidase enzyme electrodes were used for human serum analysis and glucose determination in two brands of orange juices. Polyphenol oxidase enzyme electrodes were used for the determination of phenolics in red wines of Turkey.     

IMMOBILIZATION OF YEAST CELLS IN SEVERAL CONDUCTING POLYMER MATRICES

ABSTRACT

Immobilization of yeast cells (Saccharomyces cerevisiae) in different polymer matrices was performed by constant potential electrolysis. These matrices were polypyrrole (PPy); poly(methyl methacrylate)/polypyrrole (PMMA/PPy) and thiophene-capped poly(methyl methacrylate)/polypyrrole (TPMMA/PPy). The characterization of PMMA/PPy copolymer was achieved by Fourier-transform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC) and Scanning Electron Microscopy (SEM). The invertase activity of immobilized yeast cells was determined. Optimum temperature, Michaelis-Menten constants and maximum reaction rates of the enzyme electrodes were compared with those of free yeast cells. The operational and storage stabilities of three different immobilization systems were analyzed.     

Immobilization of manganese peroxidase fromLentinula edodes on azlactone-functional polymers and generation of Mn3+ by the enzyme-polymer complex

Manganese peroxidase (MnP) purified fromLentinula edodes was covalently immobilized on 3M’s azlactone-functional copolymer, 3M EmphazeTM AB1 Biosupport Medium. Tethered MnP is capable of generating Mn³⁺ from Mn²⁺ and H₂O₂. Mn₃₊, properly chelated, can be used as a nonspecific oxidant of organopollutants. A variety of conditions designed to maximize coupling efficiency while maintaining Mn³⁺ -generating catalytic activity were tested. Biochemical characteristics of the MnP enzyme, including amino acid composition, pH and temperature stability, and concentration of its Mn²⁺ substrate, influenced chemical conditions necessary for the coupling reaction. The physical parameters of immobilization reaction time, protein concentration, ionic conditions, pH, and temperature were examined. Results of these experiments indicated maximum coupling efficiency and enzyme activity were achieved by immobilizing at MnP concentrations < 2 mg/mL for at least 2 h using pH 7.0 buffer containing 1.0M sodium sulfate and 1.0 mM Mn²⁺. Increasing coupling reaction temperature also improved coupling efficiency. A synthesis of these optimized immobilizations yielded MnP coupling efficiencies of 40–50% with 35% of the coupled protein retaining enzymatic activity. Results of MnP immobilizations on nonporous azlactone-functional dispersion polymers more hydrophobic than Emphaze are also reported, and coupling efficiencies > 65% with 100% of the coupled enzyme active have been measured.     

A new method to prepare macroporous copolymer of glycidyl methacrilate and ethylene dimethacrilate as enzyme carrier

Macroporous copolymer of glycidyl methacrylate and ethylene dimethacrylate containing surface epoxy groups was firstly synthesized with dodecyl alcohol and cyclohexanol as liquid pore-forming agents and nanosize calcium carbonate as solid porogenic agent. The scanning electron microscopy was used to characterize their surface structure. Under the optimum conditions, β-galactosidase from Aspergillus oryzae was immobilized on the support obtained above, and the basic property and the kinetic data of the reaction on immobilized enzyme were determined. These data were compared with those obtained for the enzyme immobilized on the support prepared only with the liquid solution as pore-forming agent. Satisfactory results were obtained in enzyme activity, immobilization yield, pH stability, thermal stability, operational stability, and Michaelis constants K _M. The results indicated that the copolymer of glycidyl methacrilate and ethylene dimethacrylate newly prepared was more suitable to immobilize enzyme than the carrier synthesized with traditional method.     

Investigation of Yeast Invertase Immobilization onto Cupric Ion-Chelated,Porous, and Biocompatible Poly(Hydroxyethyl Methacrylate-<Emphasis Type="Italic">n</Emphasis>-Vinyl Imidazole) Microspheres

Cupric ion-chelated poly(hydroxyethyl methacrylate-n-vinyl imidazole) (poly(HEMA-VIM)) microspheres prepared by suspension polymerization were investigated as a specific adsorbent for immobilization of yeast invertase in a batch system. They were characterized by scanning electron microscopy, surface area, and pore size measurements. They have spherical shape and porous structure. The specific surface area of the p(HEMA-VIM) spheres was found to be 81.2 m²/g with a size range of 70–120 μm in diameter, and the swelling ratio was 86.9%. Then, Cu(II) ion chelated on the microspheres (546 μmol Cu(II)/g), and they were used in the invertase adsorption. Maximum invertase adsorption was 51.2 mg/g at pH 4.5. Cu(II) chelation increases the tendency from Freundlich-type to Langmuir-type adsorption model. The optimum activity for both free and adsorbed invertase was observed at pH 4.5. The optimum temperature for the poly(HEMA-VIM)/Cu(II)-invertase system was found to be at 55 °C, 10 °C higher than that of the free enzyme at 45 °C. V _max values were determined as 342 and 304 U/mg enzyme, for free and adsorbed invertase, respectively. K _m values were found to be same for free and adsorbed invertase (20 mM). Thermal and pH stability and reusability of invertase increased with immobilization.     

Production and Characterization of a Novel Yeast Extracellular Invertase Activity Towards Improved Dibenzothiophene Biodesulfurization

The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml^?1) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5–6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5–6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l^?1 sucrose and 250 μM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l^?1 sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its μ_max from 0.035 to 0.070 h^?1 and attained a 2-hydroxybiphenyl productivity of 5.80 μM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.     

Partitioning invertase between a dilute water solution and generated droplets

Water droplets or mist occur naturally in the air at seashores. These water droplets carry inorganic and organic substances from the sea to the land via the air, creating fertile land in sandy coastal areas (1). The same phenomenon occurs in an air-fluidized bed bioreactor (2). In an air-fluidized bed reactor, proteins can be transferred from the bioreactor semisolid bulk phase to an enriched droplet phase. This protein transfer process (droplet fractionation) can be experimentally simulated by shaking a separatory funnel containing a dilute solution of a given protein, which can be an enzyme like invertase. The created droplets become richer in invertase (protein) than that of the original dilute solution. The droplets can then be coalesced by tranpping them and recovering the concentrated protein in the new liquid phase. Typically, in such a droplet fractionation process a collected enzyme can be degraded in its ability tocatalyze a chemical reaction. In this article, we explore whether the initial solution pH control variable can be adjusted to minimize the decrease of enzyme activity in this process. The protein droplet recovery problem is one in which the recovered amount of desired protein (enzyme) in the droplet is maximized, subject to the minimization of the enzyme activity loss. The partition coefficient, which is the ratio between the protein concentration in the droplets and the residual solution, is maximized at approx 4.8 and occurs at pH 3.0. Here, the partition coefficient for invertase decreases as the initial solution pH increases, between pH 3.0 and 8.0. Interestingly, the initial solution surface tension seems to be inversely proportional to the partition coefficient. The partition coefficien treachesa maximum value at a surface tension value of approx 63 mN/m at pH 3.0. The enzymatic activity of the initial, the residual, and the droplet solutions all decrease as the bulksolution pH increases. Adecrease of enzymatic activity was observed in the residual bulk solution when compared with that in the initial bulk solution at all pH levels. Also, up to 90% of the invertase activity was lost in the droplets when compared to the initial bulk solution.     

Concanavalin a immobilized affinity adsorbents for reversible use in yeast invertase adsorption

Concanavalin A (Con A) immobilized poly(2-hydroxyethyl methacrylate) (PHEMA) beads were investigated for specific adsorption of yeast invertase from aqueous solutions. PHEMA beads were prepared by a suspension polymerization technique with an average size of 150-200 microm, and activated by epichlorohydrin. Con A was then immobilized by covalent binding onto these beads. The maximum Con A immobilization was found to be 10 mg/g. The invertase-loading capability of the PHEMA/Con A beads was 107 mg/g. The maximum invertase adsorption capacity on the PHEMA/Con A adsorbents was observed at pH 5.0. The values of the Michaelis constant K(m) of invertase were significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V(max) was smaller for the adsorbed invertase. Adsorption improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with adsorption. The adsorbed enzyme activity was found to be quite stable in repeated experiments. Storage stability of adsorbed invertase.     

Poly(pyrrole) versus poly(3,4-ethylenedioxythiophene): amperometric cholesterol biosensor matrices

Two conducting polymers, poly(pyrrole) (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT) were used as immobilization matrices for cholesterol oxidase (ChOx). The amperometric responses of the enzyme electrodes were measured by monitoring oxidation current of H₂O₂ at +0.7 V in the absence of a mediator. Kinetic parameters, such as K _m and I _max, operational and storage stabilities, effects of pH and temperature were determined for both entrapment supports. K _m values are found as 7.9 and 1.3 mM for PPy and PEDOT enzyme electrodes, respectively; it can be interpreted that ChOx immobilized in PEDOT shows higher affinity towards the substrate.     

Invertase immobilization onto radiation-induced graft copolymerized polyethylene pellets

The graft copolymer poly(ethylene-g-acrylic acid) (LDPE-g-AA) was prepared by radiation-induced graft copolymerization of acrylic acid onto low density polyethylene (LDPE) pellets, and characterized by infrared photoacoustic spectroscopy and scanning electron microscopy (SEM). The presence of the grafted poly(acrylic acid) (PAA) was established. Invertase was immobilized onto the graft polymer and the thermodynamic parameters of the soluble and immobilized enzyme were determined. The Michaelis constant, K_m, and the maximum reaction velocity, V_max, were determined for the free and the immobilized invertase. The Michaelis constant, K_m was larger for the immobilized invertase than for the free enzyme, whereas V_max was smaller for the immobilized invertase. The thermal stability of the immobilized invertase was higher than that of the free enzyme.     

Highly Efficient Method Towards In Situ Immobilization of Invertase Using Cryogelation

A novel method was developed for the immobilization of Saccharomyces cerevisiae invertase within supermacroporous polyacrylamide cryogel and was used to produce invert sugar. First, the cross-linking of invertase with soluble polyglutaraldehyde (PGA) was carried out prior to immobilization in order to increase the bulkiness of invertase and thus preventing the leakage of the cross-linked enzyme after immobilization by entrapment. And then, in situ immobilization of PGA cross-linked invertase within cryogel synthesis was achieved by free radical polymerization in semi-frozen state. The method resulted in 100 % immobilization and 74 % activity yields. The immobilized invertase retained all the initial activity for 30 days and 30 batch reactions. Immobilization had no effect on optimum temperature and it was 60 °C for both free and immobilized enzyme. However, optimum pH was affected upon immobilization. Optimum pH values for free and immobilized enzyme were 4.5 and 5.0, respectively. The immobilized enzyme was more stable than the free enzyme at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes and microorganisms.     

Importance of C-Terminal Region for Thermostability of GH11 Xylanase from Streptomyces lividans

The amino acid sequences of xylanase B (XlnB) and xylanase C (XlnC) from Streptomyces lividans show significant homology. However, the temperature optima and stabilities of the two enzymes are quite different. XlnB exhibits an optimum temperature of 40 °C and retains 50% of its maximum activity at 43 °C, whereas the corresponding values for XlnC are 60 and 70 °C. To analyze these properties further, as well as to study the effect of the exchange of homologous segments in the C-terminal region, four chimeras designated as BSC, BFC, CSB, and CFB were constructed by substituting segments from the C-terminal homologous region of XlnB gene with that of XlnC and in turn substituting XlnC gene with that of XlnB. The purified chimeric enzymes were characterized with respect to pH/temperature activity, stability, and kinetic parameters. Most of enzymatic properties of chimeras were admixtures of those of the two parents. The chimeric enzymes were optimally active at 45–55 °C and pH 7.0. Both K _m and k _cat values of chimeric enzymes for p-nitrophenyl-β-d-cellobioside were admixtures of both parental enzymes, except that the k _cat value of chimeric BFC (2.79 s⁻¹) was higher than that of parental XlnC (1.99 s⁻¹). Notably, thermal stability of chimeric BSC and BFC was increased by 25 and 13 °C separately, as compared to one of parental XlnB, whereas the thermal stability of chimeric CSB and CFB was decreased by 23 and 21 °C, respectively, as compared to another parental XlnC. These results suggest that homologous C-terminal region in S. lividans GH11 xylanase appears to play an important role in determining enzyme characteristics, and exchanging of different segments of gene in this region might significantly alter or improve the enzymatic properties such as thermal stability.     

Study of immobilized and extracellular invertase of lemon balm

Cell suspensions of lemon balm (Melissa officinalis L.) were permeabilized by Tween 20, Tween 80, ethanol, hexadecyltrimethylammonium bromide, and hexadecylpyridinium chloride, and immobilized by glutaraldehyde. The invertase pH optimum was 4.5 at temperature 50°C. The hydrolysis of substrate was linear for 4 h, reaching 60% conversion. The cells had high invertase activity and good stability, and in longterm storage they showed good physicomechanical properties. The culture medium (without cells) was used for the identification and determination of extracellular enzyme activity. Intracellular activity was estimated from the cell suspension. For the lemon balm cell suspension, the intracellular activity accounted for 83.7% of the total activity, and the extracellular one for 12.7%. The intracellular specific activity is 4.2 times higher. Our method permits the rapid, simple, and specific identification and determination of plant invertase. Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 611-615, November-December, 2008.