Continuous chitosan hydrolyzate production by immobilized chitosanolytic enzyme from Enterobacter sp. G-1.

Chitosanolytic enzymes from Enterobacter sp. G-1 were immobilized on various carriers to continuously hydrolyze chitosan. Four different carriers were tested: FE-3901 (strong basic anion exchange resin, ionic binding), glutaraldehyde-treated FE-4612 (weak basic anion exchange resin, cross-linking), Chitopearl (chitosan beads), and alginate calcium. Glutaraldehyde-treated FE-4612 and Chitopearl immobilized more protein than the others. The enzyme immobilized on FE-3901 had the greatest activity. The activity of enzyme immobilized on FE-3901 decreased rapidly when exposed to a continuous flow of 1% chitosan. The enzyme immobilized with Chitopearl retained more than 50% of its original activity after 17 days, and the activity was fully restored by re-immobilization.     

Immobilization of levan fructotransferase for the production of di-fructose anhydride from levan

Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was immobilized on various carriers of which Chitopearl BCW2501 beads showed the higher activity of 320 U g^–1 for the formation of di-fructose anhydride compounds. The immobilized enzyme retained about 60% of its initial activity after being used for 20 cycles.     

Continuous production of maltotetraose using a dual immobilized enzyme system of maltotetraose-forming amylase and pullulanase

In order to produce a product with a high content of maltotetraose, dual-enzyme systems composed of immobilized maltotetraose-forming amylase (G(4)-forming amylase) and pullulanase were studied. The thermostability of individually immobilized enzymes was examined in continuous operation; studies revealed that the enzyme immobilized on "Chitopearl" was much more stable than that immobilized on Diaion HP-50. The effects of operating conditions on the stability of G(4) forming amylase immobilized on "Chitopearl" were examined to confirm that the apparent half-life data could be arranged using the immobilized enzyme stability factor, f(s). As for the dual immobilized enzyme system, six methods of usage were considered, with five yielding a 7-10% (w/w) higher content of maltotetraose product than the single-enzyme system. The effects of operating conditions on the maltotetraose production reaction were examined to confirm that the maltotetraose content of the products could be analyzed using the specific space velocity,SSV. In dual immobilized enzyme systems, pullulanase immobilized on the same carrier as the G(4)-forming amylase was found to be more stable than pullulanase immobilized on separate carriers. The effectiveness of using immobilized pullulanase along with the G(4)-forming amylase was confirmed from constant-conversion operations in which the maltotetraose content in the product was kept at 50% (w/w) in laboratory-scale experimentation.     

On the Activities of Glucoamylase Chemically Fixed to Cyanogen Bromide-activated Cellulose

N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than that of strain KNK712.     

Amberlite IRA 900 versus calcium alginate in immobilization of a novel,engineered β-fructofuranosidase for short-chain fructooligosaccharide synthesis from sucrose

The immobilization of β-fructofuranosidase for short-chain fructooligosaccharide (scFOS) synthesis holds the potential for a more efficient use of the biocatalyst. However, the choice of carrier and immobilization technique is a key to achieving that efficiency. In this study, calcium alginate (CA), Amberlite IRA 900 (AI900) and Dowex Marathon MSA (DMM) were tested as supports for immobilizing a novel engineered β-fructofuranosidase from Aspergillus japonicus for scFOS synthesis. Several immobilization parameters were estimated to ascertain the effectiveness of the carriers in immobilizing the enzyme. The performance of the immobilized biocatalysts are compared in terms of the yield of scFOS produced and reusability. The selection of carriers and reagents was motivated by the need to ensure safety of application in the production of food-grade products. The CA and AI900 both recorded impressive immobilization yields of 82 and 62%, respectively, while the DMM recorded 47%. Enzyme immobilizations on CA, AI900 and DMM showed activity recoveries of 23, 27, and 17%, respectively. The CA, AI900 immobilized and the free enzymes recorded their highest scFOS yields of 59, 53, and 61%, respectively. The AI900 immobilized enzyme produced a consistent scFOS yield and composition for 12 batch cycles but for the CA immobilized enzyme, only 6 batch cycles gave a consistent scFOS yield. In its first record of application in scFOS production, the AI900 anion exchange resin exhibited potential as an adequate carrier for industrial application with possible savings on cost of immobilization and reduced technical difficulty.     

Preparation of d-sorbose from d-tagatose by immobilized d-tagatose 3-epimerase

d-Tagatose 3-epimerase (d-TE) from Pseudomonas sp. ST-24 was immobilized on various types of Chitopearl beads. The highest activity was found in d-TE immobilized on Chitopearl beads of BCW 2503, the yield being about 80% of free enzyme applied. Maximum activity of the immobilized enzyme was obtained at pH 7–9 and around 60°C. The enzyme was stable in a pH range of 7–10, and below 60°C. In a high concentration (30%) of substrate, the reaction progressed without substrate inhibition. Two grams of d-sorbose crystals could be obtained from 3 g d-tagatose. Furthermore, in a batch reaction repeated five times, about 70% of d-tagatose was converted to d-sorbose each time.     

Synthesis of glyceryl ferulate by immobilized ferulic acid esterase

Glyceryl ferulate was synthesized by the condensation of ferulic acid with glycerol using Pectinase PL “Amano” from Aspergillus niger, which contained ferulic acid esterase, to improve the water-solubility of ferulic acid. The optimum reaction medium was glycerol/0.1 M acetate buffer, pH 4.0, (98:2 v/v). The enzyme immobilized onto Chitopearl BCW3003 exhibited the highest activity among the those immobilized onto various kinds of Chitopearl BCW resins. The optimum temperature for the immobilized enzyme was 50°C, and it could be reused at least five times without a significant loss in activity for the synthesis of glyceryl ferulate in batch reaction. Storage of the reaction mixture at 25°C improved the molar fraction of glyceryl ferulate relative to the dissolved ferulic residues.     

Immobilization of Aspergillus oryzae tannase and properties of the immobilized enzyme

Tannase enzyme from Aspergillus oryzae was immobilized on various carriers by different methods. The immobilized enzyme on chitosan with a bifunctional agent (glutaraldehyde) had the highest activity. The catalytic properties and stability of the immobilized tannase were compared with the corresponding free enzyme. The bound enzyme retained 20·3% of the original specific activity exhibited by the free enzyme. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme. The optimum temperature of the reaction was determined to be 40 °C for the free enzyme and 55 °C for the immobilized form. The stability at low pH, as well as thermal stability, were significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilized enzyme retained about 85% of the initial catalytic activity, even after being used 17 times.     

Amino acid modified chitosan beads: Improved polymer supports for immobilization of lipase from Candida rugosa

Amino acid modified chitosan beads (CBs) for immobilization of lipases from Candida rugosa were prepared by activation of a chitosan backbone with epichlorohydrin followed by amino acid coupling. The beads were analyzed by elemental analysis and solid state NMR with coupling yields of the amino acids ranging from 15 to 60%. The immobilized lipase on unmodified chitosan beads showed the highest immobilization yield (92.7%), but its activity was relatively low (10.4%). However, in spite of low immobilization yields (15–50%), the immobilized lipases on the amino acid modified chitosan beads showed activities higher than that of the unmodified chitosan beads, especially on Ala or Leu modified chitosan beads (Ala-CB or Leu-CB) with 49% activity for Ala-CB and 51% for Leu-CB. The immobilized lipases on Ala-CB improved thermal stability at 55 °C, compared to free and immobilized lipases on unmodified chitosan beads and the immobilized lipase on Ala-CB retained 93% of the initial activity when stored at 4 °C for 4 weeks. In addition, the activity of the immobilized lipase on Ala-CB retained 77% of its high initial activity after 10 times of reuse. The kinetic data (k_cat/K_m) supports that the immobilized lipase on Ala-CB can give better substrate specificity than the unmodified chitosan beads.     

Adsorption of BSA on strongly basic chitosan: Equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a new adsorbent, a strongly basic crosslinked chitosan (Chitopearl 2503), which is hard and is not compressed by pressure in a column, have been presented and compared with diethylaminoethyl (DEAE) Sepharose Fast Flow (hard gel). In Chitopearl 2503, when only buffer existed in the BSA solution, the isotherm was not affected by the initial concentration of BSA but it was affected by pH considerably. The isotherm was favorable when pH >/= pl ( congruent with 4.8). When NaCl existed in the BSA solution, the amount of BSA absorbed on the resin decreased with increasing concentration of NaCl. When the concentration of NaCl was 200 mol/m(3), the resin did not adsorb BSA at all. The equilibrium data were correlated by the Langmuir equation reasonably well. The BSA may be adsorbed mainly by electrostatic attraction between negatively charged BSA and positively charged quanternary ammonium groups at pH > pl and by protonation reaction of the primary ammonium groups by weak acid groups of BSA at pH = pl. These are confirmed by measuring the amount of inorganic ion exchanged for BSA. In DEAE Sepharose Fast Flow, the isotherm was favorable when pH > pl but unfavorable ar pH = pl. The saturation capacity of BSA on Chitopearl 2503 is about 1.3 to 2.2 times larger than that on DEAE Sepharose Fast Flow. (c) 1994 John Wiley & Sons, Inc.     

Immobilization of urease on nanostructured polymer membrane and preparation of urea amperometric biosensor

A new matrix for enzyme immobilization of urease was obtained by incorporating rhodium nanoparticles (5% on activated charcoal) and chemical bonding of chitosan with different concentration (0.15%; 0.3%; 0.5%; 1.0%; 1.5%) in previously chemically modified AN copolymer membrane. The basic characteristics of the chitosan modified membranes were investigated. The SEM analyses were shown essential morphology change in the different modified membranes. Both the amount of bound protein and relative activity of immobilized enzyme were measured. A higher activity (about 77.44%) was measured for urease bound to AN copolymer membrane coated with 1.0% chitosan and containing rhodium nanoparticles. The basic characteristics (pH(opt), T(opt), thermal, storage and operation stability) of immobilized enzyme on this optimized modified membrane were also determined. The prepared enzyme membrane was used for the construction of amperometric biosensor for urea detection. Its basic amperometric characteristics were investigated. A calibration plot was obtained for urea concentration ranging from 1.6 to 23 mM. A linear interval was detected along the calibration curve from 1.6 to 8.2mM. The sensitivity of the constructed biosensor was calculated to be 3.1927 μAmM(-1)cm(-2). The correlation coefficient for this concentration range was 0.998. The detection limit with regard to urea was calculated to be 0.5mM at a signal-to-noise ratio of 3. The biosensor was employed for 10 days while the maximum response to urea retained 86.8%.     

Covalent immobilization of ω-transaminase from Vibrio fluvialis JS17 on chitosan beads

Chitosan beads were prepared by emulsion method and used for the immobilization of ω-transaminase of Vibrio fluvialis. The yield of enzyme immobilization (54.3%) and its residual activity (17.8%) were higher than those obtained with other commercial beads. ω-Transaminase was effectively immobilized on the chitosan beads at pH 6.0. The optimal pH of the immobilized enzyme was pH 9.0, which is the same as that of the free enzyme. The immobilized enzyme on chitosan beads retained ca. 77% of its conversion after five consecutive reactions with the 25 mM substrate, while the immobilized enzyme on Eupergit^® C retained 12%. Also, the immobilized ω-transaminase on chitosan bead retained 70% of initial activity when it's stored at 4 °C for 3.5 weeks. Addition of the co-factor, pyridoxal 5-phosphate (PLP), was needed to maintain the stability of the immobilized ω-transaminase.     

α-<Emphasis Type="SmallCaps">l</Emphasis>-Rhamnosidase of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> immobilized on ferromagnetic supports

α-l-Rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan derivative (0.06 nkat/μg of protein). The activity–pH and activity–temperature profiles for all immobilized enzymes did not show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C. The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of 40–70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity of the preparations did not appreciably decrease after ten successive reuses. Apparent K _m of α-l-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM) were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides (0.116 and 0.014 mg narirutin after 1 h, respectively).     

Removal of aqueous phenol using immobilized enzymes in a bench scale and pilot scale three-phase fluidized bed reactor

The main objective of this work was to investigate the removal of aqueous phenol using immobilized enzymes in both bench scale and pilot scale three-phase fluidized bed reactors. The enzyme used in this application was a fungal tyrosinase [E.C. 1.14.18.1] immobilized in a system of chitosan and alginate. The immobilization matrix consisted of a chitosan matrix cross-linked with glutaraldehyde with an aliginate-filled pore space. This support matrix showed superior mechanical properties along with retaining the unique adsorptive characteristics of the chitosan. Adsorption of the o-quinone product by the chitosan reduced tyrosinase inactivation that is normally observed for this enzyme under these conditions. This approach allowed reuse of the enzyme in repeated batch applications. For the bench scale reactor (1.2-l capacity) more than 92% of the phenol could be removed from the feed water using an immobilized enzyme volume of 18.5% and a residence time of the liquid phase of 150 min. Removal rates decreased with subsequent batch runs. For the pilot scale fluidized bed (60 l), 60% phenol removal was observed with an immobilized enzyme volume of 5% and a residence time of the liquid phase of 7 h. Removal decreased to 45% with a repeat batch run with the same immobilized enzyme.     

Immobilization of <Emphasis Type="Bold">β</Emphasis>-fructofuranosidase from <Emphasis Type="Italic">Aspergillus japonicus</Emphasis> on chitosan using tris(hydroxymethyl)phosphine or glutaraldehyde as a coupling agent

A partially purified -fructofuranosidase from Aspergillus japonicus was covalently immobilized on to chitosan beads using either glutaraldehyde or tris(hydroxymethyl)phosphine (THP) as a coupling agent. Compared with the glutaraldehyde-immobilized and the free enzyme, the THP-immobilized enzyme had the highest thermal stability with 78% activity retained after 12 days at 37 ° C. The THP-immobilized enzyme also had higher reusability than that immobilized by glutaraldehyde, 75% activity was retained after 11 batches (or 11 days) at 37° C for the THP immobilized enzyme system. Less yield (48%) of fructooligosaccharides (FOS) were produced by the THP-immobilized enzyme compared with the free enzyme system (58%) from 50 (w/v) sucrose at 50 ° C.     

The simultaneous production of sorbitol from fructose and gluconic acid from glucose using an oxidoreductase of Zymomonas mobilis

Summary The production of sorbitol and gluconic acid by toluene-treated, permeabilized cells of Zymomonas mobilis has been evaluated. From a 60% total sugar solution (300 g/l glucose and 300 g/l fructose), a sorbitol concentration of 290 g/l and a gluconic acid concentration of 283 g/l were achieved after 15 h in a batch process using free toluene-treated cells. A continuous process with immobilized cells was developed and only a small loss of enzyme activity (less than 5%) was evident after 120 h. With a strongly basic anion exchange resin and an eluent of 0.11 M Na₂B₄O₇/0.11 M H₃BO₃, good separation of sorbitol and gluconic acid was achieved.     

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 proteases to porous chitosan beads and their catalysis for ester and peptide synthesis in organic solvents.

alpha-Chymotrypsin (CT), subtilisin BPN' (STB), and subtilisin Carlsberg (STC) were immobilized by adsorption to porous chitosan beads (Chitopearl, CP). The immobilized enzymes showed higher catalytic activities than free enzymes for amino acid esterification in many hydrophilic organic solvents except for methanol and DMF. In ethanol, the initial rate of the esterification increased with water content, whereas in ethyl acetate, the maximum rate was obtained at 2%-3% water. CP-immobilized CT also catalysed transesterification of Ac-Tyr-OMe in ethanol and peptide synthesis in acetonitrile from Ac-Tyr-OH or its ethyl ester and amino acid amides. The immobilized enzymes are highly stable in organic solutions, and can easily be separated from the reaction solutions. Repeated esterifications of Ac-Tyr-OH in acetonitrile by a CP-immobilized CT gave almost constant yields of the ester for more than 3 weeks.     

Immobilization of cycloisomaltooligosaccharide glucanotransferase for the production of cycloisomaltooligosaccharides from dextran

Immobilization of cycloisomaltooligosaccharide glucanotransferase (CITase) and its application in the production of cycloisomaltooligosaccharides (CIs) from dextran were studied. Among various carrier materials examined, the enzyme adsorbed physically on Chitopearl BCW-3505 showed the highest activity (1.75 U/ml carrier). The activity remaining was 35%. The maximum CI yield in batch reactions at 0.2, 2 and 10% dextran was 28, 24 and 12%, respectively. The maximum CI yield at 2% dextran (24%) was slightly less than that with the free enzyme under the same conditions (26%). The concentration of linear oligosaccharides, the byproducts in the reaction mixture, was greater with the immobilized CITase than the free enzyme. The immobilized CITase was less thermostable than the free enzyme by about 10 degrees C. The pattern of influence of Ca(2+) concentration on the thermostability differed between the free and immobilized CITase. A Ca(2+) concentration of 50-100 mM was optimum for the thermostability of the immobilized CITase, 10-50 mM for the free enzyme. CIs were produced continuously by a column system packed with the immobilized enzyme at 40 degrees C with a space velocity (SV) of 6 h(-1). The three quarters life time was 4 weeks. We think that relatively long life time at fast SV was accomplished and CI production cost by this method should be lower than the batch reaction. This is the first report on immobilization of CITase.     

Preparation and some properties of immobilized Penicillium funiculosum 258 dextranase

The production of dextranase was investigated in static cultures of Penicillium funiculosum 258. Maximal enzyme productivity was attained at pH 8.0, with 3.5% (w/v) dextran (MW, 260 000) as carbon source, NaNO₃ (1%, w/v) and yeast extract (0.2%, w/v) as nitrogen source, 0.4% (w/v) K₂HPO₄ and 0.06% (w/v) MgSO₄. It was possible to increase the productivity of dextranase to 41.8 units ml⁻¹ in the modified medium. The enzyme was immobilized on different carriers by different techniques of immobilization. The enzyme prepared by covalent binding on chitosan using glutaraldehyde had the highest activity, the immobilized enzyme retaining 63% of its original specific activity. Compared with the free dextranase, the immobilized enzyme exhibited: a higher pH optimum, a higher optimal reaction temperature and energy of activation, a higher Michaelis constant, improved thermal stability and higher values of deactivation rate constant. The immobilized enzyme retained about 80% of the initial catalytic activity even after being used for 12 cycles.