Immobilization of glucoamylase onto novel porous polymer supports of vinylene carbonate and 2-hydroxyethyl methacrylate

Glucoamylase was immobilized onto novel porous polymer supports. The properties of immobilized glucoamylase and the relationship between the activity of immobilized enzyme and the properties of porous polymer supports were investigated. Compared with the native enzyme, the temperature profile of immobilized glucoamylase was widened, and the optimum pH was also changed. The optimum substrate concentration of immobilized glucoamylase was higher than that of native enzyme. After storage for 23 d, the immobilized glucoamylase still maintained about 84% of its initial activity, whereas the native enzyme only maintained about 58% of the initial activity. Moreover, after using repeatedly seven times, the immobilized enzyme maintained about 85% of its initial activity. Furthermore, the properties of porous polymer supports had an effect on the activity of the immobilized glucoamylase.     

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone-Activated Alginate Beads: II. Enzyme Immobilization and Characterization

A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K _m values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V _m value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.     

Immobilization of single-strand specific nuclease (S1 nuclease) fromAspergillus oryzae

S1 nuclease fromAspergillus oryzae (EC 3.1.30.1) was coupled to gelatin-alginate composite matrix using the residual free aldehyde groups on the surface of glutaraldehyde crosslinked matrix. The immobilized enzyme retained approximately 10% activity of the soluble enzyme. When partially purified enzyme was bound to the matrix, the immobilized preparation did not show any detectable enzyme activity. However, the activity could be restored when the coupling was carried out in the presence of a coprotein or substrate. The optimum pH of the immobilized S1 nuclease shifted to 3.8 from 4.3 for the soluble enzyme. Also, optimum temperature increased to 65°C after immobilization. Bound S1 nuclease showed increased pH and temperature stabilities. Immobilization brought about a twofold decrease in the Michaelis-Menton constant (K _m).     

Immobilization of Phenylalanine Dehydrogenase and Its Application in Flow-Injection Analysis System for Determination of Plasma Phenylalanine

Phenylalanine dehydrogenase (l-PheDH) from Sporosarcina ureae was immobilized on DEAE-cellulose, modified initially with 2-amino-4,6-dichloro-s-triazine followed by hexamethylenediamine and glutaraldehyde. The highest activity of immobilized PheDH was determined as 95.75 U/g support with 56% retained activity. The optimum pH value of immobilized l-PheDH was shifted from pH 10.4 to 11.0. The immobilized l-PheDH showed activity variations close to the maximum value in a wider temperature range of 45–55 °C, whereas it was 40 °C for the native enzyme. The pH and the thermal stability of the immobilized l-PheDH were also better than the native enzyme. At pH 10.4 and 25 °C, K _m values of the native and the immobilized l-PheDH were determined as K _{m Phe} = 0.118, 0.063 mM and K _{m NAD}⁺ = 0.234, 0.128 mM, respectively. Formed NADH at the exit of packed bed reactor column was detected by the flow-injection analysis system. The conversion efficiency of the reactor was found to be 100% in the range of 5–600 μM Phe at 9 mM NAD⁺ with a total flow rate of 0.1 mL/min. The reactor was used for the analyses of 30 samples each for 3 h per day. The half-life period of the reactor was 15 days.     

Cloning, heterologous expression, and characterization of Thielavia terrestris glucoamylase

Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/cellular biology is poorly understood. Only a few genes have been cloned from the Thielavia genus. We detected an extracellular glucoamylase in culture filtrates of T. terrestris and cloned the corresponding glaA gene. The coding region contains five introns. Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase. Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15. The T. terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A. oryzae α-amylase promoter and an Aspergillus niger glucoamylase terminator. The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 μmol/(min·mg) with maltose as substrate. With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60°C. The enzyme was stable at 60°C for 30 min. The K _m and k _cat of the enzyme for maltotriose were determined at various pHs and temperatures. At 20°C and pH 4.0, the enzyme had a K _m of 0.33±0.07 mM and a k _cat of (5.5±0.5)×10³ min⁻¹ for maltotriose. The temperature dependence of k _cat /K _m indicated an activation free energy of 2.8 kJ/mol across the range of 20–70°C. Overall, the enzyme derived from the thermophilic fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi.     

Scanning electron microscopic observation of porous glass fibers with immobilized glucoamylase

Porous glass fibers with silane-glutaraldehyde immobilized glucoamylase have been examined by Scanning Electron Microscopy (SEM). Partial multilayer coating (“sheeting”) on the fibers’ surfaces has been observed even on gold uncoated samples by using a high resolution SEM. This “sheeting” is attributed to the fiber chemical activation treatment prior to enzyme loading. A 40% reduction of free pore area as a consequence of enzyme attachment is also observed.     

Immobilization,characterization, and laboratory-scale application of bovine liver arginase

Arginase isolated from beef liver was covalently attached to a polyacrylamide bead support bearing carboxylic groups activated by a water-soluble carbodiimide. The most favorable carbodiimide wasN-cyclohexyl-Nt’-(methyl-2-p-nitrophenyl-2-oxoethyl) aminopropyl carbodiimide methyl bromide, but for practical purposes,N-cyclohexyl-Nt’-morpholinoethyl carbodiimide methyl tosylate was used. The optimal conditions for the coupling procedure were determined. The catalytic activity of the immobilized arginase was 290–340 U/g solid or 2.9–3.4 U/mL wet gel. The pH optimum for the catalytic activity was pH 9.5, the apparent temperature maximum was at 60°C and K_mapp was calculated to be 0.37M L-arginine. Immobilization markedly improved the conformational stability of arginase. At 60°C, the pH for maximal stability was found to be 8.0. The immobilized arginase was used for the production of L-ornithine and D-arginine.     

Comparative studies on soluble and immobilized rabbit muscle pyruvate kinase

Rabbit muscle pyruvate kinase was immobilized by covalent attachment to a polyacrylamide support (Akrilex C) containing carboxylic functional groups. As a result of immobilization, the pH optimum for catalytic activity shifted into a more alkaline direction. The apparentK _m value with phosphoenolpyruvate increased, and that with ADP slightly decreased. With respect to the stability against urea and thermal inactivation, the immobilized pyruvate kinase seemed to be the more stable at lower urea concentrations and between 45 and 55°C. At 1.5 and 2.5M urea and at higher temperature, there were no marked differences between the soluble and the immobilized enzyme.     

Kinetic studies of lipase from Candida rugosa

The search for an in expensive support has motivated our group to undertake this work dealing with the use of chitosan as matrix for immobilizing lipase. In addition to its low cost, chitosan has several advantages for use as a support, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, we describe the immobilization of Canada rugosa lipase onto porous chitosan beads for the enzymatic hydrolysis of oliveoil. The binding of the lipase onto the support was performed by physicalad sorption using hexane as the dispersion medium. A comparativestudy between free and immobilized lipase was conducted in terms of pH, temperature, and thermal stability. A slightly lower value for optimum pH (6.0) was found for the immobilized form in comparison with that attained for the soluble lipase (7.0). The optimum reaction temperature shifted from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half-life of the soluble free lipase at 55°C was equal to 0.71 h (K _d=0.98 h⁻¹), whereas for the immobilized lipase it was 1.10 h (K _d=0.63 h⁻¹). Kinetics was tested at 37°C following the hydrolysis of olive oil and obeys the Michaelis-Menten type of rate equation. The K _m was 0.15 mM and the V _max was 51 μmol/(min·mg), which were lower than for free lipase, suggesting that the apparent affinity toward the substrate changes and that the activity of the immobilized lipase decreases during the course of immobilization.     

Lactose Hydrolysis by β-Galactosidase Covalently Immobilized to Thermally Stable Biopolymers

Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan (hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from 50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and temperatures. The apparent K _m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V _max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after 15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable for immobilization of other industrial enzymes.     

Twentyfold increase in alkaline phosphatase activity by sequential reversible activation of the enzyme followed by coupling with a copolyme of ethylene and maleic anhydride

Alkaline phosphatase, APase, (EC 3.1.31) from calf intestine, after shifting the equilibrium by effector molecules towards the dimeric form of the enzyme, was coupled (ratio 1:2, protein: copolymer) to a copolymer of ethylene and maleic anhydride, EMA. The water-soluble APase-EMA was separated from APase and the unbound EMA by DEAE-cellulose ion exchange chromatography. The specific activity of the APase-EMA, compared to APase, increased 26-fold at pH 7.1 and 10-fold at pH 8.6. The pH optimum of APase-EMA was shifted down from pH 9.5 (native APase) to 8.6. This change could be interpreted in terms of polyelectrolyte theory. APase-EMA retained 50–70% of its optimum activity in the pH range 7–8, while APase retained only 5–15% of its optimum activity within the same pH range. Its isoelectric point, pI, was 4.2 (APase 6.0) and it migrated on polyacrylamide gel electrophoresis in a single band, anodic movement twice as fast as APase. Parallel with the kinetic measurements, the reactive-enzyme sedimentation method was used to measure S_20,w values. S_20,w values obtained for APase-EMA, activated APase, and APase dialyzed against wafer were 6.56S, 6.46S, and 5.17S, respectively. Molecular weights, M_r, were determined by equilibrium sedimentation: the values obtained were 180,000, 160,000, and 84,500. M_r values of APase-EMA and APase (native) estimated by Sepharose-4B gel filtrations were essentially the same. The above-mentioned values remained unchanged for APase-EMA after intensive dialysis against water, whereas for the activated APase, separation from the effector molecules caused the equilibrium to shift back to the monomeric, very slightly active enzyme with concomitant changes of S_20,w to 5.15 and M_r to 82,000.     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.     

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.     

Studies of Penicillin G Acylase Immobilization Using Highly Porous Cellulose-Based Polymeric Membrane

The different ionic molecules/compounds were used as a ligand for the immobilization of penicillin G acylase on the highly porous cellulose-based polymeric membrane having buffer flux 1,746 LMH (L m⁻² h⁻¹) at 0.5 bar pressure. The immobilized enzyme activity around 250 U_App was obtained with the ligand such as proline, tryptophan, casein acid hydrolysate, and brilliant green. Comparatively, proline showed less IMY% (percentage immobilization yield—58) but higher RTA% (percentage of activity retention—71) and specific activity (145 U_App g⁻¹). However, the crosslinked preparation of brilliant green obtained using glutaraldehyde showed 82 ± 2.7% immobilized enzyme activity after the completion of successive five cycles. In comparison with the free enzyme, the enzyme immobilized on the brilliant green coupled membrane showed around 2.4-fold increase in K _m value (47.4 mM) as well as similar optimum pH (7.2) and temperature (40 °C). The immobilized enzyme retained almost 50% activity after 107 days and 50 cycles of operation. Almost 50% decrease in buffer flux after enzyme immobilization was observed. At the end of the 30 cycles, flux pattern shows around 38% decrease in buffer flux however, after 16 cycles of operation flux moves closer towards the steady state.     

Radiation-induced polymerization for the immobilization of penicillin acylase

The immobilization ofEscherichia coli penicillin acylase (EC 3.5.1.11) was investigated by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperature. A leak-proof composite that does not swell in water was obtained by adding the crosslinking agent trimethylolpropane trimethacrylate to the monomer-aqueous enzyme mixture. Penicillin acylase, which was immobilized with greater than 70% yield, possessed a higherK _m value toward the substrate 6-nitro-3-phenylacetamidobenzoic acid than the free enzyme form (K _m = 1.7 × 10⁻⁵ and 1 × 10⁻⁵M, respectively). The structural stability of immobilized penicillin acylase, as assessed by heat, guanidinium chloride, and pH denaturation profiles, was very similar to that of the free-enzyme form, thus suggesting that penicillin acylase was entrapped in its native state into aqueous free spaces of the polymer matrix.     

Immobilization of Naringinase in PVA–Alginate Matrix Using an Innovative Technique

A synthetic polymer, polyvinyl alcohol (PVA), a cheap and nontoxic synthetic polymer to organism, has been ascribed for biocatalyst immobilization. In this work PVA–alginate beads were developed with thermal, mechanical, and chemical stability to high temperatures (<80 °C). The combination of alginate and bead treatment with sodium sulfate not only prevented agglomeration but produced beads of high gel strength and conferred enzyme protection from inactivation by boric acid. Naringinase from Penicillium decumbens was immobilized in PVA (10%)–alginate beads with three different sizes (1–3 mm), at three different alginate concentrations (0.2–1.0%), and these features were investigated in terms of swelling ratio within the beads, enzyme activity, and immobilization yield during hydrolysis of naringin. The pH and temperature optimum were 4.0 and 70 °C for the PVA–alginate-immobilized naringinase. The highest naringinase activity yield in PVA (10%)–alginate (1%) beads of 2 mm was 80%, at pH 4.0 and 70 °C. The Michaelis constant (K _Mapp) and the maximum reaction velocity (V _maxapp) were evaluated for both free (K _Mapp = 0.233 mM; V _maxapp = 0.13 mM min⁻¹) and immobilized naringinase (K _Mapp = 0.349 mM; V _maxapp = 0.08 mM min⁻¹). The residual activity of the immobilized enzyme was followed in eight consecutive batch runs with a retention activity of 70%. After 6 weeks, upon storage in acetate buffer pH 4 at 4 °C, the immobilized biocatalyst retained 90% of the initial activity. These promising results are illustrative of the potential of this immobilization strategy for the system evaluated and suggest that its application may be effectively performed for the entrapment of other biocatalysts.     

Purification and characterization of an exoinulinase from Aspergillus fumigatus

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg⁺⁺, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K _m (0.25 mM) and higher V _max (333.3 IU/mg) values for inulin.     

Investigations of stabilities,pH, and temperature profiles and kinetic parameters of glucoamylase immobilized on plastic supports

The covalent immobilization of glucoamylase on new epoxide-, isocyanate-, acid chloride-, and carboxylic acid-activated plastic supports shows the viability of such supports for immobilizing enzymes (especially those reacting with 1,6-diaminohexane and glutaraldehyde) for producing side arms. The operational stability of immobilized glucoamylase could be extended by crosslinking the enzyme, by increasing the substrate concentration, or by extending the support’s side arm. The pH curves for the immobilized enzyme were in general not found to be shifted from the pH optimum of the soluble enzyme. However, the immobilized enzyme’s temperature activity profiles were shifted to a lower temperature range when compared to the soluble enzyme. The immobilized glucoamylase Michaelis constants increased, and the maximum rates and specific activities decreased with respect to the soluble enzyme kinetic parameters.     

Using of silica particles as porogen for preparation of macroporous chitosan macrospheres suitable for enzyme immobilization

Porous chitosan macrospheres were prepared at the first time by using silica particles as porogen, and the optimal weight ratio of silica to chitosan during preparation was determined. Scanning electron microscopy micrographs showed that the support with silica as porogen (support I) had a much more porous surface structure than the support without porogen (support II). Both supports were applied to immobilize β-galactosidase from Aspergillus oryzae. Much higher specific activity and yield of galactose hydrolysis products were observed for the support I. Properties of both immobilized enzyme were determined and compared with the free enzyme, satisfactory results were obtained in thermostability, pH arid operational stability, Michaelis constants K _m and in maximum velocity of hydrolysis (V _m). Suggested method allow to prepare chitosan macrospheres as immobilized enzyme carrier with moreporous surface structure and more active reaction groups.     

Glycosidases of turnip leaf tissues

Four myrosinase (β-thioglucosidase EC. 3.2.3.1) and seven disaccharase (β-fructofuranosidase, EC. 3.2.1.26) isoenzymes were isolated from turnip leaves. The most active enzymes were isolated in pure form. Myrosinase and disaccharase mol wt was 62.0 × 10³ and 69.5 × 10³ dalton, respectively, on the basis of gel filtration on Sephadex G-200. Myrosinase pH profile showed high activity between pH 5 and 7 with the optimum at pH 5.5. The purified enzyme was heat-stable for 60 min at 30°C with only loss of 24% of activity. Its activity is strongly inhibited (100%) by Pb²⁺, Ba²⁺, Cu²⁺ and Ca²⁺ ions, and activated (70%) by EDTA at 0.04M. The pure enzyme failed to hydrolyze amylose, glycogen, lactose, maltose, and sucrose. TheK _m andV _max values of myrosinase using sinigrin as specific substrate was 0.045 mM and 2.5 U, respectively. The maximal activity of disaccharase enzyme was obtained at pH 4–5 and 35–37°C. The enzyme was heat-stable at 30°C for 30 min with only 10% loss of its activity. Its activity is strongly activated (70–240%) by Ca²⁺, Ba²⁺, Cu²⁺, and EDTA at 0.01M. The enzyme activity is specific to the disaccharide sucrose and failed to hydrolyze other disaccharides (maltose and lactose). TheK _m andV _max of disaccharase were 0.123 mM and 3.33 U, respectively.