Immobilization of enzymes on spheron: 1. Trypsin and glucoamylase by the titanium-chelation method

In a preliminary study, trypsin (EC 3.4.21.4) and glucoamylase (exo-1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) were immobilized on Spheron by the titanium-chelation method. The activity of trypsin immobilized on Spheron P100 000 was higher against tosyl-l-arginine 4-nitroanilide than against casein. The variation in the specific activities of glucoamylase immobilized on Spherons of different porosities to wards substrates of different molecular weights was examined.     

Thermal Stability of Immobilized Glucoamylase Entrapped in Polyacrylamide Gels and Bound to SP-Sephadex C–50

Glucoamylase[α-1,4: 1,6-glucan-4: 6-glucohydroease, EC 3.2.1.3] from Rhizopus niveus was entrapped in polyacrylamide gels and adsorbed onto SP-Sephadex C–50 to elucidate the thermostability mechanism of immobilized enzymes. The thermal stability of immobilized glucoamylase entrapped in polyacrylamide gels was enhanced slightly compared with glucoamylase in free solution, and was independent of the acrylamide monomer concentration and N, N′-methylene-bis (acrylamide) content. To explain this phenomenon, the cellular structure of polyacrylamide gel was taken into consideration in addition to interactions between glucoamylase and gel, and a decrease in dielectric constant in the gel [S. Moriyama et al., Agric. Biol. Chem., 41, 1985 (1977)¹⁾]. On the other hand, immobilized glucoamylase bound to SP-Sephadex by ionic interaction showed lower stability than free glucoamylase, and much greater stability than glucoamylase in the presence of dextran sulfate, a constituent of SP-Sephadex. Thermal stabilities for the free and immobilized enzymes were also compared at the pH not in the bulk solution, but in the SP-Sephadex.     

Immobilization of mold and bacterial amylases on silica carriers

The immobilization of alpha-amylase and glucoamylase was investigated by several coupling methods on silica carriers, different types of Silokhroms, and silica gels. The most active immobilized mold and bacterial alpha-amylases and mold glucoamylase were obtained with titanium salts. These activities were twice the value of that obtained by glutaraldehyde or azo coupling. The half-lives of A. oryzae alpha-amylase, B. subtilis alpha-amylase, and A. niger glucoamylase, immobilized to silica carriers at 45 degrees C and under continuous operation at a high concentration of substrate, were 14, 35, and 65 days, respectively.     

Immobilization of enzymes on crosslinked gelatin particles activated with various forms and complexes of titanium(IV) species

A number of methods of activating the surface of glutaraldehyde crosslinked gelatin beads with titanium(IV) compounds, for subsequent enzyme coupling, have been investigated. Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) was so immobilized using titanium(IV)-urea, -acrylamide, -citric acid and -lactose complexes; however, immobilized enzyme preparations with low activities were obtained (0.36–1.28 U g^?1). Activation with uncomplexed titanium(IV) chloride, however, of both moist and freeze-dried crosslinked gelatin particles resulted in highly active immobilized glucoamylase preparations (1.74–26.6 U g^?1). Dual immobilized enzyme conjugates of glucoamylase and invertase (β-d-fructofuranosidase, EC 3.2.1.26) were also prepared using this method. Invertase was served on the entrapped enzyme while glucoamylase was coupled on the surface of titanium(IV)-activated gelatin pre-entrapped invertase particles. A dual gelatin coupled glucoamylase/gelatin entrapped glucoamylase was prepared (3.8 U g^?1) and ～72.5% of the total combined activity was due to the surface bound enzyme.     

Preparation,characterization and laboratory-scale application of an immobilized glucoamylase

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 5.5– 6.0 units g^?1solid. The optimum pH for catalytic activity was pH 3.8. The apparent optimum temperature was found at 60°C. With soluble starch as substrate the K_m value was 14 mg ml^?1. The pH for maximum stability was pH 4.0–4.5. In the presence of 8 m urea the immobilized glucoamylase retained most of its catalytic activity but it was more susceptible to guanidinium hydrochloride than the soluble enzyme. The practical applicability of immobilized glucoamylase was tested in batch process and continuous operation.     

A simple kinetic model for the hydrolysis of α-d-glucans using glucoamylase immobilized on titanium (IV) activated porous silica

A simple kinetic model which describes the hydrolysis of α-d-glucans by immobilized glucoamylase (exo-1,4-d-glucosidase, EC 3.2.1.3) is reported. The hydrolysis of starch, amylose, amylopectin, maltose and 40DE starch hydrolysates using glucoamylase immobilized on alkylamine derivatives of titanium(IV) activated porous silica are described by a kinetic model based on Langmuir-Hinshelwood kinetics. This model involves enzyme kinetics with or without product inhibition and reverse reactions as well as mass transfer and diffusion effects in immobilized enzyme reactors. The results of other authors are also interpreted by the model developed in this article.     

Pretreatment of starch raw materials and their enzymatic hydrolysis by immobilized glucoamylase

The pretreatment of starch raw materials such as sweet potato, potato and cassava has been carried out using various types of crusher, viz juice mixer, homogenizer and high-speed planetary mill. The effect of pretreatment of the materials on their enzymatic hydrolysis was studied. High-speed planetary mill treatment was the most effective and comparable with heat treatment (pasting). Various crushing times were used to examine the effect of crushing by mill treatment on the enzymatic hydrolysis. In the enzymatic hydrolysis of cassava, the use of both cellulase [1,4-(1,3; 1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and glucoamylase [1,4-α-d-glucan glucohydrolase, EC 3.2.1.3] enhanced the d-glucose yield. The immobilization of glucoamylase was studied by radiation polymerization of hydrophilic monomers at low temperature, and it was found that enzymatic activity of the immobilized glucoamylase particles varied with monomer concentration and particle size. Starchy raw materials pretreated with the mill can be efficiently hydrolysed by immobilized glucoamylase.     

Investigation of the binding mechanism of glucoamylase to alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) has been coupled to several porous silica matrices by a new covalent process using alkylamine derivatives of titanium(IV)-activated supports. In order to investigate the interaction of the titanium element with the silanol groups of the inorganic matrices, activation was performed at different times, using titanium(IV) chloride, either pure or as a 15% w/v solution, in 15% w/v hydrochloric acid at 25, 45 and 80°C, followed by washing with sodium acetate buffer (0.02m, pH 4.5) or chloroform. Using pure TiCl₄, the highest activities of all preparations were obtained at 80°C and with acetate buffer washing, resulting from a higher content of titanium coating of the carrier. When activation was performed in aqueous TiCl₄ solution, followed by a drying step, the highest activity was obtained with preparations washed with chloroform, with or without amination. When reacting pure TiCl₄ with controlled pore glass (CPG) and with porous silica (Spherosil), colour formation was observed after reaction of glutaraldehyde with the aminated support. This did not happen when Celite was used as the support. As a criterion for comparison of the different immobilized enzyme preparations, the concept of an ‘instability factor’, which measures the percentage of immobilized enzyme activity due to release of enzyme into solution, is introduced. Instability factors of immobilized enzyme preparations on Celite were always higher than those obtained with the other matrices, confirming that there was no covalent coupling of the enzyme to Celite. However, when the activation was performed with aqueous TiCl₄ solution with drying, Schiff's base formation was observed in all preparations and very stable immobilized enzyme preparations were obtained. The results of the activation of controlled pore glass and porous silica with pure titanium(IV) chloride suggest the existence of a true reaction between the titanium element and the silanol groups of these carriers by formation of a bridge, Si-O-Ti, while with the titanium(IV) chloride solution in hydrochloric acid, a coating of hydrous titanium(IV) oxide is obtained.     

Immobilization of glucoamylase on DEAE-cellulose activated with chloride compounds

Partially purified glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus niger NRRL 330 has been immobilized on DEAE-cellulose activated with cyanuric chloride in 0.2 m acetate buffer, pH 4.2. In the matrix-bound glucoamylase, enzyme yield was 20 mg g^?1 of support, corresponding to 40 200 units g^?1 of DEAE support. Binding of the enzyme narrows the pH optimum from 3.8–5.2 to 3.6. Thermal stability of the bound glucoamylase enzyme was decreased although it showed a higher temperature optimum (70°C) than the free form (55°C). The rate of reaction of glucoamylase was also changed after immobilization. V_max values for free and bound enzyme were 36.6 and 22.6 μmol d-glucose ml^?1 min^?1 and corresponding K_m values were 3.73 and 4.8 g l^?1 respectively. Free and immobilized enzyme when used in the saccharification process gave 84 and 56% conversion of starch to d-glucose, respectively. The bound enzyme was quite stable and in the batch process it was able to operate for about five cycles without any loss of activity.     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

Immobilization of enzymes on porous silica supports

Four silica supports differing in pore dimensions were activated by treatment with SiCl₄ and then with ethylenediamine to obtain alkylamine groups on the silica surface. Three enzymes, peroxidase from cabbage, glucoamylase from Aspergillus niger C and urease from soybean were immobilized on these supports using glutaraldehyde as coupling agent. It was found that the protein content, the retained enzymatic activity and the storage stability of the silica supported enzymes were considerably affected by support pore size and enzyme molecular weight, the factors which are supposed to alter protein distribution inside the support pores. The highest activity was found for peroxidase and glucoamylase attached to the silica with the widest pores, but their loss in activity during storage was considerable. The urease retained less activity after immobilization, but its storage stability was excellent.     

Preparation and activity of carbonic anhydrase immobilized on porous silica beads and graphite rods

Techniques for the immobilization of bovine carbonic anhydrase (BCA) on porous silica beads and graphite are presented. Surface coverage on porous silica beads was found to be 1.5 x 10(-5) mmol BCA/m(2), and on graphite it was 1.7 x 10(-3) mmol BCA/m(2) nominal surface area. Greater than 97% (silica support) and 85% (graphite support) enzyme activity was maintained upon storage of the immobilized enzyme for 50 days in pH 8 buffer at 4 degrees C. After 500 days storage, the porous silica bead immobilized enzyme exhibited over 70% activity. Operational stability of the enzyme on silica at 23 degrees C and pH 8 was found to be 50% after 30 days. Catalytic activity expressed as an apparent second-order rate constant K'(Enz) for the hydrolysis of p-nitrophenyl acetate (p-NPA) catalyzed by BCA immobilized on silica beads and graphite at pH 8 and 25 degrees C is 2.6 x 10(2) and 5.6 x 10(2) M(-1)s(-1) respectively. The corresponding K(ENZ) value for the free enzyme is 9.1 x 10(2) M(-1)s(-1). Activity of the immobilized enzyme was found to vary with pH in such a manner that the active site pK, on the porous silica bead support is 6.75, and on graphite it is 7.41. Possible reasons for a microenvironmental influence on carbonic anhydrase pK(a), are discussed. Comparison with literature data shows that the enzyme surface coverage on silica beads reported here is superior to previously reported data on silica beads and polyacrylamide gels and is comparable to an organic matrix support. Shifts in BCA-active site pK(a) values with support material, a lack of pH dependent activity studies in the literature, and differing criteria for reporting enzyme activity complicate literature comparisons of activity; however, immobilized BCA reported here generally exhibits comparable or greater activity than previous reports for immobilized BCA.     

Compositions and compositional-behavioural relationships of enzymes immobilized on porous inorganic supports via titanium(IV) species

The compositions and compositional-behavioural relationships of glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) immobilized on titanium(IV)-activated porous inorganic supports have been investigated for several transition metal activation techniques based on the metal-link/chelation method developed by our group. The highest activity (239 Ug^?1 matrix) of immobilized glucoamylase was obtained with the hydrous titanium(IV) oxide derivative of the support when this and a 15% w/v TiCl₄ solution were dried at 45°C in vacuum for 30 h. However, the immobilized enzyme preparation displayed a very unstable behaviour, as did also the preparation which was obtained by drying the mixture of support and transition metal solution at atmospheric pressure. This was mainly due to an enzyme deactivation by titanium inhibition instead of enzyme loss in substrate solution. When amination and carbonylation steps were included in the immobilization technique much more stable preparations were obtained, mainly when the support was activated by drying at 45°C with a 15% w/v TiCl₄ solution ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1495}\text{h}$) although with a lower initial activity (35.6 Ug^?1 matrix). The pure TiCl₄ support activation rather than TiCl₄/HCl solution support activation led to less stable immobilized enzyme preparations (washing and amination solvent chloroform, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 365}\text{h}$; washing and amination solvent water, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 276}\text{h}$) than the preparation obtained with the dried titanium(IV)-activated support. This was due to loss of enzyme-titanium(IV) complex in solution, as the interactions between the titanium(IV) and the silanol groups of the porous silica are weak. However, the amination (with 1,6-diaminohexane) and carbonylation (with glutaraldehyde) steps always led to immobilized enzyme preparations with constant specific activities and protein/titanium(IV) ratio. This suggests that the spacing effect introduced by these reactions removes the titanium(IV) inhibition of glucoamylase.     

Soluble and immobilized enzyme technology in bioconversion of barley starch

Homogeneous and heterogeneous biocatalysis were both investigated as tools for barley starch syrup production. Barley starch was first liquefied by soluble heat-stable Bacillus sp. α-amylase EC 3.2.1.1 (1,4-α-d-glucan glucanohydrolase) Termamyl 60 L at 95°C, pH 6.5, to obtain slurries of varying DE-values up to ≈37. Alternatively, it was extruded with a Creusot-Loire BC 45 twin-screw extruder at 25% moisture, 150°C, for denaturation. After cooling and adjusting the pH to 4.5 or grinding, respectively, the pretreated starch was saccharified either by soluble or by immobilized Aspergillus niger glucoamylase EC 3.2.1.3 (1,4-α-d-glucan glucohydrolase) at 60°C, pH 4.5, to obtain glucose syrup of up to DE 96. The course of hydrolysis was followed by automated Biogel P-2 chromatographic analysis. Glucoamylase was immobilized either on a phenol-formaldehyde resin Duolite S 761 or on silanized Spherosil porous silica beads. Barley glucose syrup obtained was further continuously converted to high fructose syrup by a packed bed reactor of Actinoplanes missouriensis whole cell glucose isomerase (EC 5.3.1.5) Maxazyme entrapped within α-cellulose beads. We could conclude that barley starch may be used as an alternative raw material for biocatalytic starch syrup production.     

Development of sensors for direct detection of organophosphates. Part I: Immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (< 1 mm2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol-gel process to create highly porous and stable silica matrices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol-gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol-gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.     

Immobilization of glucoamylase from Aspergillus niger on poly(ethylenimine)-coated non-porous glass beads

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) from A. niger was immobilized on cationic nonporous glass beads (13–44 μm) by electrostatic adsorption followed by rosslinking with glutaraldehyde. Over 80% of the enzyme's total soluble activity was expressed upon immobilization. d-Glucose production from maltodextrins was virtually complete, suggesting that the lack of pores can eliminate the problem of product reversion. Immobilized glucoamylase showed decreased stability upon heating, compared with the soluble enzyme.     

铜金属螯合载体固定糖化酶

[目的]制备出含Cu2+的琼脂糖-IDA螯合载体及对其固定糖化酶工艺条件进行优化.[方法]利用金属螯合配体(IDA-Cu2+)与蛋白质表面供电子氨基酸相互作用的原理制备载体,采用紫外分光光度法测定不同影响因素下固定化糖化酶的酶活.[结果]Cu2+的加入量和固定化过程的酸度比给酶量对固定化糖化酶的活性影响还要大,在给酶量80 mg/g载体、1.0× 10-2 mol Cu2+/g载体、pH 4.6和固定化4h的固定化条件下,固定化酶活为252.1 U/g,重复使用5次后酶活为首次固定化酶活的65.1％.[结论]该Cu2+-IDA-金属螯合琼脂糖可用于淀粉水解糖化酶的优良固定化载体材料.     

Studies on extracellular and intracellular purified amylases from a thermophilic Bacillus stearothermophilus

Extracellular and intracellular amylases have been purified from a thermophilic Bacillus stearothermophilus and further studies have been made with the purified enzyme. The molecular weights for extra- and intracellular α- and β-amylases were found to be 47 000, 58 000, 39 000 and 67 000, respectively. α-Amylase (1,4-α-d-glucan glucanohydrolase, EC 3.2.1.1) and glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) were glycoproteins, whereas β-amylase (1,4-α-d-glucan maltohydrolase, EC 3.2.1.2) had little or no carbohydrate moiety. Extracellular FI (α-amylase), FIII (glucoamylase), FIV and FV (α-amylase) had carbohydrate moieties of 14.4, 27.0, 11.0 and 12.5%, respectively, whereas intracellular amylases FI (α-amylase), FII (β-amylase) and FIII (α-amylase) contained 15.2, 0.8 and 13.4% carbohydrate, respectively. The amino acid profile of the amylase protein digest showed a total number of 16 amino acids with aspartic acid showing the highest value followed by glutamic acid and leucine plus isoleucine. Compared to other thermostable amylases, proline and histidine contents were low. Both α- and β- amylase had the - SH group at their active site, which was essential for enzyme activity. EDTA and parachloromercuribenzoate exhibited dose dependent non-competitive inhibition of enzyme activity indicating the involvement of a divalent cation and the - SH group for activity.     

Surface immobilization and entrapping of enzymes on glutaraldehyde crosslinked gelatin particles

A mild and reproducible method has been developed for the surface-immobilization of enzymes on glutaraldehyde crosslinked gelatin beads. In this method glutaraldehyde is used in a dual capacity, as crosslinking agent and as the enzyme coupling agent. Glucoamylase (exo-α-1,4-d-glucosidase, EC 3.2.1.3), β-d-fructofuranosidase (invertase, EC 3.2.1.26) and β-d-glucoside (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) have been successfully immobilized by this method, on the surface of the crosslinked gelatin particles. The method can be combined with the existing technology for the production of gelatin-entrapped enzymes. Thus, dual immobilized enzyme conjugates of glucoamylase and invertase have been prepared using this method, by entrapment of one enzyme in, and surface-binding of the other to, the gelatin matrix. The coupling of glucoamylase onto cross-linked gelatin particles by precipitation with poly(hexamethylenebiguanide hydrochloride) was also tested.     

Comparative studies on extracellular protease secretion and glucoamylase production by free and immobilized <Emphasis Type="Italic">Aspergillus niger</Emphasis> cultures

The effects of cell immobilization on the secretion of extracellular proteases and glucoamylase production by Aspergillus niger were investigated under a variety of immobilization techniques and culture conditions. Immobilization was achieved by means of cell attachment on metal surfaces or spore entrapment and subsequent growth on porous Celite beads. Free-suspension cultures were compared with immobilized mycelium under culture conditions that included growth in shake flasks and an airlift bioreactor. Cell attachment on metal surfaces minimized the secretion of proteases while enhancing glucoamylase production by the fungus. Growth on Celite beads in shake-flask cultures reduced the specific activity of the secreted proteases from 128 to 61 U g⁻¹, while glucoamylase specific activity increased from 205 to 350 U g⁻¹. The effect was more pronounced in bioreactor cultures. A reduction of six orders of magnitude in protease specific activities was observed when the fungus grew immobilized on a rolled metal screen, which served as the draft tube of an airlift bioreactor. Received 29 October 2001/ Accepted in revised form 14 June 2002