Immobilization of urease onto chemically modified acrylonitrile copolymer membranes

Poly (acrylonitrile-methylmethacrylate-sodium vinylsulfonate) membranes were subjected to seven different chemical modifications. The amounts of new groups incorporated in the membranes with the modifications were determined. Urease was covalently immobilized on the modified membranes. Both the amount of bound protein and relative activity of immobilized urease were measured. The highest activity was found for urease bound to membranes modified with hydroxylammonium sulfate (68%) and hydrazinium sulfate (67%). Optimum pH of free urease was determined to be 5.8. For positively charged membranes, pH optimum was shifted to higher values, while for negatively charged membranes-to lower pH. The charge of the matrix affected also the rate of the enzyme reaction. The highest rate was measured with urease immobilized on membranes modified with hydroxylammonium sulfate and hydrazinium sulfate. The major part of the immobilized enzyme on different modified membranes remained stable-only ca. 20% of enzyme activity was lost for 4 h at 70 degrees C while the free enzyme was totally inactivated.     

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.     

游离及固定化果糖基转移酶部分酶学性质的比较研究

 从诱变、筛选的米曲霉GX0 0 10菌株所产生的果糖基转移酶 ,经过纯化和固定化操作分别制备游离酶和固定化酶 ,对两者的酶学性质进行了比较研究 .结果表明 ,两者在蔗糖转化为蔗果低聚糖的酶促反应中 ,最适pH为 5 5,在pH5 0～ 7 5之间酶活性相对稳定 .游离酶和固定化酶的适宜温度范围分别是 4 5～ 52℃和 4 0～ 55℃ .在 55℃保温 60min ,酶活性保存率分别是 61 6%和 87 5% .固定化酶的热稳定性提高 .0 1mmol LHg2 +和 1mmol LAg+能完全抑制游离酶的活性 ,但只能部分抑制固定化酶的活性 ,1mmol L的Ti2 +能完全抑制两者的活性 .以蔗糖为底物时 ,游离酶的米氏常数Km=2 15mmol L ,而固定化酶Km =386mmol L .游离酶只能使用一次 ,固定化酶反复使用 54次后 ,剩余活力为 55 2 % .用 55% (W V)蔗糖溶液与固定化酶在pH5 0 ,4 6℃下作用 12h ,可获得61 5% (总低聚糖 总糖 )产物 ,其中蔗果五糖含量达到 7 2 % .     

环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶*

游离酶经过固定化后,稳定性和环境耐受性得到提高,在食品、医药、化工、环境和皮革等领域可以很好的提高酶的利用率并降低生产成本,具有极大的应用潜力。新型交联剂在固定化酶工艺的应用极大推进了固定化酶研究的深入。借助新型交联剂聚乙二醇二缩水甘油醚(PEGDGE),利用氨基载体LX-1000HA固定化海洋假丝酵母脂肪酶,结合单因素和正交试验优化得到交联及固定化条件为:交联温度30℃,交联2h,交联剂浓度0.75%,pH7.0,加酶量800U,载体量0.5g,固定化2h,固定化温度45℃。根据上述最佳固定化工艺,制备得到固定化酶LX-1000HA-PEGDGE-CRL在最适条件下测得酶活达到160.81U/g,约为此前制备的固定化酶LX-1000HA-GA-CRL(由LX-1000HA和戊二醛交联脂肪酶得到)和LX-1000EA-PEGDGE-CRL(由短链氨基载体LX-1000EA和PEGDGE交联脂肪酶得到)酶活的2倍,发现固定化酶LX-1000HA-PEGDGE-CRL的最适反应温度相比于游离酶提高15℃;在70℃的环境中3h后酶活仍存留70%;循环使用6次后残留65%左右的酶活;酸碱耐受性和储存稳定性也表现良好,4℃保存30天后剩余约70%的初始酶活。同时,将制备的固定化酶LX-1000HA-PEGDGE-CRL与游离酶、固定化酶LX-1000HA-GA-CRL、固定化酶LX-1000EA-PEGDGE-CRL进行了比较,发现固定化酶LX-1000HA-PEGDGE-CRL在温度耐受性和重复使用性等方面具有更好的使用效果。     

Immobilization of cane invertase on bentonite

Sugar-cane invertase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26) immobilized on bentonite clay in 0.05 m acetate buffer, pH 4.5, has been shown to be capable of hydrolysing sucrose. The bentonite-invertase (BI) complex gave 55.5% retention of enzyme activity on the surface. A further 17 and 22% increase in retention of enzyme activity was obtained using the covalent linking agents, cyanuric chloride and thionyl chloride, giving bentonite-cyanuric chloride-invertase (BCCI) and bentonite-thionyl chloride-invertase (BTCI) complexes. Concentrations of acetate buffer >0.2 M disrupt the bentonite-invertase complexes. The immobilized invertase complexes showed high temperature optima (60–65°C) and high thermal stability compared to the free enzyme. The pH profiles of the free and immobilized enzyme were the same. The rate of hydrolysis of sucrose was increased using immobilized enzymes, which required a higher substrate concentration than the free enzyme. The insoluble enzyme conjugate-carrier complexes when used for sucrose hydrolysis in a batch process showed 53.1 (BI), 57.4 (BCCI) and 59.6% (BTCI) conversions, respectively, in 12 h, compared to 42.3% conversion in 24 h with the free enzyme. The immobilized invertase complexes can be used for sucrose inversion for about five cycles. The application of this immobilization procedure may help in the removal of invertase from cane juice to reduce sugar losses in industry.     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

重组菌BL21/pET22b-argE固定化条件研究

研究了基因工程菌BL21/pET22b-argE固定化条件。最适包埋材料为海藻酸钙,优化后的包埋条件为海藻酸钠20g.L-1(含有12 g.L-1菌液)滴至2%CaCl2溶液中,固定化16 h。固定化细胞酶拆分蛋氨酸的速率比游离细胞酶慢,但其终极拆分能力与游离细胞酶相当。固定化细胞酶反复利用8批时酶活仍保持在95%以上,具有很好的工业应用优势。     

Covalent attachment of cholesterol oxidase and horseradish peroxidase on perlite through silanization: activity, stability and co-immobilization

In the present work, co-immobilization of cholesterol oxidase (COD) and horseradish peroxidase (POD) on perlite surface was attempted. The surface of perlite were activated by 3-aminopropyltriethoxysilane and covalently bonded with COD and POD via glutaraldehyde. Enzymes activities have been assayed by spectrophotometric technique. The stabilities of immobilized COD and POD to pH were higher than those of soluble enzymes and immobilization shifted optimum pH of enzymes to the lower pH. Heat inactivation studies showed improved thermostability of the immobilized COD for more than two times, but immobilized POD was less thermostable than soluble POD. Also activity recovery of immobilized COD was about 50% since for immobilized POD was 11%. The K(m) of immobilized enzymes was found slightly lower than that of soluble enzymes. Immobilized COD showed inhibition in its activity at high cholesterol concentration which was not reported for soluble COD before. Co-immobilized enzymes retained 65% of its initial activity after 20 consecutive reactor batch cycles.     

纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres , M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS 呈规则的圆球形,粒径在30nm 左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50 mg 磁性壳聚糖微球,加入20mL 0.25 mg/mL 酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05 mol/L ,pH 7.0) ,在4 ℃固定2h。M-CS 容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

Repeated batch production of agar-oligosaccharides from agarose by an amberlite IRA-900 immobilized agarase system

The production of agar-oligosaccharides from agarose by free and immobilized agarase, obtained from a Pseudomonas aeruginosa AG LSL-11 was investigated and the activity, longevity and the operational stability of immobilized enzyme was compared with that of the free enzyme. The agar hydrolyzed products of free enzyme and immobilized enzyme were neoagarobiose, neoagarotetraose and neoagarohexaose as evidenced by LC-MS analysis. The immobilization of agarase was confirmed by SEM and also by the enzymatic transformation of agarose into agaroligosaccharides. The free agarase showed maximum activity at 40°C, whereas it’s immobilized counterpart showed maximum activity at 45oC, however, the optimum pH for both systems remained unchanged (pH 8.0). The relative activities of free agarase at pH 9.0 and 10.0 were 90 and 74%, respectively, whereas, the corresponding activities of the immobilized system were determined to be 97 and 90%. The stabilities of free agarase at pH 9.0 and 10.0 were 80 and 60% respectively, but for the immobilized system the respective residual activities were estimated to be 97 and 85%. Immobilized agarase appears to be more tolerant to high temperatures in terms of its activity and stability as it is compared to that of the free enzyme which retained 74 and 50.84% of relative activity at 55 and 60°C while, free agarase retained only 40 and 16.79% of its original activity. Furthermore, the immobilized agarase could be reused in batches efficiently for eight cycles, and could be stored for 3 months at 4°C as wet beads and for more than 6 months as dry beads.     

Continuous production of fructooligosaccharides using fructosyltransferase immobilized on ion exchange resin

A continuous production of fructooligosaccharides from sucrose was investigated by fructosyltransferase immobilized on a high porous resin, Diaion HPA 25. The optimum pH (5.5) and temperature (55°C) of the enzyme for activity was unaltered by immobilization, and the immobilized enzyme became less sensitive to the pH change. The optimal operation conditions of the immobilized enzyme column for maximizing the productivity were as follows: 600 g/L of sucrose feed concentration, flow rate of superficial space velocity 2.7 h^?1. When the enzyme column was run at 50°C, about 8% loss of the initial activity of immobilized enzyme was observed after 30 days of continuous operation, during which high productivity of 1174 g/L·h was achieved. The kinds of products obtained using the immobilized enzyme were almost the same as those using soluble enzymes or free cells.     

Immobilization of Papain on the Mesoporous Molecular Sieve MCM‐48

The immobilization of papain on the mesoporous molecular sieve MCM‐48 (with a pore size of 6.2 nm in diameter) with the aid of glutaraldehyde, and the characteristics of this immobilized papain are described. The optimum conditions for immobilization were as follows: 20 mg native free enzyme/g of the MCM‐48 and 0.75 % glutaraldehyde, 2 h at 10–20 °C and pH 7.0. Under these optimum conditions for immobilization, the activity yield [%] of the immobilized enzyme was around 70 %. The influence of the pH on the activity of the immobilized enzyme was much lower compared to the free enzyme. The thermostability of the immobilized enzyme, whose half‐life was more than 2500 min, was greatly improved and was found to be significantly higher than that of the free enzyme (about 80 min). The immobilized enzyme also showed good operational stability, and the activity of the immobilized enzyme continued to maintain 76.5 % of the initial activity even after a 12‐day continuous operation. Moreover, the immobilized enzyme still exhibited good storage stability. From these results, papain immobilized on the MCM‐48 with the aid of glutaraldehyde, can be used as a high‐performance biocatalyst in biotechnological processing, in particular in industrial and medical applications.     

Immobilization of α-galactosidase on galactose-containing polymeric beads

Industrial application of α-galactosidase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free enzyme. An α-galactosidase from tomato fruit was immobilized on galactose-containing polymeric beads. The immobilized enzyme exhibited an activity of 0.62 U/g of support and activity yield of 46%. The optimum pH and temperature for the activity of both free and immobilized enzymes were found as pH 4.0 and 37 °C, respectively. Immobilized α-galactosidase was more stable than free enzyme in the range of pH 4.0–6.0 and more than 85% of the initial activity was recovered. The decrease in reaction rate of the immobilized enzyme at temperatures above 37 °C was much slower than that of the free counterpart. The immobilized enzyme shows 53% activity at 60 °C while free enzyme decreases 33% at the same temperature. The immobilized enzyme retained 50% of its initial activity after 17 cycles of reuse at 37 °C. Under same storage conditions, the free enzyme lost about 71% of its initial activity over a period of 7 months, whereas the immobilized enzyme lost about only 47% of its initial activity over the same period. Operational stability of the immobilized enzyme was also studied and the operational half-life (t_1/2 was determined as 6.72 h for p-nitrophenyl α-d-galactopyranoside (PNPG) as substrate. The kinetic parameters were determined by using PNPG as substrate. The K_m and V_max values were measured as 1.07 mM and 0.01 U/mg for free enzyme and 0.89 mM and 0.1 U/mg for immobilized enzyme, respectively. The synthesis of the galactose-containing polymeric beads and the enzyme immobilization procedure are very simple and also easy to carry out.     

Immobilization of adenosine deaminase onto agarose and casein

In the present study adenosine deaminase (ADA) was immobilized onto two different polymeric materials, agarose and casein. The factors affecting the amount of enzyme attachment onto the polymeric supports such as incubation time were investigated. The maximum amount of enzyme immobilized onto different polymeric supports occurred at incubation pH value 7.5 and ADA concentration 42 units/g and the incubation time needed for the maximum amount of enzyme attachment to the polymeric supports was found to be 8 h. Some phsicochemical properties of the free and immobilized ADA such as operational stability, optimum temperature and thermal stability, pH optimum and stability, storage stability, and the effect of gamma-radiation were studied. The operational stability of the free and immobilized enzyme showed that the enzyme immobilized by a cross-linking technique using gultaric dialdehyde showed poor durability and the relative activity decreased sharply due to the leakage after repeated washing, while the enzymes immobilized by covalent bonds to the carriers showed a slight decrease in most cases in the relative activity (around 20%) after being used 10 times. Storage for 4-6 months, showed that the free enzyme lost its activity, while the immobilized enzyme showed the opposite behavior. Subjecting the immobilized enzyme to a dose of gamma radiation of 0.5-10 Mrad showed complete loss in the activity of the free enzyme at a dose of 5 Mrad, while the immobilized enzymes showed relatively high resistance to gamma radiation up to a dose of 5 Mrad.     

Some properties of free and immobilized alpha-amylase from Penicillium griseofulvum by solid state fermentation

Alpha-amylase was produced from Penicillium griseofulvum by an SSF technique. Alpha-amylase was immobilized on Celite by an adsorption method. Various parameters, such as effect of pH and temperature, substrate concentration, operational and storage stability, ability to hydrolyze starch and products of hydrolysis were investigated; these findings were compared with the free enzyme. The activity yield of immobilization was 87.6%. The optimum pH and temperature for both enzymes were 5.5 degrees C and 40 degrees C, respectively. The thermal, and the operational and storage stabilities of immobilized enzyme were better than that of the free enzyme. Km and Vmax were calculated from Lineweaver-Burk plots for both enzymes. Km values were 9.1 mg mL(-1) for free enzyme, and 7.1 mg mL(-1) for immobilized enzyme. The Vmax of the immobilized enzyme was approximately 40% smaller than that of the free enzyme. The hydrolysis ability of the free and immobilized enzyme were determined as 99.3% and 97.9%, respectively. Hydrolysis products of the a-amylase from P. griseofulvum were maltose, unidentified oligosaccharides, and glucose.     

Preparation and characterization of UV-curable polymeric support for covalent immobilization of xylanase enzyme

The hydroxyl group of poly(ethylene glycol) monoacrylate (PEGMA) was activated by 1,1′-carbonyldiimidazole (CDI) and then a xylanase enzyme was immobilized to amine active PEGMA. UV-curable polymeric support formulation was prepared by mixing the xylanase bonded PEGMA, aliphatic polyester, 2-hydroxyethyl methacrylate (HEMA), poly(ethylene glycol) diacrylate (PEGDA) and photoinitiator. After UV irradiation, the enzymatic activity of the polymeric matrix was evaluated and compared with the corresponding free enzyme. By immobilization, the temperature resistance of the enzyme was improved and showed maximum activity at 60 °C. pH dependent activities of the free and immobilized enzymes were also investigated, and it was found that the pH of maximum activity for the free enzyme was 6.0, while for the optimal pH of the immobilized enzyme was 6.5. The immobilized enzyme retained 75% of its activity after 33 runs. The morphology of the polymeric support was characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) coupled with SEM was used to explore the chemical composition. The results have confirmed the evidence of enzyme in the structure of the polymeric material.     

Modulation of the catalytic activity of free and immobilized peroxidase by extremely low frequency electromagnetic fields: dependence on frequency

A study of the influence of electromagnetic fields (EMF) of various frequencies, from 50 up to 400 Hz, on the catalytic activity of soluble and insoluble horseradish peroxidase (POD) was carried out. To simulate the conditions in which the enzyme operates in vivo, the POD was immobilized by entrapment on a gelatin membrane or by covalent attachment on a nylon graft membrane. The rate of inactivation of the soluble POD was found to exhibit positive and negative interactions with the 1 mT applied magnetic field, with an optimum positive effect at 130 Hz. The immobilized PODs, on the contrary, do not exhibit negative interactions, but show a maximum positive interaction at 150 Hz when entrapped and at 170 Hz when covalently attached. At 50 Hz and at frequencies higher than 250 Hz no effects were observed with insoluble POD. The optimum frequency of positive interaction between the EMF and the catalytic activity of the insoluble enzymes is shifted with respect to that of the soluble enzymes towards higher frequencies, the size of the shifts being dependent on the intensity of the physical forces involved in the immobilization process.     

A comparative study of free and immobilized soybean and horseradish peroxidases for 4-chlorophenol removal: protective effects of immobilization

Horseradish peroxidase (HRP) and soybean peroxidase (SBP) were covalently immobilized onto aldehyde glass through their amine groups. The activity yield and the protein content for the immobilized SBP were higher than for the immobilized HRP. When free and immobilized peroxidases were tested for their ability to remove 4-chlorophenol from aqueous solutions, the removal percentages were higher with immobilized HRP than with free HRP, whereas immobilized SBP needs more enzyme to reach the same conversion than free enzyme. In the present paper the two immobilized derivatives are compared. It was found that at an immobilized enzyme concentration in the reactor of 15 mg l(-1), SBP removed 5% more of 4-chlorophenol than HRP, and that a shorter treatment was necessary. Since immobilized SBP was less susceptible to inactivation than HRP and provided higher 4-chlorophenol elimination, this derivative was chosen for further inactivation studies. The protective effect of the immobilization against the enzyme inactivation by hydrogen peroxide was demonstrated.     

Glucose oxidase immobilization on a novel cellulose acetate-polymethylmethacrylate membrane

Glucose oxidase (GOD) was immobilized on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane. The immobilized GOD showed better performance as compared to the free enzyme in terms of thermal stability retaining 46% of the original activity at 70 degrees C where the original activity corresponded to that obtained at 20 degrees C. FT-IR and SEM were employed to study the membrane morphology and structure after treatment at 70 degrees C. The pH profile of the immobilized and the free enzyme was found to be similar. A 2.4-fold increase in Km value was observed after immobilization whereas Vmax value was lower for the immobilized GOD. Immobilized glucose oxidase showed improved operational stability by maintaining 33% of the initial activity after 35 cycles of repeated use and was found to retain 94% of activity after 1 month storage period. Improved resistance against urea denaturation was achieved and the immobilized glucose oxidase retained 50% of the activity without urea in the presence of 5M urea whereas free enzyme retained only 8% activity.