氨基化二氧化硅颗粒固定木瓜蛋白酶研究

采用正硅酸乙酯与N-(β-氨乙基)氨丙基三乙氧基硅烷在油包水形成的微胶囊中同步水解的方法,一步法制备了氨基化的二氧化硅颗粒,得到的颗粒粒径在0.3～0.5μm之间,平均大小为0.37μm, 氨基含量和颗粒大小可控,氨基含量高达56mmol/g。此颗粒经戊二醛处理后,采用共价法固定木瓜蛋白酶,固定化最适pH6.5,最佳给酶量为15mg/g载体,固定化酶的最适反应温度为70℃,最适反应pH为6.5,固定化酶热稳定性,pH耐受性,贮存稳定性都明显高于游离酶,表明此颗粒可作为一种优良的酶固定化载体。     

Silica carriers for immobilization of thermostable α-amylase

The possibilities for immobilization of thermostable α-amylase from Bacillus licheniformis 44MB82 on silica carriers activated by different methods have been studied. Immobilization on Ti(IV)-activated CPG, Chromosorb P, quartz powder and pumice stone was sufficiently effective. The preparation immobilized on CPG showed a shift in pH and temperature-profiles of enzyme action. They were found to be changed from pH 6,0–6,5 to pH 8,5 and from 90°C to 80°C, respectively, when compared to these parameters of the soluble enzyme. Immobilization leads to enhancement of thermostability. Possibilities for batchwise use of the immobilized preparation were established.     

金属螯合载体定向固定化木瓜蛋白酶的研究

以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体（IDACu²⁺）与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu²⁺1.5×10^-2mol/g载体、固定化时间4h、固定化pH7.0、给酶量30mg/g载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4％,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。     

Immobilization of maltogenase onto polyurethane microparticles from poly(vinyl alcohol) and hexamethylene diisocyanate

A series of porous polyurethane (PU) microparticles from poly(vinyl alcohol) (PVA) and hexamethylene diisocyanate (HMDI) using different ratios of components were obtained by one step method. Molar compositions of PU microparticles were estimated by determination of nitrogen, isocyanate and hydroxyl groups. PU carriers which were synthesized using optimal initial molar ratios of PVA and HMDI were applied for immobilization of maltogenase (MG) from Bacillus stearothermophilus. Immobilized enzyme exhibited higher catalytic activity and enhanced temperature stability in comparison with the native MG. Maximal loading 7.78 mg/g wet carrier was reached when PU microparticles with initial molar ratio of PVA and HMDI = 1:3 was used as a carrier for immobilization. The high efficiency of immobilization (EI) was obtained using PU microparticles when initial molar ratio of HMDI and PVA was 1:1–1:10. High stability of MG immobilized onto PU microparticles during storage was demonstrated. Immobilized starch hydrolyzing enzyme was successfully tested in batch and column type reactors for hydrolysis of potato starch. MG immobilized onto PU enables easy separation from the reaction medium and reuse of the immobilized preparation over seven reaction cycles in bath operation and at least three cycles in column type reactor.     

Purification and immobilization of Aspergillus niger β-xylosidase

β-Xylosidase from a commercial Aspergillus niger preparation was purified by differential ammonium sulfate precipitation and either gel permeation or cation exchange chromatography, giving 16-fold purification in 32% yield for the first technique or 27-fold purification in 19% yield for the second. The second method in addition almost completely removed interfering β-glucosidase activity. Enzymes prepared by this method was immobilized to 10 different carriers, but only when it was bound to alumina with TiCl₄ and to alkylamine porous silica with glutaraldehyde were substantial efficiencies and stabilities achieved. With alumina, the variation of activation procedure, amount of β-xylosidase offered, and activation solution composition yielded maximum activities of over 40 U/g with approximately 70% immobilization efficiency. Variation of binding pH and incubation time led to a maximum immobilized activity of 1.3 U/g with 78% immobilization efficiency on silica.     

海藻酸钠包埋法制备固定化菠萝蛋白酶

以海藻酸钠为载体,包埋法固定菠萝蛋白酶,对固定化奈件进行优化,同时探讨固定化菠萝蛋白酶的部分酶学性能。结果表明：固定化菠萝蛋白酶的质量受海藻酸钠质量分数、固定化酶量、固定化时间以及CaCl2质量分数的影响,其最佳固定化条件为：海藻酸钠质量分数1．0％,CaCl2质量分数3％,固定化酶液量与海藻酸钠体积之比1：2,固定化时间60min,在此条件下,制备的固定化菠萝蛋白酶的比活力为211．8U／g（湿质量载体）,由此制得的固定化酶的最适pH为7．6,与游离酶相比,升高了0．8个pH单位,同时显示固定化菠萝蛋白酶能耐受较高的碱性环境,固定化酶最适温度与游离酶相同,均为50℃,固定化酶在较高温度范围内,仍能保持较高的相对活力。     

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.     

Immobilization of pectinase fromSclerotium rolfsii

Immobilization of pectinase fromS. rolfsii was studied on different matrices of which Amberlite XAD-7 showed maximum adsorption and expression of the enzyme. The most active preparation was obtained when XAD-7 was activated with 2% glutaraldehyde and 1.7 μkat of enzyme per g resin was used for immobilization at pH 5.5 and 28°C. Optimum pH and temperature of theS. rolfsii pectinase remain unaltered, 3.5 and 55°C, respectively, after immobilization. However, the apparentK _M value of the enzyme decreased from 1.75 g/L for soluble enzyme preparation to 1.4 g/L for immobilized enzyme preparation. Both soluble and immobilized enzyme preparations were most stable at pH 4.0. The immobilized enzyme preparation was more stable than the soluble enzyme.     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.     

Enzyme immobilization techniques on poly(glycidyl methacrylate-co-ethylene dimethacrylate) carrier with penicillin amidase as model.

Two types of bead-form macroporous carriers based on glycidyl methacrylate with ethylene dimethacrylate copolymers were used for the immobilization of penicillin amidase either directly or after chemical modification. Direct binding through oxirane groups, which is equally efficient at pH 4.2 and 7, is relatively slow and brings about an activity loss at low enzyme concentrations. The most efficient immobilization was achieved on glutaraldehyde-activated amino carrier, irrespective of whether the amino groups were formed by ammonia or 1,6-diaminohexane treatment of the original oxirane carrier. Hydrazine treatment gave lower immobilization yields. The same is true of the azide method independent of the length of the spacer. Most enzyme activity was preserved by coupling the carbodiimide-activated enzyme to the carrier with alkyl or arylamino groups at the end of a longer substituent. Immobilization on diazo-modified carrier gave average results. Rapid immobilization by a lysine-modified phosgene-treated carrier resulted in an activity loss. It is suggested that multipoint and very tight attachment of the enzyme molecule to the matrix decreased the activity. The immobilized activity is quite stable in solution and very stable upon lyophilization with sucrose.     

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Penicillium notatum

A new low-cost β-galactosidase (lactase) preparation for whey permeate saccharification was developed and characterized. A biocatalyst with a lactase activity of 10 U/mg, a low transgalactosylase activity and a protein content of 0.22 mg protein/mg was obtained from a fermenter culture of the fungus Penicillium notatum. Factors influencing the enzymatic hydrolysis of lactose, such as reaction time, pH, temperature and enzyme and substrate concentration were standardized to maximize sugar yield from whey permeate. Thus, a 98.1% conversion of 5% lactose in whey permeate to sweet (glucose-galactose) syrup was reached in 48 h using 650 β-galactosidase units/g hydrolyzed substrate. After the immobilization of the acid β-galactosidase from Penicillium notatum on silanized porous glass modified by glutaraldehyde binding, more than 90% of the activity was retained. The marked shifts in the pH value (from 4.0 to 5.0) and optimum temperatures (from 50°C to 60°C) of the solid-phase enzyme were observed and discussed. The immobilized preparation showed high catalytic activity and stability at wider pH and temperature ranges than those of the free enzyme, and under the best operating conditions (lactose, 5%; β-galactosidase, 610–650 U/g lactose; pH 5.0; temperature 55°C), a high efficiency of lactose saccharification (84–88%) in whey permeate was achieved when lactolysis was performed both in a batch process and in a recycling packed-bed bioreactor. It seems that the promising results obtained during the assays performed on a laboratory scale make this immobilizate a new and very viable preparation of β-galactosidase for application in the processing of whey and whey permeates.     

Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking

The main objective of the present work is to study the immobilization process of Aspergillus oryzae β-galactosidase using the ionic exchange resin Duolite A568 as carrier. Initially, the immobilization process by ionic binding was studied through a central composite design (CCD), by analyzing the simultaneous influences of the enzyme concentration and pH on the immobilization medium. The results indicate that the retention of enzymatic activity during the immobilization process was strongly dependant of those variables, being maximized at pH 4.5 and enzyme concentration of 16 g/L. The immobilized enzyme obtained under the previous conditions was subjected to a cross-linking process with glutaraldehyde and the conditions that maximized the activity were a glutaraldehyde concentration of 3.83 g/L and cross-linking time of 1.87 h. The residual activity of the immobilized enzyme without glutaraldehyde cross-linking was 51% of the initial activity after 30 uses, while the enzyme with cross-linking immobilization was retained 90% of its initial activity. The simultaneous influence of pH and temperature on the immobilized β-galactosidase activity was also studied through a central composite design (CCD). The results indicate a greater stability on pH variations when using the cross-linking process.     

Immobilized β-glucosidase fromCurvularia lunata

β-Glucosidase fromCurvularia lunata was immobilized in pellets of polyacrylamide, sodium alginate and agar. The activity of the enzyme was estimated at different times by measuring the absorbance of a solution into which 2-nitrophenol was released by the enzyme. The effect of pH and temperature was studied to select the optimum conditions. Thermostability of the β-glucosidase in each of the carriers was assessed over a period of 12–26 d. The immobilized enzyme on all the three carriers retained its activity longer than free enzyme did. Polyacrylamide was the best carrier both in terms of thermostability and of reusability of the immobilized enzyme preparations. The Michaelis constant (K _m) for each of the immobilized enzyme preparation was calculated.     

Laccase immobilization on the tailored cellulose-based Granocel carriers

Extracellular laccase produced by Cerrena unicolor was immobilized by adsorption or covalent bonds formation on the cellulose-based carrier Granocel. Immobilization was optimized by changing the anchor groups and the methods of activation/immobilization. On the base of measured activity and stability of immobilized preparations, the covalent method was selected. It was shown that coupling of the enzyme to the carrier via divinyl sulfone or glutaraldehyde yielded an enzyme-carrier preparation of high activity and storage stability. Further optimization of the carrier's superstructure consisted in changing pore diameters and amount of functional groups on the carriers surface. Three-fold higher activity was noted when the enzyme was immobilized on NH2-modified Granocel with the highest size exclusion limit and amino group content. Relatively low products sorption was observed on the carrier surface. The effects of protein concentration and pH-value of the coupling mixture on immobilization efficiency were evaluated also.     

Immobilized glucose dehydrogenase for cofactor regeneration in heme-catalyzed demethylation and hydroxylation reactions

Glucose dehydrogenase (E.C. 1.1.1.47) from B. megaterium M 1286 was immobilized together with mutarotase (E.C. 5.1.3.3) on several organic carriers and by different methods. The storage stability of the enzyme at pH-values > 6 is slightly improved by immobilization and the pH-optimum is shifted from 8.3 to 8.0. Kinetic constants of the immobilized enzyme are: K′_M(NAD⁺) = 5.36 × 10^?4 mol/l K′_M(glucose) = 3.76 · 10^?2 mol/l and V′_max = 5.54 · 10^?5 mol/(l min g carrier) for the most active preparation (2.16 mg enzyme/g carrier). In reactor experiments the immobilized glucose dehydrogenase was used with glucose to regenerate NADPH in NADPH-dependent iron-III-protoporphyrin-IX-imidazole catalyzed hydroxylation and demethylation of model substrates of cytochrome P-450. The advantages of the coupling of both reactions with cofactor recycling are shown and discussed.     

Preparation and characterization of Rhizopus amyloglucosidase immobilized on poly(o-toluidine)

The starch hydrolyzing enzyme amyloglucosidase (AMG) from Rhizopus was immobilized onto the protonated salt (TS) and basic (TB) forms of chemically synthesized poly(o-toluidine) (POT) using adsorption and covalent binding. The polymers were activated with glutaraldehyde prior to covalent bonding. The immobilization efficiency was affected by the pH of the immobilization medium, contact time and amount of enzyme. After immobilization, the pH and temperature were changed to conditions under which the enzyme is most active. Immobilized AMG was more stable with respect to changes in pH and increases in temperature compared to free AMG. The immobilized enzyme retained high catalytic activity after multiple uses and showed enhanced stability with storage compared to free enzyme.     

活性炭吸附固定化粪产碱杆菌青霉素G酰化酶

目的：以活性炭为载体固定化粪产碱杆菌来源的青霉素G酰化酶,考察固定化酶的性质。方法：对影响酶固定化的因素优化筛选,确定有显著影响的因素：pH、离子强度、酶量、固定化时间进行L934的正交实验,获得最佳固定化条件,并对固定化酶的最适反应温度、pH及批次稳定性进行研究。结果：最佳固定化条件为：载体0.3g,酶量5mL,总反应体系为12mL,离子强度1mol/L,温度4℃,pH 7.0,固定化40h;最高固定化酶活性为135.9U/g湿载体。固定化酶性最适反应温度为55℃,最适pH为10,重复使用12次后没有活性损失。结论：活性炭吸附固定化青霉素G酰化酶的活性高,批次反应稳定,具有工业应用潜力。     

树脂载体固定S-腺苷甲硫氨酸合成酶的研究

目的:筛选一种适合S-腺苷甲硫氨酸合成酶固定化的树脂载体,进行固定化工艺优化及固定化酶性质研究。方法:以固定化率和表观酶活回收率为指标,筛选固定化效果最佳的一种树脂,采用单因素实验对固定化条件进行优化。结果:阴离子交换树脂载体ESR-2表现出最优的固定化率(94.03%)和酶活回收率(47.45%);最佳固定化条件为加酶量4U/g、pH 8.0、15℃吸附10h,最佳条件下固定化酶表观酶活为2.1U/g,表观酶活回收率达51.6%。固定化酶的最适pH为8.5,最适温度为35℃,连续反应10批次后酶活剩余77.92%。结论:树脂载体ESR-2固定化S-腺苷甲硫氨酸合成酶酶活及稳定性较好,能够用于S-腺苷甲硫氨酸的工业化大规模生产。     

Fabrication of an organic–inorganic nanocomposite carrier for enzyme immobilization based on metal–organic coordination

An organic–inorganic nanocomposite which combined mesoporous silica SBA-15 and chitosan using a carboxyl functionalized ionic liquid as the bridging agent (SBA@CS) was successfully fabricated, and was used to immobilize porcine pancreas lipase (PPL) by physical adsorption, cross-linking and metal–organic coordination, respectively. The as-prepared carriers were characterized by scanning electron microscopy, Fourier transform infrared and energy-dispersive X-ray spectroscopy. Compared with immobilization onto the pure mesoporous silicon material SBA-15, all the batches of PPL immobilized onto organic–inorganic nanocomposites showed higher activity, improved stability and reusability as well as better resistance to pH and temperature changes. Among the immobilized PPLs, immobilization based on Co²⁺ coordination (SBA@CS-Co-PPL) produced the best enzymatic properties. The maximum immobilization efficiency and specific activity of 79.6% and 1975.8 U g⁻¹ were obtained with SBA@CS-Co, separately. More importantly, the activity of immobilized enzyme can still maintain 84.0% after 10 times of reuse. These results demonstrated that thus prepared organic–inorganic nanocomposite could be an ideal carrier for enzyme immobilization by metal–organic coordination.     

Optimized immobilization of endoglucanase Cel9A onto glutaraldehyde activated chitosan nanoparticles by response surface methodology: The study of kinetic behaviors

Immobilization of enzyme onto nanoparticles such as chitosan can have biotechnological importance. In this study, chitosan nanoparticles (ChNPs) were prepared by Ionic gelation method and Endoglucanase Cel9A from Alicyclobacillus acidocaldariius (AaCel9A) immobilized on the nanoparticles. The FTIR results showed that the enzymes were immobilized on the ChNPs. The dynamic light scattering and scanning electron microscope (SEM) results illustrated that the AaCel9A-ChNPs approximately had 40 nm diameters. For optimizing enzyme immobilization, response surface methodology was employed using different variables (pH, enzyme immobilization time, and enzyme to ChNPs ratio [E/Cs]). The results showed that the high immobilization efficiency was achieved in pH 7, E/Cs of 0.4 in 2.63 hr. The enzyme activity results showed that, immobilization increased optimum pH for activity (from 6.5 to 7.5) and the enzyme K_m (from 3.703 to 12.195 [mg/ml]), which make it suitable to use in some industries such as detergents.