尼龙网固定化果胶酶的制备及其性质研究

用尼龙网作载体,经3-二甲氨基丙胺活化,用戊二醛将果胶酶固定化。所得固定化酶Km值与自然酶接近;对温度的稳定性有较大的提高,100℃保温30min才能使其失活。固定化酶在较宽的pH范围内能保持其正常活力,它对金属离子抑制剂的耐受性有较显著的提高,用0.5％果胶溶液作底物,重复使用10次后酶活力保留44％。固定化果胶酶与自然酶相比较,对不同果汁的澄清效果不同。固定化果胶酶在无保护剂存在的条件下,室温放置四个月活力不减少。     

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。     

Studies on the properties of immobilized urokinase: effects of pH and temperature

Human urokinase was immobilized on an ethylene vinyl acetate copolymer surface. Soluble urokinase showed its maximum activity at pH 8.5, while the immobilized enzyme was most active at pH 9.0. Apparently, the shift in optimal pH was due to the polyanionic nature of the carrier surface on which the enzyme was immobilized. Optimal temperatures of soluble urokinase and immobilized enzyme were identical, i.e., 37 degrees C. The stability of immobilized enzyme against thermal degradation was several times higher than that of the soluble enzyme. Its stability at higher temperatures is one of the main reasons for the clinical use of immobilized urokinase as an antithrombotic material.     

Kinetic studies of sepharose-and CH-sepharose-immobilized dihydrofolate reductase

Dihydrofolate reductase, purified to homogeneity from amethopterin-resistant Lactobacillus casei, was immobilized by coupling to cyanogen bromide-activated Sepharose or carbodiimide-activated CH-Sepharose. Coupling yields were determined by amino acid analysis following the hydrolysis of the gel. Enzyme activity was measured by the conventional spectrophotometric procedure, thus permitting the facile characterization of the immobilized enzyme. The pH optimum of the immobilized enzyme was shifted to 5.8 compared with pH 5.5 for the soluble enzyme. The immobilized enzyme retained greater than 90%of the initial activity over a six-month period and could be reused as many as ten times without loss of activity. As observed with the soluble enzyme, the activity of immobilized enzyme, which was lost on denaturation with 4M guanidine hydrochloride, was recovered rapidly and completely by washing the gel with buffer. The K(m) (app) values for dihydrofolate and NADPH for the immobilized enzyme were increased 15-164-fold over the K(m) values measured for soluble dihydrofolate reductase. Scatchard analysis of the interaction of amethopterin with the immobilized enzyme yielded linear plots and a K(d) (app) value of 0.56 x10(-8)M, and revealed that all of the immobilized enzyme molecules were capable of binding the ligand.     

Immobilization of Bacillus licheniformis 5A1 milk-clotting enzyme and characterization of its enzyme properties

Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated 5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting enzyme. The activation energy (E _A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E _A = 1.59 and 1.99 Kcal mol⁻¹ respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0 for both free and immobilized enzyme. The Michaelis constant K _m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme.     

多聚半乳糖醛酸内切酶的固定化及其性质研究

棉花枯萎病菌多聚半乳糖醛酸内切酶在pH大于7时不稳定,故对它进行多种化学修饰而又不影响其活性,必须在pHd小于7的体系中进行。本文报道将PGAUase在还原剂存在下,与稀酸处理的Sepharose 4B交联,获得较高活力的固定化酶。固定化酶催化动力学表明,最适pH为4,4,最适温度为55℃,在pH1至8.0范围内稳定。和溶液酶比较,对热稳定性提高,但对碱稳定性下降。以多聚半乳糖醛酸为底物,Km为0.27mmol/L,Vmax为66.67nmol/L·min,均大于溶液酶(Km=0.07mmol/L,Vmax=28.00nmol/L·min)。在pH4.8,30℃,聚半乳糖醛酸在固相酶的柱中循环水解不同的时间降解产物经圆盘电泳和等电聚焦测定,得到不同大小的寡糖片段混合物,证明固相酶和溶液酶的作用方式相同,同时使以酶解法制备一定大小的有生物活性的寡糖分子成为可能。     

强化海藻酸钠凝胶制备固定化酶

为了确定生产帕拉金糖（异麦芽酮糖）的固化酶最佳条件,在固化材料和固化方法上进行多项试验。结果表明：使用添加剂强化海藻酸钙凝胶包埋整个细胞酶,固化效果最佳,固化酶的酶活为３０～６０ｕ／ｇ,蔗糖平均转化率８５％,最高转化率９５％,实验室中固化酶连续转化半衰期长达４５ｄ。     

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.     

Immobilization of glucose oxidase on silica-based supports

Glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) was covalently coupled to silica-based supports containing aldehyde functional groups. The activity of the immobilized enzyme was about 1000 U/g support. The optimum pH of the catalytic activity was 5.5 for the soluble enzyme and 6.0 for the immobilized enzyme. With glucose as a substrate the Km value of the immobilized enzyme was higher than in case of the soluble enzyme. The immobilized enzyme was found to be more thermostable than the soluble one. The immobilization did not affect the stability of glucose oxidase against the denaturing effect of urea.     

Enhanced thermostability of silica-immobilized lipase from Bacillus coagulans BTS-3 and synthesis of ethyl propionate

A lipase from the thermophilic isolate Bacillus coagulans BTS-3 was produced and purified. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The purified lipase was immobilized on silica and its binding efficiency was found to be 60%. The enzyme took 60 min to bind maximally onto the support. The pH and temperature optima of immobilized lipase were same as those of the free enzyme, i.e. 8.5 and 55 degrees C, respectively. The immobilized enzyme had shown marked thermostability on the elevated temperatures of 55, 60, 65 and 70 degrees C. The immobilized enzyme was reused for eigth cycles as it retained almost 80% of its activity. The catalytic activity of immobilized enzyme was enhanced in n-hexane and ethanol. The immobilized enzyme when used for esterification of ethanol and propionic acid showed 96% conversion in n-hexane in 12 h at 55 degrees C.     

胰蛋白酶的固定化及其性质研究

 以自制的脱乙酰壳多糖作载体,戊二醛为交联剂,对胰蛋白酶的固定化条件及其固定化酶的性质进行了研究。考查了交联剂的用量、pH值、以及载体与酶的比例等因素对胰蛋白酶固定化的影响。在所选择的固定化条件下,固定化酶的活性回收可达50％以上。同时研究了固定化胰蛋白酶的一些性质;最适温度60℃,最适PH8.0,Km值比可溶性酶升高,热稳定性、pH贮存稳定性以及在乙醇水溶液中的稳定性明显高于可溶性胰蛋白酶。在柱式反应器内,以2％酪蛋白为底物对,操作半衰期为40天。     

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.     

Site-directed and random immobilization of subtilisin on functionalized membranes: activity determination in aqueous and organic media

Kinetic comparisons have been made between a randomly immobilized and a site-specifically immobilized subtilisin BPN' on microfiltration membranes of varying hydrophilicities in both aqueous and organic media. Site-directed mutagenesis was employed to introduce a single cysteine into the amino acid sequence of subtilisin at a location away from the active site. Immobilization of this mutant enzyme was then carried out using the single cysteine residue to orient the active site of the enzyme away from the membrane surface. Kinetic comparison of the immobilized mutant enzyme with the randomly immobilized wild-type enzyme in aqueous media showed an activity enhancement on both hydrophilic silica-containing and hydrophobic poly(ether)sulfone membranes. Higher loading efficiencies were observed for the site-directed enzyme on immobilization. Optimal enzyme loading values were calculated for the randomly immobilized enzyme. An enhancement of activity was also observed for the site-directed immobilized systems using nearly anhydrous hexane as the solvent.     

无花果蛋白酶在阴离子交换树脂上的固定化

无花果蛋白酶通过８％戊二醛活化载体,共价结合到聚苯乙烯阴离子交换树脂ＧＭ２０１上,固定化作用在ｐＨ７．７,酶浓度０．８ｍｇ／ｇ树脂,４℃下进行６ｈ。得到的固定化酶表观Ｋ_ｍ值（酪蛋白,１．１１×１０~（－４）ｍｏｌ／Ｌ）小于溶液酶Ｋ_ｍ值（１．９６×１０~（－４）ｍｏｌ／Ｌ）;固定化酶活性在ｐＨ６～８保持稳定,溶液酶最适ｐＨ为７．２;固定化酶最适温度由溶液酶的５０～６０℃移至３７℃;固定化酶２５℃保持７ｄ,重复水解酪蛋白７次后,保留８３．３％活性。固定化酶对酪蛋白水解度达４７．５％,对大豆球蛋白达１１．６％。     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.     

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.     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。     

Batch production of deacetyl 7-aminocephalosporanic acid by immobilized cephalosporin-C deacetylase

Bacillus subtilis SHS0133 cephalosporin-C deacetylase (CAH) overexpressed in Escherichia coli was immobilized on an anion-exchange resin, KA-890, using glutaraldehyde. The activity yield of immobilized enzyme was approximately 55% of the free enzyme. The pH range for stability of the immobilized enzyme (pH 5–10) was broader than that for free enzyme. The K_m^app value of immobilized enzyme for 7-aminocephalosporanic acid (7-ACA) was similar to that of the free enzyme. This immobilized enzyme obeyed Michaelis–Menten kinetics similar to those of the free enzyme. A batch-type reactor with a water jacket was employed for deacetylation of 7-ACA using CAH immobilized on KA-890. Ten kilograms of 7-ACA were completely converted to deacetyl 7-ACA at pH 8.0 within 90 min. The reaction kinetics agreed well with a computer simulation model. Moreover, the immobilized enzyme exhibited only a slight loss of the initial activity even after repeated use (52 times ) over a period of 70 days. This reaction will thus be useful for the production of cephalosporin-type antibiotics.     

Immobilization of catalase on cotton fabric oxidized by sodium periodate

Cotton fabric was first oxidized with sodium periodate, and then employed to immobilize catalase. Optimization studies for oxidation of the fabric and immobilization of the enzyme were performed. The properties of the immobilized catalase were examined and compared with those of the free enzyme. A high activity of the immobilized enzyme was obtained when the fabric was oxidized at 40°C and pH 6.0 for 8h in a bath containing 0.20 mol L^?1 sodium periodate and the enzyme was immobilized at 4°C for 24h with a catalase dosage of 120.0 U mL^?1. The immobilized enzyme exhibited optimum activity at 40°C, while the free enzyme had optimal temperature of 30°C, suggesting that the immobilized catalase could be used in a broader temperature range. Both the immobilized and free enzyme had pH optima of 7.0. The staining test and reusability showed that the catalase was fixed covalently on the oxidized cotton fabric.     

Degradation of raffinose oligosaccharides in soymilk by immobilized alpha-galactosidase of Aspergillus oryzae

Alpha-galactosidase was immobilized in a mixture of k-carrageenan and locust bean gum. The properties of the free and immobilized enzyme were then determined. The optimum pH for both the soluble and immobilized enzyme was 4.8. The optimum temperature for the soluble enzymes was 50 degrees C, whereas that for the immobilized enzyme was 55 degrees C. The immobilized enzyme was used in batch, repeated batch, and continuous modes to degrade the raffinose-family sugars present in soymilk. Two hours of incubation with the free and immobilized alpha-galactosidases resulted in an 80% and 68% reduction in the raffinose oligosaccharides in the soymilk, respectively. In the repeated batch, a 73% reduction was obtained in the fourth cycle. A fluidized bed reactor was also designed to treat soymilk continuously and the performance of the immobilized alpha-galactosidase tested at different flow rates, resulting in a 90% reduction of raffinose-family oligosaccharides in the soymilk at a flow rate 40 ml/h. Therefore, the present study demonstrated that immobilized alpha-galactosidase in a continuous mode is efficient for reducing the oligosaccharides present in soymilk, which may be of considerable interest for industrial application.