共查询到20条相似文献,搜索用时 31 毫秒
1.
尼龙网固定化果胶酶的制备及其性质研究 总被引:2,自引:0,他引:2
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。 相似文献
2.
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. 相似文献
3.
Immobilization of Aspergillus oryzae tannase and properties of the immobilized enzyme 总被引:1,自引:0,他引:1
M. A. Abdel-Naby A. A. Sherif A. B. El-Tanash A. T. Mankarios 《Journal of applied microbiology》1999,87(1):108-114
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 Km value and a lower energy of activation. The immobilized enzyme retained about 85% of the initial catalytic activity, even after being used 17 times. 相似文献
4.
Mona A. Esawy Yannick Combet-Blanc 《World journal of microbiology & biotechnology》2006,22(3):197-200
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. 相似文献
5.
固定化青霉素V酰化酶的制备及性质 总被引:2,自引:0,他引:2
尖镰孢(Fusarium oxysporum)FP941青霉素V酰化酶经γ氧化铝吸附洗脱、硫酸铵沉淀和脱盐处理后,固定在环氧丙烯聚合物载体上,湿固定化酶表现活力为217 IU/g,固定化产率为53%。固定化酶作用最适温度为55℃,最适pH为80;在pH50~110及50℃以下稳定;37℃使用25次后,酶活力保留90%。 相似文献
6.
K. H. Jang K. B. Song J. S. Kim C. H. Kim B. H. Chung S. K. Rhee 《Bioprocess and biosystems engineering》2000,23(1):89-93
Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 °C. 相似文献
7.
Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six-carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy. 相似文献
8.
Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six-carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy. 相似文献
9.
以超临界二氧化碳(SCCO2)为分散介质在聚偏氟乙烯(PVDF)微孔膜表面和孔内进行马来酸酐和苯乙烯的接枝共聚,合成出超高分子量的苯乙烯/马来酸酐交替共聚物(SMA)基微孔PVDF膜。以SMA基PVDF膜为载体通过酸酐基和酶分子上的氨基偶联,制备出具有酶催活性的功能性分离膜。考察了影响酶固定化的因素,确定其最佳固定化条件为: 温度,4oC;pH,8.2; 酶/膜,1:10;反应时间,6h。固定化酶膜的最适温度为55oC,最适pH为7.8,均比自由酶稍高;Km(0.3mM/L)与自由酶接近。固定化酶膜活力达13.5 U/cm2 膜, 比活为280.0 U/mg 蛋白,蛋白载量为68.2 g/cm2 膜,相对活力为89.0%。固定化酶膜表现出良好的操作稳定性和储存稳定性,SMA基PVDF微孔酶膜超滤制备低乳糖牛奶实验表明该技术应用前景广阔。 相似文献
10.
Singh AK Flounders AW Volponi JV Ashley CS Wally K Schoeniger JS 《Biosensors & bioelectronics》1999,14(8-9):703-713
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. 相似文献
11.
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。 相似文献
12.
尼龙经CaCl_2和H_2O的甲醇溶液处理,稀HCl水解用戊二醛交联以制备固定化木瓜蛋白酶。在溶液酶浓度为1mg/mL pH7.5—8.0、4—15℃条件下固定3h,活力回收42.5%,相对活力46%,偶联效率52%,半衰期72天。溶液酶Km值和固定化酶K_m~(aPP)值(底物酪蛋白W/V,%)分别为0.28%和0.35%。溶液酶和固定化酶分别在pH6.5和pH8.0以下活力稳定;最适pH分别为7.0和8.0;在65℃处理30min活力分别为原有活力的89%和66%。当酪蛋白浓度为1.5%和2.5%以上活力分别受到抑制。固定化酶在6mol/L脲中连续浸洗5次共6h其活力稳定,仍有原活力的44.4%;用以处理啤酒浊度比对照下降了2-11倍;蛋白质含量下降了55%;冷藏(4℃)120天,无冷混浊发生;同时各项理化指标和风味不变。 相似文献
13.
Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme. 相似文献
14.
Kozo Yamamoto Tetsuya Tosa Kiyokazu Yamashita Ichiro Chibata 《Biotechnology and bioengineering》1977,19(8):1101-1114
The kinetics of the reversible fumarase reaction of immobilized Brevibacterium ammoniagenes cells and the decay behavior of enzyme activity were investigated in a plug flow system. The time course of the reaction in the immobilized cell column was well explained by the time-conversion equation including the apparent kinetic constants of the immobilized cell enzyme. The decay rate of fumarase activity was faster in the upper sections of the column (inlet side of the substrate solution) compared with the lower sections when 1M sodium fumarate (pH 7.0) was continuously passed through the column at 37°C. It was shown that the decay rate of the fumarase activity in the immobilized cell column depends on the flow rate of the substrate solution. The effect of flow rate on the decay rate of enzyme activity was considered to be related to the rate of contamination of enzyme with poisonous substances derived from the substrate solution or to the rate of leakage of enzyme stabilizers and/or enzyme itself from the immobilized cells. 相似文献
15.
Summary Fluid immobilized cellulase was prepared using polyethyleneglycols and hexamethylene diisocyanate, and its properties studied. The cellulase activity of the immobilized enzymes varied with monomer composition and molecular weight of polyethyleneglycols. The enzyme activity was affected by the viscosity of the carrier. A solid substrate (cellulose powder) can be hydrolyzed with the fluid immobilized enzyme. 相似文献
16.
Shigeaki Fujikawa Tomoko Yokota Kunimasa Koga Jun-ichi Kumada 《Biotechnology letters》1987,9(10):697-702
Summary The reaction velocity of immobilized -glucosidase was approximated by the first-order reaction kinetics. A plug flow reactor was used for continuous hydrolysis of geniposide with this immobilized enzyme. The activity of this immobilized enzyme was retained 100% for 600 h. The amount of genipin formed by using the immobilized enzyme was 17 fold that formed using the native enzyme without reuse. Using immobilized enzyme, purity and yield of genipin, which is a hydrolyzate of geniposide, was improved comparing with the native enzyme. 相似文献
17.
J Tramper S A Angelino F Müller H C van der Plas 《Biotechnology and bioengineering》1979,21(10):1767-1786
Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis. 相似文献
18.
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。 相似文献
19.
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%。 相似文献
20.
纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究 总被引:1,自引:0,他引:1
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。 相似文献