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1.
用多孔强碱性三乙醇胺基聚苯乙烯树脂作为载体,用CNBr与载体上多羟基作用共价偶联葡萄糖异构酶(GI)。最适偶联条件表明:CNBr量增多,蛋白载量增加,但比活下降。固定化葡萄糖异构酶(IGI)最适反应温度比天然酶提高15℃。并系统地研究了影响IGI活力-pH的曲线的各种因素:用具有不同平均孔径的载体(R=137A,185A,230A,365A)固定化GI,在低离子强度条件下(0.0064mol/L),测定其最适pH值分别7.76,7.56,7.50,8.20。选择平均孔径为230A且具有不同数量三乙醇胺基的载体(0.94,1.05,1.13,1.37mmol/g干胶)分别固定化GI,其最适pH值分别为7.70,7.50,7.46,7.36。 相似文献
2.
作者合成了阴离子型和阳离子型葡聚糖,以此为载体,用CNBr活化其剩余羟基,固定化了葡萄糖淀粉酶和葡萄糖异构酶。就离子型载体对固定化酶的蛋白载量、最适pH和热稳定性等的影响做了考察。发现固定化酶的蛋白载量不仅与载体的电性质有关,也与酶分子自身的电性质有关。当载体电性质与酶蛋白电性质相反时,固定化酶的蛋白载量增加,热稳定性提高、载体电性质与酶蛋白电性质相同时,固定化酶的蛋白载量不变或下降,其热稳定性不变。作者还发现当离子型载体孔度和体系缓冲液浓度一定时,酶分子能否进入多孔性载体内部,对其最适pH是否变化影响极大。若酶分子仅被连接在载体的外表层,其最适pH不发生变化,反之亦然。作者还观察到当多糖类载体引入氨基或羧基后,大大增强了其抵抗微生物侵蚀的能力。 相似文献
3.
将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上.制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35%.研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性.结果表明:过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25℃;但固定化酶的热稳定性显著提高,在75℃保存5 h后,仍能保留30%的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88%以上,而自由酶在此条件下则完全失活;此外,固定化过氧化氢酶还表现出了良好的操作稳定性,在室温下连续反应26次后,相对活力为57%.该研究表明胶原纤维可作为固定化过氧化 相似文献
4.
接枝淀粉载体固定化糖化酶的研究 总被引:6,自引:0,他引:6
合成了淀粉接枝丙烯腈及烯酰胺的两亲性高分子化合物,并以此为载体,用物理吸附方法固定化了糖化酶,最适偶联条件研究表明:缓冲液的浓度,PH值及吸附时间和加酶量都对固定化酶活力,比活有一定的影响,有最适固定化条件下,固定化酶的活力为1500U/g干胶,蛋白载量为25mg/g干胶,比活为60U/mg蛋白,比天然酶的比活提高6倍,最适反应温度比天然酶提高10℃。无底物存在下,固定化酶在55℃的半衰期为24h 相似文献
5.
将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上。制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35%。研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性。结果表明:过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25 ℃;但固定化酶的热稳定性显著提高,在75 ℃保存5 h后,仍能保留30%的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88%以上,而自由酶在此条件下则完全失 相似文献
6.
以葡聚糖为载体,在葡聚糖上引入了不同量的羧甲基,采用CNBr法活化葡聚糖上剩余的羟基,使之与葡萄糖淀粉酶(GA)共价结合,从而观察载体羧甲基定量变化对固定化葡萄糖淀粉酶pH——活性曲线的影响。分别使用了两种不同孔度的葡聚糖(SephadxG100、G200)作为载体,首次发现在载体带有离子基因的情况下,载体的孔度变化对固定化酶的pH——活性曲线影响极大,甚至可以完全消除载体静电荷对此带来的影响。这些结果表明:固定化酶的微环境不仅与载体的静电势有关,也与载体分子空间结构的疏密程度有关。本文对固定化葡萄糖淀粉酶(IGA)的热稳定性也作了评价。 相似文献
7.
共价结合法是重要的工业酶固定化方法,利用稳定的共价键固定化工业酶,在载体和酶间形成多点共价连接,可以制备稳定性较好的固定化酶,更具有实际应用价值。利用氨基载体共价结合固定化海洋假丝酵母脂肪酶,采用较为廉价的戊二醛进行辅助交联,通过单因素和正交试验,确定最佳固定化条件为:25℃、pH5. 0、0. 1%戊二醛、0. 25g载体、交联0. 5h、固定化1h、加酶量为800U,最终得到的固定化酶酶活达到83. 01U/g。固定化脂肪酶的最适pH较游离酶向碱性方向偏移,最适反应温度提高10℃,固定化酶的热稳定性和酸碱稳定性比游离酶好且重复使用性和储存稳定性明显优于游离酶。同时发现交联剂是制备固定化脂肪酶的重要因素,因此探索新型交联剂对于固定化效果的提高具有重要意义,为海洋假丝酵母脂肪酶的固定化工艺技术和工业应用奠定了良好基础。 相似文献
8.
“构象记忆”的辣根过氧化物酶的微水相共价固定化 总被引:1,自引:0,他引:1
本研究利用酶在微水溶剂中的"构象记忆"特性,以壳聚糖微球为载体,以辣根过氧化物酶(Horseradish peroxidase,HRP)为研究对象,将HRP于活性构象下冻干"固定"后,在二氧六环:水=99:1(V/V)微水介质中与载体进行共价交联,同时与传统水介质中共价交联固定化的HRP进行比较。结果发现,两种介质中固定化HRP的最适温度都提高到60°C,最适pH均为6.5,而微水相中固定的酶活力损失较低,酶活比传统水相中固定的酶高6倍以上;70°C保温30min后,微水相中固定的酶保留75.42%的活力,而水相中固定的HRP仅存15.4%的活力;微水相中固定的HRP具有更好的操作稳定性和热稳定性,60°C下连续操作5次之后,微水相固定的HRP保留77.69%的酶活,而水相固定的HRP仅存16.67%的酶活;微水相中固定的HRP在苯酚的去除中表现得更具优势;微水相中共价交联制备的CS-HRP-SWCNTs/Au酶修饰电极对H2O2的响应信号比水相中共价固定的酶电极强2.5倍,灵敏度更高。本研究表明利用酶的"构象记忆"在微水介质中进行共价交联是固定化酶的一种可行方法,所制备的固定化酶具有更优良的性质。 相似文献
9.
葡萄糖氧化酶的有机相共价固定化 总被引:1,自引:0,他引:1
将葡萄糖氧化酶(GOD)在最适pH条件下冻干后,以戊二醛活化的壳聚糖为载体,分别在传统水相和1,4-二氧六环、乙醚、乙醇三种不同的有机相中进行共价固定化。通过比较水相固定化酶和有机相固定化酶的酶比活力、酶学性质及酶动力学参数,考察酶在有机相中的刚性特质对酶在共价固定化过程中保持酶活力的影响。结果表明,戊二醛浓度为0.1%、加酶量为80 mg/1 g载体、含水1.6%的1,4-二氧六环有机相固定化GOD与水相共价固定化GOD相比,酶比活力提高2.9倍,有效酶活回收率提高3倍;在连续使用7次后,1,4-二氧六环有机相固定化GOD的酶活力仍为相应水相固定化酶的3倍。在酶动力学参数方面,不论是表观米氏常数,最大反应速度还是转换数,1,4-二氧六环有机相固定化的GOD(Kmapp=5.63 mmol/L,Vmax=1.70μmol/(min.mgGOD),Kcat=0.304 s-1)都优于水相共价固定化GOD(Kmapp=7.33 mmol/L,Vmax=1.02μmol/(min.mg GOD),Kcat=0.221 s-1)。因此,相比于传统水相,GOD在合适的有机相中进行共价固定化可以获得具有更高酶活力和更优催化性质的固定化酶。该发现可能为酶蛋白在共价固定化时因构象改变而丢失生物活性的问题提供解决途径。 相似文献
10.
聚乙二醇二缩水甘油醚(PEGDGE)作为双功能环氧试剂,在实验中被用于交联氨基载体LX-1000EA共价固定化海洋脂肪酶,经过处理后的载体共价固定化脂肪酶具有良好的效果。实验经过单因素初筛和正交试验,得到最佳的交联及固定化条件为0.75%交联剂浓度、交联温度35℃、交联时间3h、载体量1.25g、pH9.0、固定化温度55℃、固定化时间1h。对LX-1000EA-PEGDGE固定化酶与游离酶、戊二醛(GA)交联LX-1000HA-GA的固定化酶进行酶学性质的比较,发现LX-1000EA- PEGDGE固定化酶较游离酶最适反应温度未改变,与LX-1000HA-GA相同的是最适反应pH都由7.0提高为8.0。在最适条件中所测LX-1000EA-PEGDGE酶活达到78.84U/g,固定化改变了游离酶的酸碱耐受性,热稳定性和操作稳定性较游离酶和LX-1000HA-GA固定化酶均有提高。LX-1000EA-PEGDGE的热稳定表现优异,在60℃孵育3h后保留90%酶活;使用5次后仍能残余50%酶活;保存30天酶活仍保留60%。首次使用新型双环氧交联剂PEGDGE交联有机氨基载体共价结合固定化脂肪酶,为更有效的固定化方法提供了技术支持,同时也发现交联剂对固定化酶的性质存在较大影响。 相似文献
11.
G N Rudenskaia V A Isaev T S Kalebina V M Stepanov K V Mal'tsev S V Shvets N A Luk'ianova Iu A Kislitsin A I Miroshnikov 《Bioorganicheskaia khimiia》1998,24(2):112-118
Trypsin PC from the hepatopancreas of the king crab Paralithodes camtschatica was isolated and purified to apparent homogeneity by ion-exchange chromatography on Aminosilochrom and DEAE-Sephadex and affinity chromatography on arginine-agarose. The yield of the enzyme was 37.7%, and the purification degree was 21. Trypsin PC has a molecular mass of 29 kDa and pI < 2.5. It hydrolysis N-benzoyl-L-arginine p-nitroanilide at the optimum pH of 7.5-8.0 and at the temperature optimum of 55 degrees C (K(m) = 0.05 mM). Trypsin PC retained its activity within the pH range of 5.8-9.0 in the presence of Ca2+. The enzyme was inhibited by the specific inhibitors of serine proteases diisopropyl fluorophoshate and phenylmethylsulfonyl fluoride, by the trypsin inhibitor N-tosyl-L-lysylchloromethylketone, and by the trypsin inhibitors from soybean and potato. Trypsin PC was found to hydrolyze amide bonds formed by carboxylic groups of lysine and arginine in peptide substrates. The N-terminal sequence of this enzyme is IVGGTEVTPG. 相似文献
12.
Trypsin was immobilized on sand using five different methods. Attempts were made to attach amino-functional groups onto sand using 3-aminopropyltriethoxysilane, hexamethylenetetramine, hexamethylenediamine, and melamine. Glutaraldehyde was used as a bifunctional agent in all the methods. Methods for the estimation of the proteolytic 1activity and protein content of immobilized trypsin were standardized. The maximum retained activity was observed for trypsin immobilized on sand via 3-aminopropytriethoxysilane and glutaraldehyde. Immobilized trypsin showed a shift in the pH optimum toward the acidic side over that of soluble trypsin in all five cases. The optimum temperature for both native and immobilized trypsin prepared by the silane-glutaraldehyde method was found to be 45°C. However, the pH and thermal stabilities of immobilized trypsin were observed to be better than that of the native enzyme. 相似文献
13.
Summary Trypsin, pronase and subtilisin were immobilized on chitosan by glutaraldehyde coupling. Significant retention of activity was observed when synthetic substrates as well as casein were used. The specific activities of the bound proteinases ranged from 38% to 79% of their initial specific activities. The pH-activity profile of trypsin was slightly shifted toward alkaline values, and its thermal stability was increased. Immobilized trypsin was found to be less sensitive to its natural inhibitors than the soluble enzyme.A preliminary report of this work has already been presented at the First European Congress on Biotechnology (September 25–30, 1978, Interlaken, Switzerland). 相似文献
14.
Silica gel bead coated with macroporous chitosan layer (CTS-SiO2) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu2+, Zn2+, Ni2+ ions, respectively on CTS-SiO2, and trypsin could be adsorbed on the IMAC adsorbent through metal–protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu2+-CTS-SiO2 with the value of 4980 ± 125 IU g−1 of the adsorbent, while the maximal adsorption capacity for trypsin on Zn2+ and Ni2+ loaded adsorbent was 3762 ± 68 IU g−1 and 2636 ± 53 IU g−1, respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5–10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO2 beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 °C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO2 could effectively digest BSA revealed by HPLC peptide mapping. 相似文献
15.
D Bharadwaj A Mukherjee M S Roy R N Hati R N Mukherjee 《Indian journal of biochemistry & biophysics》1992,29(4):375-377
Trypsin immobilized by covalent coupling to silanized silica shows significant activity (30-38%) and greater thermostability as compared to soluble trypsin. Proteolytic processing of albumin at varying periods suggest that the enzyme matrix can be used efficiently for limited proteolysis. Repeated use of the immobilized enzyme in protein digestion produces similar products as seen by electrophoretic analysis. Also, digestion of albumin by the immobilized enzyme follows similar pattern as that by soluble enzyme. The enzyme matrix can be easily removed from the incubation mixture. The results indicate the possibility of the immobilized enzyme for its effective application as analytical tool in peptide mapping and limited proteolytic processing. 相似文献
16.
Trypsin was covalently immobilized on porous glass in the presence and absence of a specific substrate and reacted in various organic solvents of different dielectric constants. Optimum solvent concentration, pH profile, Km(app), Vmax(app), productivity versus temperature, activity, and reaction rates were determined. Reaction rates of six lysyl dipeptides were compared. Crystalline trypsin was dansylated for studies by nanosecond fluorescence techniques to determine the effects of introducing high concentrations of organic solvents on the molecule. The results indicated that greater reaction rates were observed with dipeptides having more acidic carboxyl terminal groups. The data also indicated that greater reaction rates were observed in higher concentrations of solvents of lower dielectric constants. Nanosecond fluorescence spectroscopy of trypsin in high concentrations of a low dielectric constant solvent indicated major dehydration even though maximal enzyme activity was achieved under these conditions. 相似文献
17.
Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG2) showed the highest immobilization capacity (8.2 mg SBP g-1 PANIG2). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the Km was conserved while the specific Vmax dropped from 14.6 to 11.4 µmol min-1 µg-1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP. 相似文献
18.
Soybean callus succinyl CoA synthetase (succinate: CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose-succinyl CoA synthetase can be stored at 4 degrees C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme. 相似文献
19.
Marques D Pessela BC Betancor L Monti R Carrascosa AV Rocha-Martin J Guisán JM Fernandez-Lorente G 《Biotechnology progress》2011,27(3):677-683
The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pK(a) amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pK(a) amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins. 相似文献
20.
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