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1.
以硅藻土为载体,采用吸附法,对脂肪酶进行固定化,研究了固定化条件对固定化脂肪酶的催化活性的影响,得到最佳的固定化条件:给酶量为33374U/g,固定化温度为35℃,pH值为7.5,时间为4h,此时固定化酶的活力约为5833U/g载体。固定化酶的热稳定性较游离酶有了很大的提高,其在80℃以下能保持80%以上的酶活,而游离酶60℃残余酶活仅为5%。最适反应温度和最适pH值也分别由游离酶的40℃上升至50℃和由7上升到7.5。对固定化中的中性脂肪酶在生物柴油合成中的应用也进行了初步研究。  相似文献   

2.
β-D-Galactosidase (BGAL) from Kluyveromyces lactis was covalently immobilized to functionalized silicon dioxide nanoparticles (10-20 nm). The binding of the enzyme to the nanoparticles was confirmed by Fourier transform-infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Functionalized nanoparticles showed 87% immobilization yield. Soluble and immobilized enzyme preparation exhibited pH-optima at pH 6.5 and 7.0, respectively, with temperature optima at 35 and 40°C, respectively. Michaelis constant (K(m)) was 4.77 and 8.4mM for free and immobilized BGAL, respectively. V(max) for the soluble and immobilized enzyme was 12.25 and 13.51 U/ml, respectively. Nanoparticle immobilized BGAL demonstrated improved stability after favoring multipoint covalent attachment. Thermal stability of the immobilized enzyme was enhanced at 40, 50 and 65°C. Immobilized nanoparticle-enzyme conjugate retained more than 50% enzyme activity up to the eleventh cycle. Maximum lactose hydrolysis by immobilized BGAL was achieved at 8h.  相似文献   

3.
The yeast cells of Pichia farinosa Y-118 were immobilized in polyacrylamide gel and used for 17 beta-oxidoreduction of secondione to secol. The loss of hydroxysteroid oxidoreductase activity of cells was found to be insignificant during immobilization. The preparation exhibited greater temperature stability as compared to free cells. The ratio of reaction volume to the volume of immobilized biocatalyst in the range 1.4-1.9 was found to be satisfactory for the reaction conditions studied. This ratio played a significant role in the stability of the catalyst particle, since beyond a critical value the disintegration of gel granules was rapid resulting in sharp decline of activity. The immobilized cell preparation could be used 50 times over a period of 100 days without loss of activity. However, the activity declined in further reuses, leaving the preparation 50 and 35% active after its 60th and 70th uses, respectively.  相似文献   

4.
Glucose oxidase (GOD) was covalently immobilized onto Fe3O4/SiO2 magnetic nanoparticles (FSMNs) using glutaraldehyde (GA). Optimal immobilization was at pH 6 with 3-aminopropyltriethoxysilane at 2% (v/v), GA at 3% (v/v) and 0.143 g GOD per g carrier. The activity of immobilized GOD was 4,570 U/g at pH 7 and 50°C. The immobilized GOD retained 80% of its initial activity after 6 h at 45°C while free enzyme retained only 20% activity. The immobilized GOD maintained 60% of its initial activity after 6 cycles of repeated use and retained 75% of its initial activity after 1 month at 4°C whereas free enzymes retained 62% of its activity.  相似文献   

5.
A new low-cost glucoamylase preparation for liquefaction and saccharification of starchy raw materials in a one-stage system was developed and characterized. A non-purified biocatalyst with a glucoamylase activity of 3.11 U/mg, an alpha-amylase activity of 0.12 WU/mg and a protein content of 0.04 mg protein/mg was obtained from a shaken-flask culture of the strain Aspergillus niger C-IV-4. Factors influencing the enzymatic hydrolysis of starchy materials such as reaction time, temperature and enzyme and substrate concentration were standardized to maximize the yield of glucose syrup. Thus, a 90% conversion of 5% starch, a 67.5% conversion of 5% potato flour and a 55% conversion of 5% wheat flour to sweet syrups containing up to 87% glucose was reached in 3 h using 1.24 glucoamylase U/mg hydrolyzed substrate. The application of such glucoamylase preparation and a commercially immobilized glucose isomerase for the production of glucose-fructose syrup in a two-stage system resulted in high production of stable glucose/fructose blends with a fructose content of 50%. A high concentration of fructose in obtained sweet syrups was achieved when isomerization was performed both in a batch and repeated batch process.  相似文献   

6.
An experimental design was carried out to evaluate the effect of the concentrations of sodium alginate, glutaraldehyde and activated coal on the immobilization of inulinase from Kluyveromyces marxianus NRRL Y-7571. The experimental condition of 20?g/L of sodium alginate, 50?mL/L of glutaraldehyde and 30?g/L of activated coal led to the highest specific activity (2,063.5?U/mg of protein), corresponding to an enhancement of about 26 times compared to the activity of the free enzyme (79.1?U/mg of protein). The effect of pH and temperature on the immobilized enzyme activity was also evaluated, showing optimal activities at pH of 5.5 and 55?°C. The study of storage of immobilized inulinase in different temperatures showed that the extract kept its initial activity after 43?days of storage at 40 and 50?°C and after 138?days of storage either at 4 or 25?°C.  相似文献   

7.
The present work deals with maltodextrin hydrolysis by glucoamylase immobilized onto corn stover in a fluidized bed reactor. An industrial enzyme preparation was covalently grafted onto corn stover, yielding an activity of up to 372 U/g and 1700 U/g for support particle sizes of 0.8 and 0.2 mm, respectively. A detailed kinetic study, using a differential reactor, allowed the characterization of the influence of mass transfer resistance on the reaction catalyzed by immobilized glucoamylase. A simple and general mathematical model was then developed to describe the experimental conversion data and found to be valid.  相似文献   

8.
Penicillin acylase (PA) is known to regulate the microenvironment of nanospores. In this study, nanopores containing chemically-modified macromolecules co-assembled with immobilized PA were constructed. We also investigated the various types of functionalized mesocellular siliceous foams (MCFs) commonly used for the immobilization of PA by measuring the catalytic performance and stability of each PA preparation. Amino-MCF activated by p-benzoquinone was chosen as the optimum support for PA immobilization. Successful modification of macromolecules was verified by FT-IR and ultraviolet (UV) spectroscopy. The specific activity of PA co-assembled with dextran 10 k was 99.1 U/mg, which was 1.5-fold that of pristine immobilized PA, while the optimum pH was shifted to neutral. Compared to pristine immobilized and free PA, the optimum temperatures for the modified PA were 5 and 10°C higher, respectively. The residual activity of the ficoll derivative of PA after treatment at 50°C for 6 h was 70%, and this was later increased to 214.5% compared to that of pristine immobilized PA. The dextran 10 k derivative of PA exhibited 90.2% residual activity after 25 times of continuous use. The results show that chemically-modified macromolecules co-assembled with PA in amino-MCF provided a suitable microenvironment for enzyme stability.  相似文献   

9.
鹰嘴豆孢克鲁维酵母(Kluveromyces cicerisporus Y-179)分泌的糖基化菊粉外切酶经高碘酸钠氧化其分子表面的糖链产生醛基,再共价结合于氨基型固定化载体ZH-HA上,固定化酶活力达到4 000 U/g湿载体。所制备的固定化酶在pH 3.5和70℃温度下表现出最大反应活性,该固定化酶pH稳定性和热稳定性较游离酶明显提高。固定化酶在分批式反应器中重复水解菊粉50批次,活力没有明显损失,表现出良好的工作稳定性。  相似文献   

10.
alpha-Chymotrypsin was immobilized with a high coupling yield (up to 80%) to tresyl chloride activated Sepharose CL-4B.The immobilized enzyme was tested for its ability to synthesize soluble peptides from N-acetylated amino acid esters as acyl donors and amino acid amides as acceptor amines in water-water-miscible organic solvent mixtures. It was found that the yield of peptide increased with increasing concentration of organic cosolvent. Almost complete synthesis (97%) of Ac-Phe-Ala-NH(2) was obtained from Ac-Phe-OMe using a sixfold excess of Ala-NH(2). The rate of peptide formation in aqueous-organic solvent mixtures was good. Thus, 0.1M peptide was formed in less than 2 h in 50 vol% DMF with 0.1 mg immobilized chymotrypsin/mL reaction mixture. The immobilized enzyme distinguished between the L and D configurations of acceptor amino acid amides even in high concentration of nonaqueous component (90% 1,4-butanediol). The effect of temperature was studied. It was found that both the yield of peptide and the stability of immobilized enzyme increased when the temperature was lowered. Experiments could be performed at subzero temperatures in the aqueous-organic solvent mixtures resulting in very high yield of peptide. After three weeks continuous operation at 4 degrees C in 50% DMF, the immobilized enzyme retained 66%of its original synthetic activity. The activity of the immobilized enzyme was better conserved with a preparation made from agarose with a higher tresyl group content compared to a preparation made from a lower activated agarose, indicating that multiple point of attachment has a favorable effect on the stability of the enzyme in aqueous-organic solvent mixtures. The major advantage of using water-miscible instead of water-immiscible organic solvents to promote peptide syntheses appears to be the increased solubility of substrates and products, making continuous operation possible.  相似文献   

11.
Abstract

We have developed an improved and effective method to immobilize lipase on hydrophobic polyurethane foam (PUF) with different modifications. PUF was treated with hydrochloric acid to increase the active sites and then the active carboxyl groups and amino groups were exposed. Enzyme activity of lipase immobilized on PUF-HCL (8000?U/g) was 50% higher than that of lipase immobilized on PUF (5300?U/g). There is an increase in the activity of the immobilized lipase on AA/PEI-modified support (115,000?U/g), a 2.17-fold increase compared to lipase immobilized on the native support was observed. The activity of immobilized lipases was dependent on the PEI molecular weight, with best results from enzyme immobilized on PUF-HCL-AA/PEI (MW 70,000?Da, 12,800?U/g)), which was 2.41 times higher compared to that of the same enzyme immobilized on PUF. These results suggest that the activity of immobilized lipase is influenced by the support surface properties, and a moderate support surface micro-environment is crucial for improving enzyme activity. Finally, the immobilized lipase was used for the production of vitamin A palmitate. The immobilized lipase can be reused for up to 18 times with a conversion rate above 90% for 12?h in a 3?L bioreactor.
  • Research highlights
  • An efficient immobilization protocol on polyurethane foam was developed

  • Polyethyleneimine and acetic acid were used to regulate the micro-environment concurrently

  • The activity of lipase immobilized on PUF-HCL-AA/PEI was improved by 2.41 times

  • Immobilized lipase exhibited excellent operational stability for vitamin A palmitate synthesis

  相似文献   

12.
Tomato pomace and pectin were used as the sole carbon sources for the production of polygalacturonase from a strain of Coriolus versicolor in submerged culture. The culture of C. versicolor grown on tomato pomace exhibited a peak of polygalacturonase activity (1,427 U/l) on the third day of culture with a specific activity of 14.5 U/mg protein. The production of polygalacturonase by C. versicolor grown on pectin as a sole carbon source increased with the time of cultivation, reaching a maximum activity of 3,207 U/l of fermentation broth with a specific activity of 248 U/mg protein. The levels of different isoenzymes of polygalacturonase produced during the culture growth were analysed by native PAGE. Differential chromatographic behaviour of lignocellulosic enzymes produced by C. versicolor (i.e. polygalacturonase, xylanase and laccase) was studied on immobilized metal chelates. The effect of ligand concentration, pH, the length of spacer arm and the nature of metal ion were studied for enzyme adsorption on immobilized metal affinity chromatography (IMAC). The adsorption of these lignocellulosic enzymes onto immobilized metal chelates was pH-dependent since an increase in protein adsorption was observed as the pH was increased from 6.0 to 8.0. The adsorption of polygalacturonase as well as other enzymes to immobilized metal chelates was due to coordination of histidine residues which are available at the protein surface since the presence of imidazole in the equilibration buffer abolished the adsorption of the enzyme to immobilized metal chelates. A one-step purification of polygalacturonase from C. versicolor was devised by using a column of Sepharose 6B-EPI 30-IDA-Cu(II) and purified enzyme exhibited a specific activity of about 150 U/mg protein, final recovery of enzyme activity of 100% and a purification factor of about 10. The use of short spacer arm and the presence of imidazole in equilibration buffer exhibited a higher selectivity for purification of polygalacturonase on this column with a high purification factor. The purified enzyme preparation was analysed by SDS-PAGE as well as by "in situ" detection of enzyme activity.  相似文献   

13.
A cold-active beta-galactosidase of Antarctic marine bacterium Pseudoalteromonas sp. 22b was synthesized by an Escherichia coli transformant harboring its gene and immobilized on glutaraldehyde-treated chitosan beads. Unlike the soluble enzyme the immobilized preparation was not inhibited by glucose, its apparent optimum temperature for activity was 10 degrees C higher (50 vs. 40 degrees C, respectively), optimum pH range was wider (pH 6-9 and 6-8, respectively) and stability at 50 degrees C was increased whilst its pH-stability remained unchanged. Soluble and immobilized preparations of Antarctic beta-galactosidase were active and stable in a broad range of NaCl concentrations (up to 3 M) and affected neither by calcium ions nor by galactose. The activity of immobilized beta-galactosidase was maintained for at least 40 days of continuous lactose hydrolysis at 15 degrees C and its shelf life at 4 degrees C exceeded 12 months. Lactose content in milk was reduced by more than 90% over a temperature range of 4-30 degrees C in continuous and batch systems employing the immobilized enzyme.  相似文献   

14.
Bromelain is a basic, 23.8 kDa thiol proteinase obtained from the stem of the pineapple plant (Ananas comosus) and is unique for it contains a single histidine residue (His-158) in the polypeptide. Based on the technology of protein separation with immobilized metal ion affinity chromatography (IMAC), a method for oriented immobilization of bromelain was selected. Bromelain was successfully immobilized on iminodiacetic acid carrier Sepharose 6B. Cu2+ complexed with iminodiacetate (IDA) was used as the chelating ligand to bind the lone histidine on bromelain. Simultaneously, preparation of a high affinity immobilized preparation was attempted using a soluble cross-linked preparation of bromelain on Cu-IDA-Sepharose. However this second method proved unsuccessful, possibly due to poor histidine accessibility in the cross-linked preparation. The immobilized preparation obtained using uncrosslinked bromelain was more resistant to thermal inactivation, as evidenced by retention of over enzyme 50% activity after incubation at 60 °C, as compared to 20% retained by the native enzyme. The immobilized preparation also exhibited a broader pH-activity profile in acidic range. The native, immobilized and soluble cross-linked bromelain showed apparent Michaelis constant (Km) values of 1.08, 0.42, 1.56 mg/ml, respectively, using casein as the substrate. While the maximum velocity (Vmax) values of the soluble and immobilized preparations were comparable, cross-linked preparation showed a 20% decrease, suggesting inactivation. The mild conditions used for predominantly oriented immobilization exploiting the unique property of single histidine, the high recovery of immobilized preparations, the stability, reusability and the regenerability of the matrix are the main features of the method reported here.  相似文献   

15.
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 (t1/2 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 Km and Vmax 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.  相似文献   

16.
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.  相似文献   

17.
Summary -Glucosidase from sweet almond was immobilized onto a nylon support and used to hydrolyze picrocrocin, the glycoside precursor of the saffron essential volatile oil, safranal. The nylon support was derivatized as hydrazide and the enzyme attached through Schiff base to bonds. The coupling efficiency was 46.8%, the immobilization yield 29.5%, and the derivative showed 24.2 and 4.0 U/mg activity for p-nitrophenylglucoside and picrocrocin, respectively, as substrates. Kinetic parameters of the immobilized derivative were determined, with picrocrocin as substrate, showing KM=7.2 mM and Vmax=4,0 U/mg. Glucose behaved as a competitive inhibitor (Ki=95.0 mM). The immobilized derivative was thermally stable up to 45°C; from that temperature onwards thermoinactivation occured. The operational deactivation showed a biphasic pattern, t1/2 being 4.2 days for the first four days of continuous operation, and 20.1 days from that point on. The immobilized enzyme lost a 50% of its initial activity after 30.7 days of storage at 4°C.  相似文献   

18.
Porcine pepsin was immobilized by chemical aggregation using glutaraldehyde as a bifunctional crosslinking agent. The immobilzed pepsin followed Michaelis-Menten kinetics (K(m) = 5.3 x 10(-5) M) and the yield of immobilization was 91%. The activation energy of the immobilized preparation was 90,613 cal/mol as compared to 67,532 cal/mol for native pepsin. Using acid-denatured hemoglobin and N-acetyl phenyl-alanyl-3, 5-diiodotyrosine (APDT) as substrates, the activities shown by the immobilized pepsin were, respectively, 67 and 79% that of the soluble pepsin. The immobiized pepsin showed marked stabilization against pH, temperature, urea, and guanidine hydrochloride. The activity of the immobilized preparation in the presence of urea was greater when hemoglobin was used as the substrate than when APDT was used as substrate. Storage of the preparation under refrigerated conditions for 160 days showed 58% retention in enzyme activity. The immobilized pepsin can be removed from the reaction mixture volume easily, retaining nearly 100% of its activity even after being used in seven consecutive assays.  相似文献   

19.
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   

20.
In our previous work, a method of pretreating lipase was developed to prevent loss of its activity during covalent immobilization. In this study, Rhizopus oryzae lipase was pretreated before immobilization and then immobilized on a silica gel surface. The effects of the various materials and conditions used in the pretreatment stage on the activity of immobilized lipase were investigated. Immobilized lipase pretreated with 0.1% of soybean oil had better activity than those pretreated with other materials. The optimal temperature, agitation speed, and pretreating time for lipase pretreatment were determined to be 40 degrees C, 200 rpm, and 45 min, respectively. The activity of immobilized soybean oil pretreated lipase was 630 U/g matrix, which is 20 times higher than that of immobilized non-pretreated lipase. In addition, immobilized lipase activity was maintained at levels exceeding 90% of its original activity after 10 reuses.  相似文献   

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