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31.
Enzyme immobilization on magnetic nanoparticles (MNPs) has been a field of intense studies in biotechnology during the past decade. The present study suggests MNPs negatively charged by docusate sodium salt (AOT) as a support for pectinase immobilization. AOT is a biocompatible anionic surfactant which can stabilize MNPs. Electrostatic adsorption can occur between enzyme with positive charge and oppositely charged surface of MNPs (ca. 100 nm). The effect of three factors, i.e. initial enzyme concentration, aqueous pH and AOT concentration in different levels was investigated on pectinase immobilization. Maximum specific activity (1.98 U/mg enzyme) of immobilized pectinase and maximum enzyme loading of 610.5 mg enzyme/g support was attained through the experiments. Initial enzyme concentration is significantly important on both loading and activity of immobilized enzyme, while pH and AOT concentration only affect the amount of immobilized enzyme. Immobilized enzyme on MNPs was recovered easily through magnetic separation. At near pH of immobilization, protein leakage in reusability of immobilized enzyme was low and activity loss was only 10–20% after six cycles. Since pH is associated with immobilization by electrostatic adsorption, the medium pH was changed to improve the release of protein from the support, as well. MNPs properties were investigated using Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FT-IR) spectroscopy, and Dynamic Light Scattering (DLS) analysis. 相似文献
32.
采用试剂y-氯丙基三乙氧基硅烷(cvrEs)对介孔硅材料SBA-15进行表面改性,并通过红外图谱(FT-IR)和N2吸附脱附等温图(BET)对其进行表征。结果表明:改性前原材料的比表面积为460.9m2/g,改性后材料比表面积提高到512.0m2/g。利用改性前和改性后的SBA-15对猪胰脂肪酶进行固载实验,并对实验结果进行比较,发现改性后的SBA-15在脂肪酶活性、pH环境适应性、热耐受性和可操作性都优于改性前的SBA-15,在最优条件下的酶活力提高超过60%。 相似文献
33.
Kaoru Nakamura Takehiko Miyai Kiyoko Inoue Seiji Kawasaki Shinzaburo Oka Atsuyoshi Ohno 《Biocatalysis and Biotransformation》1990,3(1):17-24
Esters of 3-methyl-2-oxobutanoic acid are reduced with bakers' yeast by three methods: free bakers' yeast in water, immobilized bakers' yeast in water, and immobilized bakers' yeast in hexane. Although (R)-hydroxy esters are obtained in all cases, the enantiomeric excess varies from 3% (reduction of the methyl ester with free bakers' yeast in water) to 93% (reduction of the butyl ester with immobilized bakers' yeast in hexane) depending on the structure of substrate and on the reaction conditions. The mechanism of the present stereochemical control is discussed. 相似文献
34.
Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl4 solution, displayed very stable behaviour, with half-lives of ~60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent Km increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion. 相似文献
35.
Tau Benned-Jensen Christoffer Norn Stephane Laurent Christian M. Madsen Hjalte M. Larsen Kristine N. Arfelt Romain M. Wolf Thomas Frimurer Andreas W. Sailer Mette M. Rosenkilde 《The Journal of biological chemistry》2012,287(42):35470-35483
Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors. 相似文献
36.
采用多种固定化方法及载体,进行了微生物絮凝剂产生菌M09固定化方法及条件的研究,并对其所产絮凝剂在不同存储条件下絮凝活性的稳定性进行探讨。结果表明,选用粒径为0.5 cm×0.5 cm×0.5 cm的多孔聚氨酯泡沫为固定化载体,4 g/L的固液比,使用初始蔗糖浓度为2%,NaNO3为0.4%的培养基,28℃振荡培养60 h可获得较高活性的固定化细胞,发酵上清液能够保持较高的絮凝率。研究还发现,在室温条件下,利用此工艺所产高絮凝活性发酵液在含有菌体的反应器内自然静置,发酵液絮凝率仍可缓慢持续上升,维持较高水平达数日,具有较高的稳定性。 相似文献
37.
38.
Methyl mercury uptake in free cells and different immobilizates of the cyanobacteriumNostoc calcicola has been examined. The general growth of the immobilized cyanobacterial cells could be negatively correlated with methyl mercury uptake. Alginate spheres proved most efficient in terms of uptake rate (0.48 nmol mg protein–1 min–1, 10 min) and total bioaccumulation (10.71 nmol mg protein–1, 1 h) with a bioconcentration factor of 3.3×103. Alginate biofilms showed a faster methyl mercury accumulation rate (0.83 nmol mg protein–1 min–1, 10 min) with a saturation of 10.28 nmol mg protein–1 reached within only 30 min (bioconcentration factor, 3.1×103). Foam preparations with a slow initial uptake approximated biofilms but were characterized by a lower bioconcentration factor (2.8×103). Free cells, in comparison, maintained the initial slow rate of uptake (0.62 nmol mg protein–1 min–1, 10 min), saturating at 30 min (8.81 nmol mg protein–1), and the resultant lowest bioconcentration factor (2.7×103). Cell ageing (30 days) brought a drastic reduction (3-fold) in organomercury uptake by free cells while alginate spheres maintained the same potential. Foam preparations of the same age showed a significant improvement in methyl mercury uptake followed by only a marginal decline in alginate biofilms. Data are discussed in the light of the physiological efficiency and longevity of immobilized cells. 相似文献
39.
青霉菌胞外半纤维素酶的固定化研究 总被引:2,自引:0,他引:2
青霉菌m8胸外半纤维素酶经过离子交换和分子筛层析两步纯化,纯化的酶固定在戊二醛交联的壳聚精上,残活力为45.6%。固定化酶的最适pH为3.6,最适温度为65℃,且固文化酶在65~75℃活力都较高。该酶的耐热性比较强,固定化酶热稳定性优于原酶;以半纤维素为底物,固定化酶的表观米氏常数为3.58×10-2g/L。 相似文献
40.
Summary Xylanase from Scytalidium thermophilum was immobilized on Eudragit L-100, a pH sensitive copolymer of methacrylic acid and methyl methacrylate. The enzyme was non-covalently
immobilized and the system expressed 70% xylanase activity. The immobilized preparation had broader optimum temperature of
activity between 55 and 65 °C as compared to 65 °C in case of free enzyme and broader optimum pH between 6.0 and 7.0 as compared
to 6.5 in case of free enzyme. Immobilization increased the t1/2 of enzyme at 60 °C from 15 to 30 min with a stabilization factor of 2. The Km and Vmax values for the immobilized and free xylanase were 0.5% xylan and 0.89 μmol/ml/min and 0.35% xylan and 1.01 μmol/ml/min respectively. An Arrhenius plot showed an increased value of activation energy for immobilized xylanase (227 kcal/mol)
as compared to free xylanase (210 kcal/mol) confirming the higher temperature stability of the free enzyme. Enzymatic saccharification
of xylan was also improved by xylanase immobilization. 相似文献