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61.
Nils Siegbahn Mats-Olle Maånsson Klaus Mosbach 《Applied biochemistry and biotechnology》1987,12(2):91-105
A soluble, bifunctional enzyme complex has been prepared by crosslinking lactate dehydrogenase and alcohol dehydrogenase with
glutaraldehyde. The crosslinking was performed on a solid phase while the active sites of alcohol dehydrogenase and lactate
dehydrogenase were held adjacent to one another with the aid of a bis-NAD analog. Subsequently, the enzyme complex was released
from the solid phase. The soluble enzyme complex was then purified by using NAD-Sepharose as an affinity adsorbent. Based
on gel filtration experiments, the complex was estimated to consist of one of each dehydrogenase.
By using a third enzyme, lipoamide dehydrogenase, which competes with lactate dehydrogenase for NADH produced by alcohol dehydrogenase,
the effect of site-to-site orientation was studied. It was found that about 83% of the NADH produced by alcohol dehydrogenase
was oxidized by site-to-site oriented lactate dehydrogenase compared to a figure of only about 61% obtained in an identical
system of separate enzymes. This indicates that given two alternative routes, the preference for the one to lactate dehydrogenase
over the one to lipoamide dehydrogenase is enhanced when lactate dehydrogenase and alcohol dehydrogenase are site-to-site
oriented. 相似文献
62.
固定化过氧化物酶在过氧化物测定中的应用 总被引:4,自引:0,他引:4
采用聚丙烯酰胺凝胶迭氮法固定了辣根过氧化物酶,其作为催化剂用于荧光法测定过氧化物,并探讨了固定酶测定过氧化物的最佳条件,如溶液pH值、温度、反应时间、荧光剂用量等。结果表明:酶固定化后,反应pH值范围变宽,为pH5.0-7.0和7.5-9.0,最佳pH为7.8左右;酶的热稳定性与储存稳定性也都得到提高,在室温下便可用固定酶进行长时间测定,且可较长时间保存。采用固定酶制成的酶柱用于HPLC测定过氧化物,固定酶可反复使用,简化了测定操作,并降低了成本。 相似文献
63.
镧对Rhizoctonia solani的毒力及其致病酶活性的影响 总被引:2,自引:1,他引:2
采用琼脂平板生长速率法及液体培养基培养测定了La对立枯丝核病菌(Rhizoctoniasolani)的抑制作用和毒力,并测定了其对病菌胞外的果胶酶、蛋白酶和纤维素酶等几种致病酶的活性的影响。结果表明,随着La浓度升高,对病菌菌丝生长的抑制作用增强,固体培养上所测定的对病菌的EC50和EC95分别为171.9和667.7mg·L-1;在液体培养基中所测定的EC50和EC95分别为111 4和500 7mg·L-1。在一定浓度范围内,La提高了单位量菌丝所产生3种致病酶的活性,但由于对菌丝生长量的强烈抑制,使病菌胞外3种致病酶的总量或总活性受到抑制,减低了病菌的致病力。 相似文献
64.
固定化酶不对称合成(S)+—(+)—布洛芬的研究 总被引:3,自引:0,他引:3
能够不对称水解布洛芬乙酯的酵母菌T158固定化在壳聚糖珠中,固定化酵母菌的活力回收达60%,最适温度为45℃,半衰期为75d以上。 相似文献
65.
In this study, a series of beadlike and hydrophilic supports containing reactive cyclic carbonate groups for enzyme immobilization
were prepared via reverse-phase suspension copolymerization of the aqueous solutions of vinylene carbonate (VCA), acrylamide
(AA), and N,N′-methylene bisacrylamide in paraffin oil. The supports were used as a matrix for immobilization of trypsin and showed a considerable
capacity to couple with trypsin and reasonable retention of activity for the immobilized trypsin, depending on the immobilization
conditions, such as the content of VCA structural units, reaction time, and pH of the medium. 相似文献
66.
Various computational approaches, using molecular mechanics (Amber), semiempirical (AM1), density functional (B3LYP), and various ONIOM methods, have been comparatively investigated for the structure of Escherichia coli NifS CsdB protein. The structure of the entire monomer containing 407 amino acid residues and 579 surrounding water molecules has been optimized. The full geometry optimization in the "active site-only" approach (including only active site atoms) has been found to give the largest root-mean-square (RMS) deviation from the X-ray structure; a much better agreement has been achieved by keeping the atoms leading to the backbones of some amino acids frozen in their positions in the X-ray structure. The best agreement has been attained by including the surrounding protein in the calculations using the two-layer ONIOM (B3LYP:Amber) approach. The results presented in this study conclusively demonstrate the importance of the protein/active-site interaction on the active-site structure of the enzyme. The present theoretical study represents the largest system studied at the ONIOM level to date, containing 7992 atoms, including 84 atoms in the QM region and rest in the MM region. 相似文献
67.
A simple and automated methodology for a sensitive electrochemical detection of enzyme immunoassays that employ alkaline phosphatase (AP) as label has been developed. A flow injection system with programmable pump, valve and cell functions, amperometric detection of indigo and screen-printed electrodes (SPEs) are responsible for the advantages of this methodology. Amperometric detection at a low potential of indigo, the product of the enzymatic hydrolysis of the substrate 3-indoxyl phosphate (IP), is combined with a flow injection system. This incorporates in the flow cell a disposable screen-printed board provided with a graphite working electrode. No electrode pretreatment is necessary to obtain reproducible signals. The system was applied to the determination by an enzyme-linked immunosorbent assays (ELISA) of pneumolysin (PLY), a toxin related to respiratory infections. Linear calibration curves for low and high concentration ranges were obtained. These were also performed in a proteic matrix and linearity was also obtained. 相似文献
68.
Neutron diffraction studies of hydrogen positions in small molecules of biological interest at Trombay have provided valuable
information that has been used in protein and enzyme structure model-building and in developing hydrogen bond potential functions.
The new R-5 reactor is expected to provide higher neutron fluxes and also make possible smallangle neutron scattering studies
of large biomolecules and bio-aggregates. In the last few years infrastructure facilities have also been established for macromolecular
x-ray crystallography research. Meanwhile, the refinement of carbonic hydrases’ and lysozyme structures have been carried
out and interesting results obtained on protein dynamics and structure-function relationships. Some interesting presynaptic
toxin phospholipases have also been taken up for study. 相似文献
69.
V. Slavinska Dz. Sile G. I. Chipens Yu. Balodis G. Rozenthal K. Venteris E. Lukevics 《Chemistry of Heterocyclic Compounds》2003,39(12):1579-1583
A method was developed for the synthesis of N-[1-(S)-(ethoxycarbonyl)-3-phenylpropyl]alanylproline (enalapril) by reductive alkylation of alanylproline with ethyl 2-oxo-4-phenylbutenoate under the conditions of hydrogenation in the presence of palladium black and 1.6% Pd/C. The yield of enalapril amounted to 65%. With the ethyl ester of the -oxo acid the diastereoselectivity of formation of the S,S,S-diastereomer was higher than with the saturated synthon. It is assumed that with ethyl 2-oxo-4-phenylbutenoate as synthon a conformationally restricted surface complex is formed between the unsaturated synthon and the active centers of the catalyst. During reductive alkylation of alanylproline by ethyl 2-oxo-4-(2-thienyl)butenoate poisoning of the catalyst occurs. 相似文献
70.
Atkinson CE Aliev AE Motherwell WB 《Chemistry (Weinheim an der Bergstrasse, Germany)》2003,9(8):1714-1723
A binding site optimisation protocol for the design of artificial enzymes based on "small molecule-small molecule" binding studies by diffusion NMR is presented. Since the reaction chosen was the hydrolysis of ester 1 ([4-(4-carboxy-1-oxobutyl)-aminobenzyl]-phenethyl ester), an analogous phosphonate ester 2 ([4-(4-carboxy-1-oxobutyl)-aminobenzyl]-phosphonic phenethyl ester) was selected as a suitable transition state analogue (TSA). The key objective of the NMR studies was to find a unit with functional groups capable of binding to the acidic sites of the TSA. Nine dipeptides, mainly with basic and hydroxyl groups, were used and their affinity to the TSA was studied by measuring the change in the diffusion coefficient, D(pep), upon binding by pulse field gradient NMR. The value of D(pep) at 298 K in D(2)O at pD 5, 7 and 10 was measured both in free solution, and mixtures containing one dipeptide and the TSA. As both components are low molecular weight species with M < 500, a TSA-to-dipeptide ratio of 10:1 was used to detect significant changes in D(pep). The results revealed that dipeptides with basic residues show higher affinity to the TSA than those with hydroxyl or aliphatic side chains in aqueous solutions. The dipeptide showing the most significant relative change in D(pep) was H-Arg-Arg-OH, and the binding constant was estimated to be 86 L M(-1) by measuring D(pep) at varying concentrations of the TSA. In addition, binding of the TSA to a new water-soluble polymer with a polyallylamine backbone and randomly distributed Arg-Arg binding sites was examined, and the binding constant was estimated to be > or =1500 L M(-1). As confirmed by further catalytic activity tests, polymers containing Arg-Arg as a binding site are capable of significant rate accelerations in the hydrolysis of ester 1. 相似文献