共查询到20条相似文献,搜索用时 15 毫秒
1.
Cheong KW Leow TC Rahman RN Basri M Rahman MB Salleh AB;Enzyme Microbial Technology Research Group 《Applied biochemistry and biotechnology》2011,164(3):362-375
A thermostable lipase from Geobacillus zalihae strain T1 was chemically modified using propionaldehyde via reductive alkylation. The targeted alkylation sites were lysines,
in which T1 lipase possessed 11 residues. Far-UV circular dichroism (CD) spectra of both native and alkylated enzyme showed
a similar broad minimum between 208 and 222 nm, thus suggesting a substantial amount of secondary structures in modified enzyme,
as compared with the corresponding native enzyme. The hydrolytic activity of the modified enzymes dropped drastically by nearly
15-fold upon chemical modification, despite both the native and modified form showed distinctive α-helical bands at 208 and
222 nm in CD spectra, leading us to the hypothesis of formation of a molten globule (MG)-like structure. As cooperative unfolding
transitions were observed, the modified lipase was distinguished from the native state, in which the former possessed a denaturation
temperature (T
m) in lower temperature range at 61 °C while the latter at 68 °C. This was further supported by 8-anilino-1-naphthalenesulfonic
acid (ANS) probed fluorescence which indicated higher exposure of hydrophobic residues, consequential of chemical modification.
Based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, a small number
of lysine residues were confirmed to be alkylated. 相似文献
2.
Novel cinchonine ammonium salt derivatives have been prepared by 1,3‐dipolar cycloaddition. Their chiral catalytic efficacy was investigated in the asymmetric alkylation of N‐diphenylmethyleneglycine t‐butyl ester in the water phase. As the special structure of the catalyst, its asymmetric alkylation catalytic behavior both in organic solvents and in water is satisfactory, which is environmentally friendly. 相似文献
3.
Polypropylene powders as the adsorbent for organic solution containing n-hexadecane and olive oil were employed as the carbon source for producing an alkaline lipase from Acinetobacter radioresistens. The best volumetric ratio of n-hexadecane to olive oil around 5 for lipase production was determined from shake-flask and fermentation cultivations. The
existence of a maximum time course lipase activity of the aqueous phase was attributed to the compensation effects of olive
oil on cell growth and lipase production, repression of lipase synthesis by oleic acid, and lipase adsorption on the supports.
A linear relationship between the average cell growth rate in the exponential phase and the ratio of surface areas of the
supports was found. The benefits of using the present fermentation process include less foaming and emulsion of the broth,
less organic phase used, higher lipase production, and easy recovery of the lipase in the centrifugation step. 相似文献
4.
The application of small angle neutron scattering (SANS) to the characterisation of sol–gel hosts containing biomolecules offers the opportunity to explore the relationship between gel structure and catalyst. A model system involving the immobilisation of Candida antarctica lipase B (CALB) was investigated.Gels were produced by fluoride-catalysed hydrolysis of fixed ratios of tetramethylorthosilicate (TMOS) and methyltrimethoxysilane (MTMS). Phase separation between the enzyme and the evolving sol–gel matrix was minimised by incorporating glycerol into the sol–gel precursor solution. The potential stabilising effect of the NaF catalyst upon the enzyme was also investigated. Scattering studies were conducted on both immobilised lipase, and lipase in free solution. Scattering studies on free enzyme provided evidence of multiple populations of enzyme aggregates and showed that choice of solvent affected the degree of aggregation. Both NaF and glycerol affected neutron scattering, indicating changes in lipase conformation. Increasing glycerol concentration increased the degree of aggregation and produced differences in solvent packing on the surface of protein molecules. Initial evidence from SANS data indicated that the presence of the enzyme during gel formation conferred structural changes on the gel matrix. Modelling the effect of sol–gel encapsulation on lipase requires comparison of data from free enzyme to the immobilised form. Removal of the enzyme from the sol–gel structure, post gelation, is necessary to better characterise the modified matrix. This methodological problem will be the subject of future investigations. 相似文献
5.
Moniruzzaman M Talukder MR Hayashi Y Kawanishi T 《Applied biochemistry and biotechnology》2007,142(3):253-262
The activity and conformation of Chromobacterium viscosum lipase-pretreated with various organic solvents were investigated. The pretreatment of lipase led to a substantial increase
of enzyme activity in AOT (sodium bis [2-ethyl -1-hexyl] sulfosuccinate)/isooctane/water reverse micelles. Among the organic
solvents used, n-hexane was found to be most effective. It was observed that higher hexane content with shorter agitation time and vice versa
had almost the same effect on the initial activity of lipase. The kinetic study showed that the Michaelis constant (K
m) and the substrate adsorption equilibrium constant (K
ad) were reduced by the pretreatment of lipase with hexane, whereas the change in the maximum reaction rate (V
max) was insignificant. The two spectroscopic techniques (Fluorescence spectra of lipase encapsulated in RMs and Fourier transform
infrared [FTIR] spectra of lipase powders) were performed to detect possible conformational changes in the enzyme caused by
the pretreatment. A correlation between the maximum fluorescence intensity and the activity of treated lipase was found as
a function of agitation time. The FTIR spectrum of lipase showed a new shape peak corresponding to 1,500 cm−1 as a result of pretreatment with organic solvents. 相似文献
6.
Covalent modification of mushroom tyrosinase with different amphiphic polymers for pharmaceutical and biocatalysis applications 总被引:1,自引:0,他引:1
Margherita Morpurgo Oddone Schiavon Paolo Caliceti Francesco M. Veronese 《Applied biochemistry and biotechnology》1996,56(1):59-72
Two different poly(ethylene glycol) derivatives (linear, mol wt 5000 and a branched form, mol wt 10000) and a new polymer
(poly [acryloylmorfoline], mol wt 5500) were covalently bound to the enzyme tyrosinase. The polymer-protein conjugates were
studied with a view to their potential pharmaceutical application and to their use for the bioconversion of phenolic substrates
in organic solvents.V
max
andK
m
for the dopa-dopaquinone conversion, thermostability, stability toward inactivation by dopa oxidation products, half-life
in blood circulation, and behavior in organic solvents for the different adducts were investi gated. Arrhenius plots for the
dopa-dopaquinone conversion were also obtained in order to study the effects of temperature on the different enzyme forms.
Covalent attachment of the polymers increased enzyme stability in aqueous solution and the solubility in organic solvents.
However, organic solvent solubilization brought about loss of enzyme conformation as assessed by CD measurements, which is
accompanied by a nonreversible loss of catalytic activity. 相似文献
7.
Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of
(R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted
from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized
lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C
and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid. 相似文献
8.
《Analytical letters》2012,45(6):1417-1428
Abstract A thermal assay probe (enzyme thermistor) was used to study the performance of enzymic analyses by immobilized enzymes in organic solvents and in mixtures of organic solvents and aqueous buffer. It was found that rather small amounts of alcohols (around 5 %, v/v) in the buffer approximately doubled the registered temperature changes for glucose oxidase and catalase, mainly by increasing the total enthalpy change of the reaction. the heat production for peroxidase catalyzed reactions was as much as 45 times higher in toluene compared to buffer. the advantage of increased substrate solubility in organic solvents was demonstrated by operating a lipase column in cyclohexane. All enzymes studied showed good stability in the organic solvents used. 相似文献
9.
Su-Xia Li Qiang Ma Kang Lin Jiao-Jiao Wu Yi-Xin Wu Jian-He Xu 《Applied biochemistry and biotechnology》2014,172(6):2945-2954
A novel lipase lipB from Serratia marcescens ECU1010 is highly stable in the presence of organic solvents. By sequence and structure comparison with homologous lipase lipA, three amino acid residues were found to be different between them. To identify the residues which increase the organic solvent stability of lipB, residues that potentially provide this stability were mutated to the ones of lipA at equivalent positions. The replacement of Gly at position 33 by Asp obviously decreased its stability in organic solvents. Molecular modeling and structural analysis also suggested that the Gly33 residue is important for the organic solvent stability of lipB. 相似文献
10.
利用原子转移自由基聚合以及随后的磺化反应合成了一系列具有不同含氟量和磺化度的嵌段共聚物P(SSt b FNEMA) (PSSF) .通过溶液粘度和表面张力的测定 ,研究了该含氟嵌段共聚物独特的溶液行为 .实验结果表明 ,PSSF能显著降低N 甲基吡咯烷酮溶液的表面张力 .然而 ,与N 甲基吡咯烷酮溶液的表面性能不同 ,PSSF不能显著地降低水溶液的表面张力 ;当PSSF中和成钠盐后 ,水溶液的表面张力趋于一致 .进一步通过TEM、1 H NMR考察了PSSF在不同溶液中的形态 ,发现PSSF在水溶液中以核壳结构存在 ,而在N 甲基吡咯烷酮溶液中主要以胶束的形式存在 .初步考察了PSSF对聚氨酯脲 丙烯酸酯 (PUA)复合水分散液的改性效果 ,发现只需加入少量的PSSF ,就可使PUA膜的吸水率从原来的 10wt%下降到 4wt% . 相似文献
11.
Debamitra Chakravorty Saravanan Parameswaran Vikash Kumar Dubey Sanjukta Patra 《Applied biochemistry and biotechnology》2012,167(3):439-461
Organic solvent-stable lipases have pronounced impact on industrial economy as they are involved in synthesis by esterification, interesterification, and transesterification. However, very few of such natural lipases have been isolated till date. A study of the recent past provided few pillars to rely on for this work. The three-dimensional structure, inclusive of the surface and active site, of 29 organic solvent-stable lipases was analyzed by subfamily classification and protein solvent molecular docking based on fast Fourier transform correlation approach. The observations revealed that organic solvent stability of lipases is their intrinsic property and unique with respect to each lipase. In this paper, factors like surface distribution of charged, hydrophobic, and neutral residues, interaction of solvents with catalytically immutable residues, and residues interacting with essential water molecules required for lipase activity, synergistically and by mutualism contribute to render a stable lipase organic solvent. The propensity of surface charge in relation to stability in organic solvents by establishing repulsive forces to exclude solvent molecules from interacting with the surface and prohibiting the same from gaining entry to the protein core, thus stabilizing the active conformation, is a new finding. It was also interesting to note that lipases having equivalent surface-exposed positive and negative residues were stable in a wide range of organic solvents, irrespective of their LogP values. 相似文献
12.
Molecular imprinting of enzymes with water-insoluble ligands for nonaqueous biocatalysis 总被引:1,自引:0,他引:1
Rich JO Mozhaev VV Dordick JS Clark DS Khmelnitsky YL 《Journal of the American Chemical Society》2002,124(19):5254-5255
Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques. 相似文献
13.
An in-water dehydrative alkylation with a novel heterobimetallic polymeric catalyst is described. Thus, a boron-iridium heterobimetallic polymeric catalyst was prepared by ionic convolution of a poly(catechol borate) and an iridium complex. The alkylation of ammonia and amines with alcohols, alkylating agents, was performed with 1 mol % Ir of the heterogeneous catalyst in water without the use of organic solvents under aerobic conditions to give the corresponding alkylated amines. 相似文献
14.
I. V. Eremeev 《Russian Journal of General Chemistry》2016,86(6):1229-1239
Mechanism of metals alkylation with an organic halide in the presence of binary systems has been defined in more detail. It has been shown that the passivating film on the surface of zinc and cadmium is partially preserved in the course of the process, and the reaction in diethyl ether is decelerated due to the competitive adsorption of the organyl halide and diethyl ether on the surface of the reacting metal. The ratelimiting stages of the studied alkylation process have been elucidated basing on the experimental data on the effect of the reagents (organyl halide and alkylated metal) nature on the rate of the steady-state reaction and modeling of the suggested catalytic cycle. 相似文献
15.
Polizelli PP Facchini FD Cabral H Bonilla-Rodriguez GO 《Applied biochemistry and biotechnology》2008,150(3):233-242
A new lipase from seeds of Pachira aquatica was purified to homogeneity by SDS-PAGE obtaining an enzyme with a molecular weight of approximately 55 kDa. The purified lipase exhibited maximum activity at 40 degrees C and pH 8.0, for an incubation time of 90 min. Concerning temperature stability, at the range from 4 to 50 degrees C, it retained approximately 47% of its original activity for 3 h. The enzyme activity increased in the presence of Ca(++) and Mg(++), but was inhibited by Hg(++), Mn(++), Zn(++), Al(+++) and various oxidizing and reducing agents. The lipase was highly stable in the presence of organic solvents, and its activity was stimulated by methanol. The values of K(m) and V(max) were 1.65 mM and 37.3 micromol mL(-1) min(-1), respectively, using p-nitrophenylacetate as substrate. The enzyme showed preference for esters of long-chain fatty acids, but demonstrated significant activity against a wide range of substrates. 相似文献
16.
Purification and partial characterization of a lipase from Bacillus coagulans ZJU318 总被引:1,自引:0,他引:1
An extracellular lipase was purified from the fermentation broth of Bacillus coagulans ZJU318 by CM-Sepharose chromatography, followed by Sephacryl S-200 chromatography. The lipase was purified 14.7-fold with
18% recovery and a specific activity of 141.1 U/mg. The molecular weight of the homogeneous enzyme was (32 kDa), determined
by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme activity was maximum at pH 9.0 and was stable
over a pH range of 7.0–10.0, and the optimum temperature for the enzyme reaction was 45°C. Little activity loss (6.2%) was
observed after 1 h of incubation at 40°C. However, the stability of the lipase decreased sharply at 50 and 60°C. The enzyme
activity was strongly inhibited by Ag+ and Cu2+, whereas EDTA caused no inhibition. SDS, Brij 30, and Tween-80 inhibited lipase, whereas Triton X-100 did not significantly
inhibit lipase activity. 相似文献
17.
Naoyuki Masuda Osamu Yamamoto Masahiro Fujii Tetsuro Ohgami Ayako Moritomo Toru Kontani 《合成通讯》2013,43(17):2305-2316
Various N‐3‐alkylated thiazolidenesulfonamide derivatives were efficiently prepared by the direct endo‐selective alkylation of thiazolylsulfonamides. The effects of different bases and solvents were investigated, and the NaH–THF combination was found to be the most effective at conferring high yields and endo‐selectivity. 相似文献
18.
Jinyong Yan Lifan Li Qianli Tang Manzhou Jiang Shenzhou Jiang 《Applied biochemistry and biotechnology》2010,162(3):757-765
Geotrichum sp. lipase modified with a combined method composed of crosslinking and bioimprinting was employed to selectively hydrolyze
waste fish oil for enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in glycerides. Crosslinked polymerization
by monomer (polyethylene glycol 400 dimethyl acrylate), crosslinker (trimethylolpropane trimethylacrylate), and photoinitiator
(benzoin methyl ether) coupled to bioimprinting using palmitic acid as imprint molecule, resulted in much more effective enzyme
preparation used in aqueous hydrolysis reaction. Since the crosslinked polymerization modification maintained bioimprinted
property and gave good dispersion of enzyme in reaction mixture, the crosslinked bioimprinted enzyme exhibited higher hydrolysis
temperature, enhanced specific activity, shorter hydrolysis time, and better operational stability compared to free lipase.
Crude fish oil was treated at 45 °C with this crosslinked bioimprinted lipase for 8 h, and 46% hydrolysis degree resulted
in the production of glycerides containing 41% of EPA and DHA (EPA+DHA), achieving 85.7% recovery of initial EPA and DHA.
The results suggested that bioimprinted enzymes did not lose their induced property in aqueous environment when prepared according
to the described crosslinking–bioimprinting method. It could also be seen that the crosslinked bioimprinted lipase was effective
in producing glycerides that contained a higher concentration of polyunsaturated fatty acid with better yield. 相似文献
19.
Jia Wang Ryoji Nomura Takeshi Endo 《Journal of polymer science. Part A, Polymer chemistry》1995,33(17):2901-2908
The selective reductive homo-coupling polymerization of aromatic diisocyanates via one-electron transfer promoted by samarium iodide in the presence of hexamethylphosphoramide (HMPA) produced the corresponding poly(oxamide)s ( 1 ) nearly quantitatively. The ob-tained polymers were insoluble in common organic solvents. The alkylation of 1 with methyl iodide or allyl bromide in the presence of potassium tert-butoxide provided the highly soluble alkylated polymer in good yields. In either case, the alkylation was almost complete, and both N-and O-alkylation proceeded. The ratio of N-and O-methylation was found to be 64 : 36 by 1H-NMR spectrum, and that of N- and O-allylation was 3 : 1 from 13C-NMR analysis. The present polymerization system could be applied to a variety of diisocyanates, including diphenylmethanediisocyanate (MDI), tolylene 2,6-diisocyanate (TDI), 2,6-naphthyl diisocyanate (NDI) and o-tolidine diisocyanate (TODI). The molecular weights of the polymers were estimated by GPC and found to be 2000–9000. The TGA measurement of the corresponding polymers showed Td10 at 248–320°C. © 1995 John Wiley & Sons, Inc. 相似文献
20.
Improved preparation and use of room-temperature ionic liquids in lipase-catalyzed enantio- and regioselective acylations. 总被引:17,自引:0,他引:17
Polar organic solvents such as methanol or N-methylformamide inactivate lipases. Although ionic liquids such as 3-alkyl-1-methylimidazolium tetrafluoroborates have polarities similar to these polar organic solvents, they do not inactivate lipases. To get reliable lipase-catalyzed reactions in ionic liquids, we modified their preparation by adding a wash with aqueous sodium carbonate. Lipase-catalyzed reactions that previously did not occur in untreated ionic liquids now occur at rates comparable to those in nonpolar organic solvents such as toluene. Acetylation of 1-phenylethanol catalyzed by lipase from Pseudomonas cepacia (PCL) was as fast and as enantioselective in ionic liquids as in toluene. Ionic liquids permit reactions in a more polar solvent than previously possible. Acetylation of glucose catalyzed by lipase B from Candida antarctica (CAL-B) was more regioselective in ionic liquids because glucose is up to one hundred times more soluble in ionic liquids. Acetylation of insoluble glucose in organic solvents yielded the more soluble 6-O-acetyl glucose, which underwent further acetylation to give 3,6-O-diacetyl glucose (2-3:1 mixture). However, acetylation of glucose in ionic liquids yielded only 6-O-acetyl glucose (>13:1 and up to >50:1). 相似文献