Amidination Reaction Research Articles

Laccase–Polyacrylonitrile Nanofibrous Membrane: Highly Immobilized, Stable, Reusable, and Efficacious for 2,4,6-Trichlorophenol Removal

Increasing attention has been given to nanobiocatalysis for commercial applications. In this study, laccase was immobilized on polyacrylonitrile (PAN) nanofibrous membranes through ethanol/HCl method of amidination reaction and successfully applied for removal of 2,4,6-trichlorophenol (TCP) from water. PAN membranes with fiber diameters from 200 nm to 300 nm were fabricated via electrospinning and provided a large surface area for enzyme immobilization and catalytic reactions. Images of scanning electron microscope demonstrated the enzyme molecules were aggregated on the nanofiber surface. The immobilized laccase exhibited 72% of the free enzyme activity and kept 60% of its initial activity after 10 operation cycles. Moreover, the storage stability of the immobilized laccase was considered excellent because they maintained more than 92% of the initial activity after 18 days of storage, whereas the free laccase retained only 20%. The laccase-PAN nanofibrous membranes exhibited high removal efficiency of TCP under the combined actions of biodegradation and adsorption. More than 85% of the TCP was removed under optimum conditions. Effects of various factors on TCP removal efficiency of the immobilized laccase were analyzed. Results suggest that laccase-PAN nanofibrous membranes can be used in removing TCP from aqueous sources and have potential for use in other commercial applications.

Electrospun polyacrylonitrile nanofibrous membranes for chitosanase immobilization and its application in selective production of chitooligosaccharides

Polyacrylonitrile nanofibrous membranes (PANNFM) were prepared by electrospinning from 10wt.% of PAN solution and its surface was modified by amidination reaction. A new chitosan degrading enzyme from Aspergillus sp. was covalently immobilized on PANNFM. Immobilization efficiency of 80% was achieved by activating PANNFM surface for 30min followed by 2h treatment with enzyme solution. The optimum temperature and pH for immobilized enzyme were 50°C and 5.8, respectively. The immobilized chitosanase retained >70% activity after ten repeated batch reaction and could be stored up to 60days at 4°C with minor loss in activity. Chitosan hydrolysis using different substrates were studied using immobilized chitosanase in batch conditions. Continuous selective production of chitooligosaccharides (dimer to hexamer) by changing the temperature was achieved by PANNFM-chitosanase.

Immobilization of Pseudomonas cepacia lipase onto the electrospun PAN nanofibrous membranes for transesterification reaction

Abstract Pseudomonas cepacia lipase was immobilized onto the electrospun polyacrylonitrile (PAN) nanofibrous membrane with covalent binding by activating the nitrile groups of PAN using the amidination reaction successfully. The fiber diameters of initial PAN nanofibers were between 150 and 300 nm. The lipase molecules (1–2 μm) were observed to be attached onto the nanofiber surfaces. After 5 min activation and 30 min reaction with lipase solution, the protein loading has reached saturation point and the immobilized lipase retained 79% activity compared to that of free lipase. The kinetic parameters, K m and V max , were determined to be 56.7 mM and 22.5 U/mg for free lipase and 88.4 mM and 18.3 U/mg for immobilized lipase. To compare with free lipase, the pH stability of immobilized lipase was improved significantly in the acidic conditions and the thermal stability of immobilized lipase still maintained 90% of initial specific activity when the temperature achieved 70 °C. After being stored at 30 °C for 20 days, the immobilized lipase kept almost 100% of its initial specific activity. For reusability test, the immobilized lipase still retained 98% of its original specific activity after 10 batch cycles. These results show that the proposed scheme for lipase immobilization has potential in industrial transesterification reaction such as biodiesel production.

Pseudomonas cepacia lipase immobilized onto the electrospun PAN nanofibrous membranes for biodiesel production from soybean oil

Abstract Lipase from Pseudomonas cepacia has good transesterification activity and can be employed for biodiesel production. For recycled uses, polyacrylonitrile nanofibrous membrane was fabricated by electrospinning method and activated by amidination reaction for immobilizing P. cepacia lipase. Fourier transform infrared spectroscopy (FTIR) spectra confirmed that the lipases were covalently attached to the nanofibers. After enzyme immobilization, the immobilized P. cepacia lipase was used as the catalyst for transesterification reaction to convert the soybean oil to biodiesel by adding methanol as the reactant. The effects of methanol concentration, methanol-to-water ratio, reaction temperature, fatty acid content and the reusability were investigated by using the immobilized P. cepacia lipase. Under the optimal reaction conditions, the biodiesel conversion of soybean oil achieved 90% after reacting 24 h. In examining the reusability, the immobilized P. cepacia lipase still retained 91% of its initial conversion capability after 10 times reused.

Hydrolysis of microalgae cell walls for production of reducing sugar and lipid extraction

In this study, cell walls of microalgae were hydrolyzed for the production of reducing sugar by cellulase, which was immobilized onto an electrospun polyacrylonitrile (PAN) nanofibrous membrane. Since the nitrile groups of the PAN membrane were activated by the amidination reaction and covalent binding to the amino groups of the cellulase, electrospun PAN nanofibrous membranes with a high specific surface area were applied as supports for the immobilization. Under the optimal hydrolysis conditions, the immobilized cellulase performed its hydrolyzing conversion at 62%, and the hydrolysis yield remained at 40% after five times of reuse. Additionally, microalgal lipid extraction efficiency increased to around 56% from 32% dramatically after cell wall hydrolysis. These results demonstrate the efficacy and feasibility of the proposed applications in hydrolysis process followed with lipid extraction.

Lipase-immobilized electrospun PAN nanofibrous membranes for soybean oil hydrolysis

Polyacrylonitrile (PAN) nanofibrous membranes were prepared by electrospinning and activated by amidination reaction for immobilizing Candida rugosa lipase with covalent binding. The diameters of initial PAN nanofibers ranged from 150 to 300 nm. The lipase, which aggregated and formed small particles that attached onto the nanofiber surfaces, was observed using field emission scanning electron microscopy. The activity retention of the immobilized lipase was 87.5% of the free enzyme. The kinetic parameters, K m and V max, of the immobilized lipase were determined to be 0.548 mM and 31.2 U/mg, respectively. The effects of oil-to-water ratio, temperature, and pH on hydrolysis of soybean oil were investigated to determine the optimal reaction conditions. Under these optimal reaction conditions, the hydrolysis conversion of soybean oil was 72% after 10 min and 85% after 1.5 h. In examining reusability, the immobilized lipase retained 65% of its initial conversion after 20 additional batch reactions. These results suggest that the proposed scheme for immobilization of lipase has potential in industrial applications for oil hydrolysis.

Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization

Polyacrylonitrile (PAN) nanofibers could be fabricated by electrospinning with fiber diameter in the range of 150–300 nm, providing huge surface area for enzyme immobilization and catalytic reactions. Lipase from Candida rugosa was covalently immobilized onto PAN nanofibers by amidination reaction. Aggregates of enzyme molecules were found on nanofiber surface from field emission scanning electron microscopy and covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After 5 min activation and 60 min reaction with enzyme-containing solution, the protein loading efficiency was quantitative and the activity retention of the immobilized lipase was 81% that of free enzyme. The mechanical strength of the NFM improved after lipase immobilization where tensile stress at break and Young's modulus were almost doubled. The immobilized lipase retained >95% of its initial activity when stored in buffer at 30 °C for 20 days, whereas free lipase lost 80% of its initial activity. The immobilized lipase still retained 70% of its specific activity after 10 repeated batches of reaction. This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters.

Immobilization of glucoamylase onto alternative acrylonitrile‐butadiene copolymer by amidination reaction

Recently, copolymers have been often used as favorable supports for immobilizing enzymes, which are more stable against physical abrasion or chemical corrosion and exhibited more functional importance than homopolymers. Hence, it is expected that the enzymic properties of the immobilized amylases are improved by using a copolymer-support. This communication is concerned with the preparation and properties of immobilized GA covalently attached to alternative acrylonitrile-butadiene (AB) copolymer

Action pattern of .ALPHA.-amylase immobilized on granular polyacrylonitrile by amidination reaction.

An α-amylase from Bacillus subtilis (liquefying type) was immobilized on partially imido esterized granular polyacrylonitrile (PAN) by an amidination reaction . The action patterns in the hydrolysis of soluble starch were compared between the native and the immobilized α-amylase . The hydrolysis character of immobilized α-amylase was not basically changed except for retardation in the production of oligosaccharides due to diff usional limitation and the deficiency of maltohexaose production potency. The amount of glucose production by a combination of the enzymatic reactions by the immobilized α-amylase with the native glucoamylase was also studied at 40°C. Approximately the same glucose amount was attained between the native and the immobilized α-amylase in the successive hydrolysis by glucoamylase.

Alkaline isomerization of ferricytochrome c: Lysine is not replacing methionine at the sixth co-ordination site of the haem iron

Oxidized cytochrome c is known to undergo a restricted conformational refolding of its haem area at around pH 9. Methionine 80, the sixth ligand of the ferric haem iron in the biologically active neutral conformational state, is replaced by a new strong-field ligand in the biologically inactive alkaline state of the molecule. It had been proposed that a lysine residue, possibly lysine 79. is the new haem ligand. We have tested this proposition by a more direct approach than hitherto employed, namely by measuring the relative chemical reactivity of lysines in the oxidized eytochrome c and in fragment 66–80 cut out of the native molecule. The relative rates of acetylation of lysine 79, measured between pH 7 and pH 11, are virtually identical in the intact molecule and in the haem-free fragment 66–80. Similarly, the rates are also the same for the amidination reaction with isethionylacetimidate. When the relative rates of acetylation and amidination of lysines 72 + 73 were compared there was again no significant difference between the intact molecule and fragment 66–80. These results contradict the involvement of any of the three lysines in the alkaline isomerization, as a haem-bound ϵ-amino group would be much less reactive than its freely accessible counterpart in fragment 66–80. To corroborate the above finding, the p K value and absolute rate constant of acetylation of lysine 79 were determined and compared with the respective values for lysines 39 and 60. The latter two residues are on the side opposite to the haem pocket and hence unable to bind to the haem iron. The three p K values and rate constants k obey the Brønsted relationship: log κ = α + βp K with β = 0.48, a value characteristic of the acetylation of freely accessible primary amino groups. Taken together, these results oppose an ϵ-amino: haem iron co-ordination in the alkaline state of oxidized eytochrome c.

Dynamic organization of mitochondrial membranes: A crosslinking study with dimethylsuberimidate

Rat liver mitochondria were treated with dimethylsuberimidate, a bifunctional alkylating agent, and the effects were evaluated kinetically. Concurrently with the modification of amino groups, mitochondrial proteins were crosslinked and the organelles lost their osmotic response. When the dimethylsuberimidate reaction was performed in the presence of succinate, more primary amino groups were available when compared with a sucrose medium. Concomitantly, osmotic stabilization and crosslinking of mitochondrial proteins were accelerated. The activity of aspartate aminotransferase was also studied in crosslinked mitochondria. The enzyme activity was only slightly modified when mitochondria were amidinated in a sucrose medium and solubilized thereafter with Triton X-100 or cetyltrimethylammonium bromide. In contrast, in the presence of succinate, 60% of activity was lost after solubilization with Triton X-100, but not after solubilization with cetyltrimethylammonium bromide. This finding was correlated with the changes in intramitochondrial localization of the enzyme (A. Waksman and A. Rendon, 1974, Biochimie 54, 907–924). When carbonylcyanide- p-trifluoromethoxyphenylhydrazone was added in both cases (sucrose or sucrose plus succinate), the rates of osmotic stabilization, amidination reaction, crosslinking of proteins, and aspartate aminotransferase activity were similar to those observed in a sucrose medium alone. The present results suggest that organizational changes of the mitochondrial membranes induced by succinate, including intramitochondrial protein movement, are prevented by carbonylcyanide- p-trifluoromethoxyphenylhydrazone.

Synthesis, plasma clearance, and in vitro stability of protein containing a conjugated indium-111 chelate

A new bifunctional chelating agent, N′-(p-diazoniumbenzyl)-N,N N″,N″-diethylenetri-aminetetraacetic acid (DTTA) was synthesized. The compound as coupled to methyl p-hydroxybenzimidate and the resulting azoimidate was attached to lysine residues of monomeric human serum albumin (HSA) via the amidination reaction. Blood clearnace of111In-labelled DTTA conjugated to HSA (AAHSA) in rabbits was biphasic. The first phase has a clearance indistinguishable from that of125I-labeled HSA. During the second phase, the111In-labeled AAHSA was cleared more rapidly so that between 24 hours and 48 hours the percent of the injected dose of111In-labeled AAHSA in the blood was significantly lower than that of125I-labeled HSA. Highly conjugated111In-labeled AAHSA (0.9 DTTA/HSA) showed accelerated clearance at 24 and 48 hours compared to lightly conjugated protein (<0.9 DTTA/HSA). As a result we postulate that the level of conjugation is the critical parameter controlling the blood clearance.

Augmentation of auto-anti-idiotypic antibody production by hapten-reactive helper T lymphocytes

Augmented auto-anti-idiotypic antibody production was effectively achieved by immunization of mice with haptenated myeloma protein in the presence of hapten-reactive helper T lymphocytes. Hapten-reactive helper T-lymphocyte activities were raised in BALB/c mice by immunization with para-azobenzoate (PAB)-derived mouse gamma globulin (MGG) prepared by amidination reaction (PAB im-MGG). Helper T cell activity was effectively enhanced by pretreatment of mice with a PAB-derived nonimmunogenic copolymer of D-glutamic acid and D-lysine ( D-GL) (PAB- D-GL) 3 days before priming with PAB im-MGG; PAB- D-GL is a potent tolerogen of both PAB-specific suppressor T lymphocytes and PAB-specific B cells. After induction of these enhanced PAB-reactive helper T lymphocytes, mice were immunized with PAB-coupled TEPC-15 myeloma protein (PAB im-T-15), which was also prepared by amidination reaction. Mice immunized in this way manifested strikingly enhanced titers of auto-anti-idiotypic antibodies, specific for the T-15 idiotype, as compared to control mice which had not been preimmunized with PAB im-MGG. The ability of PAB im-MGG preimmunization to facilitate auto-anti-idiotypic antibody production was due to the activity of PAB-reactive helper T cells since PAB-specific B cells had been abolished by prior treatment with PAB- D-GL. The implications of this model for future studies on immunological engineering the analysis of idiotype network phenomena are discussed.

PROTAMINES

The synthesis of peptides containing blocks of arginyl residues is proposed through amidination of the corresponding ornithyl analogs. In order to test this strategy the ornithyl analog of the C-terminal sequence 52--65 of galline was synthesized by the conventional method. The amidination reaction, performed on fragments of different length and ornithyl-residue content, quantitatively converts ornithines into arginines. The strategy proposed may represent a powerful tool for the synthesis of protamines and other basic proteins.

IMMOBILIZATION OF ENZYME TO GRAFT-POLYMER ON GLASS SURFACE

Abstract The immobilized glucoamylase (from Rhizopus niveus) was prepared by amidination reaction to partially imidoesterized polyacrylonitrile which had been grafted onto flaky glass surface preirradiated by electron-accelerator.

粒状固定化酵素カラムによる可溶性でんぷんの加水分解

Guucoamylase was immobilized by amidination reaction on the granular polyacrilonitrile resin which had been preliminarily treated with dry HCl in methanol. The granular polyacrilonitrile resin was made by drop-wise addition of the dimethylf ormamide solution of polyacrilonitrile to methanol. Continuous hydrolysis of soluble starch was operated in a column which was packed with the immobilized glucoamylase (granularity : 1 mmφ). Estimated values of apparent MICHAELIS constant (Km) with the immobilized glucoamylase depended on flow rates in the column and were found to be higher than that with the native glucoamylase in the batch system. The immobilized glucoamylase column was connected in a series with a column of the immobilized a-amylase (granularity : 30-200 μmφ). And, a soluble starch solution was firstly passed through the immobilized α-amylase column and then through the immobilized glucoamylase column. The effect of the pretreatment of soluble starch in the immobilized α-amylase column on the rate of hydrolysis was recognized with regard to the balance between amount of immobilized α-amylase and the degree of the hydrolysis in terms of reducing sugar unit. A similar effect was observed in the pretreatment of soluble starch with native α-amylase in a batch system. The molecular weight distribution of the reaction product of α-amylase processing was analyzed by the gel filtration (Sephadex G-75)method for the higher molecular weight fraction and by the thin layer chromatographic method for the lower molecular weight fraction up to DP 12. On the basis of these results, the pretreatment with α-amylase was thought to be significant particularly in that the effective concentration of the substrate was increased through the fission of the glucoside chains. The endurance test of the immobilized glucoamylase column was pursued by the continuous operation for two weeks with a flow rate of 0.5 ml/min at room temperature and 40°C. Similar situations were referred to in the previous study was obtained.

Inter- and intramolecular interactions of alpha-lactalbumin. V. The effect of amidination on association and aggregation.

Abstract 1. 1. α-Lactalbumin exists as the monomeric unit at pH values alkaline to the isoelectric point (4.25-4.50). However, below pH 4.00 the protein undergoes a confirmational change which results in strong association and aggregation. To determine if hydrophobic interactions play a role in the association and aggregation, new nonpolar groups have been introduced into the protein by the amidination reaction using ethyl acetimidate hydrochloride and ethyl butyrimidate hydrochloride. 2. 2. A comparison of the properties of the modified proteins with α-lactalbumin by sedimentation velocity, ultraviolet fluorescence, optical rotatory dispersion and electrophoresis showed that they retain most of the characteristics of α-lactalbumin indicating that no major structural changes have resulted from the modification. It was found, however, that the modified proteins were much more susceptible to association and aggregation than α-lactalbumin. This was evident not only at pH 2.00 but on the alkaline side of the isoelectric point at pH 6.00 and 8.50. These observations lend support to the hypothesis that hydrophobic interactions play a role in the association and aggregation of α-lactalbumin and that the difference in behavior of the “alkaline” and “acid” forms of the protein are due to a higher density of non-polar groups at the molecular surface of the “acid” form.

Effects of the amidination reaction on antibody activity and on the physical properties of some proteins.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEffects of the Amidination Reaction on Antibody Activity and on the Physical Properties of Some Proteins*Leon Wofsy and S. J. SingerCite this: Biochemistry 1963, 2, 1, 104–116Publication Date (Print):January 1, 1963Publication History Published online1 May 2002Published inissue 1 January 1963https://doi.org/10.1021/bi00901a019RIGHTS & PERMISSIONSArticle Views115Altmetric-Citations163LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (5 MB) Get e-Alerts Get e-Alerts

スルファイソオキサゾール合成に関する研究 (第6報)

2-Acetylpropionimidate hydrochloride, an intermediate in the synthesis of sulfisoxazole, does not form the corresponding amidine by ethanolic ammonia but forms a free imido ester. Examination of this amidination reaction showed that the objective 2-acetylpropionamidine (II) can be obtained by warming the free 2-acetylpropionimidate (I) with ammonia water, in the presence of a small amount of ammonium chloride, for 1 hour at 30°. Extension of the period of heating to 4 hours results in the formation of 2-(1-acetylethyl)-4, 5-dimethyl-6-aminopyrimidine (III) and the heating for 4 hours at 60°, in the formation of 2-(1-methyl-2-iminopropyl)-4, 5-dimethyl-6-aminopyrimidine (IV).