Production of L-lysine by immobilized trypsin. Study of DL-lysine methyl ester resolution

The possibility of producing L-lysine from chemically synthesized DL-lysine has been investigated. Optical resolution of racemic DK-lysine may be achieved by using the stereospecific esterasic activity of trypsin on DL-lysine methyl ester, which gives L-lysine and unchanged D-lysine methyl ester. SL-lysine methyl ester spontaneous hydrolysis may be neglected when operating at pH 5.5 and 30 degrees C. Effect of pH and substrate concentration on hydrolysis rate has been investigated when using as a catalyst either soluble or immobilized trypsin. For this purpose, trypsin was coupled onto an amine porous silica, Spherosil, activated with glutaraldehyde. The optimal pH is 5.8 for soluble trypsin and 6.0 for immobilized trypsin. It was yet possible to lower the parent optimal pH of immobilized trypsin, and thus increase its activity at 5.5, by co-grafting onto Spherosil an aminosilane, for enzyme coupling via glutaraldehyde activation and a positively charged diethyl amino ethyl (DEAE) silane, for decreasing the pH of trypsin microenvironment.     

Immobilization of a keratinolytic protease from Purpureocillium lilacinum on genipin activated-chitosan beads

Keratinase from Purpureocillium lilacinum LPSC # 876 was immobilized on chitosan beads using two different cross-linking agents: glutaraldehyde and genipin. For its immobilization certain parameters were optimized such as cross-linker concentration, activation time and activation temperature. Under optimum conditions, enzyme immobilization resulted to be 96 and 92.8% for glutaraldehyde and genipin, respectively, with an activity recovery reaching up to 81% when genipin was used. The immobilized keratinase showed better thermal and pH stabilities compared to the soluble form, retaining more than 85% of its activity at pH 11 and 74% at 50 °C after 1 h of incubation. The residual activity of immobilized keratinase remained more than 60% of its initial value after five hydrolytic cycles. The results in this study support that glutaraldehyde could be replaced by genipin as an alternative cross-linking eco-friendly agent for enzyme immobilization.     

Chemically surface modified gel (CSMG): an excellent enzyme-immobilization matrix for industrial processes

Invertase from S. cerevisiae has been immobilized on porous silica matrix, formed using sol-gel chemistry, with surface area of approximately 650 m(2)/g. The co-condensation of silica sol with 3-aminopropyl(triethoxy)silane produced an amino-chemically surface modified silica gel (N-CSMG) with a very high ligand loading of 3.6 mmol/g SiO(2); significantly higher than commercially available matrices. Surface amine groups were activated with glutaraldehyde to produce GA-N-CSMG, and invertase covalently attached by the aldehyde. Invertase was used as a model enzyme to measure the immobilizing character of the GA-N-CSMG material. Using an optimized immobilization protocol, a very high loading of 723 mg invertase per gram GA-N-CSMG is obtained; 3-200-fold higher than values published in literature. The reproducible, immobilized activity of 246,000 U/g GA-N-CSMG is also greater than any other in literature. Immobilized invertase showed almost 99% retention of free enzyme activity and no loss in catalytic efficiency. The apparent kinetic parameters K(M) and V(M) were determined using the Michealis-Menten kinetic model. K(M) of the free invertase was 1.5 times greater than that of the immobilized invertase--indicating a higher substrate affinity of the immobilized invertase. These findings show considerable promise for this material as an immobilization matrix in industrial processes.     

氨基化硅胶载体固定化纤维素酶的研究

用硅胶作载体,戊二醛作交联剂,制备了固定化的纤维素酶。对制备固定化纤维素酶的偶联剂浓度、pH、给酶量3个影响因素进行了研究,通过正交试验优化得出最佳的固定化条件:交联剂戊二醛浓度为1%,固定化pH值为5,固载量为每克载体100mg纤维素酶。     

The interaction of glutaraldehyde with poly(α,L-lysine), n-butylamine,and collagen. I. The primary proton release in aqueous medium

The interaction between poly(α,L -lysine) (DP = 180) and glutaraldehyde was investigated in dilute aqueous solution by measurement of the kinetics of proton release at constant pH and temperature and at various concentrations of the reaction components. Under various conditions, the release of protons at constant pH appeared kinetically to be composed of at least two steps: an initial zero-order reaction, followed by a slower reaction. At excess of polylysine amino groups, the pH optimum for the rates of reaction was at pH 9–10 (24–25°C). Under the conditions used and at pH 8, the initial rate of the second kinetic step was proportional to the glutaraldehyde concentration and was practically independent of polylysine concentration at pH 8 and 8.6, at an excess of amino groups. At pH values of 7, 8, and 8.6 the apparent overall energy of activation for the second kinetic step was 18–19 kcal/mole (temp. range 4–40°C). Comparing acetaldehyde with the difunctional glutaraldehyde, it was found that the rate of proton release was much smaller in the case of acetaldehyde. Comparing n-butylamine with the macromolecular polylysine at equal concentrations of amino groups, the rates of proton release were much smaller in the case of n-butylamine. Collagen in aqueous medium also interacted with glutaraldehyde in a manner analogous to polylysine, although the conditions were not quite comparable. In the case of collagen, the initial fast proton liberation step was relatively much larger than in the case of polylysine. A reaction scheme for the initial reaction steps is being proposed which includes primary complex formation between glutaraldehyde and polylysine. This dialdehyde–polyamino acid system is considered to serve as a model for tanning processes of hides and for fixation procedures.     

Immobilized enzymes: the catalytic properties of lactate dehydrogenase convalently attached to glass beads

Chick LDH (H₄ and M₄) has been covalently attached to aryl and alkyl amine glass using sodium nitrite and glutaraldehyde respectively. These immobilized enzymes remain active for months at 0°C and exhibit Km values similar to those of the soluble enzyme; however, they have pH-rate profiles that are independent of pH and show decreased substrate inhibition. Disaggregation followed by reassociation indicate the enzymes are bound by all four subunits and the resulting activity restored to the native, aryl amine and glutaraldehyde bound enzyme are 33, 25 and 90% respectively. At a pH of 3.2 and 25°, the soluble and aryl amine glass LDH's are rapidly denatured while the glutaraldehyde bound enzyme shows no loss of activity for at least 35 days.     

Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (K_m, V_max, k_cat, kcat/K_m), thermal stability, storage stability and reusability of immobilized enzyme were determined.The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The K_m value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.     

氨基化二氧化硅颗粒固定木瓜蛋白酶研究

采用正硅酸乙酯与N-(β-氨乙基)氨丙基三乙氧基硅烷在油包水形成的微胶囊中同步水解的方法,一步法制备了氨基化的二氧化硅颗粒,得到的颗粒粒径在0.3～0.5μm之间,平均大小为0.37μm, 氨基含量和颗粒大小可控,氨基含量高达56mmol/g。此颗粒经戊二醛处理后,采用共价法固定木瓜蛋白酶,固定化最适pH6.5,最佳给酶量为15mg/g载体,固定化酶的最适反应温度为70℃,最适反应pH为6.5,固定化酶热稳定性,pH耐受性,贮存稳定性都明显高于游离酶,表明此颗粒可作为一种优良的酶固定化载体。     

Properties of soluble and immobilized Aspergillus niger β-xylosidase

Aspergillus niger β-xylosidase was characterized when in soluble form and when immobilized to alkylamine porous silica with glutaraldehyde and to alumina with titanium tetrachloride. Energies of activation averaged 13.4 KcaL/mol for the soluble enzyme, 9.0 Kcal/mol when immobilized to alumina, and 8.0 Kcal/mol when bound to silica. The highest activity of all forms of β-xylosidase was found near pH 3. The soluble enzyme was highly stable at pH 4, where lowest rates of decay occurred, and temperature of 65°C and below. The decay rates of alumina-bound β-xylosidase and pH 4 and equivalent temperatures were approximately 10 times as high. Michaells constants were 0.200 and 0.262mM for o-nitrophenyl-β-D -xylopyranoside with soluble and alumina-bound β-xylosidase, respectively.     

Kinetics of appearance of sulfhydryl groups in alpha 2-macroglobulin on reaction of the inhibitor with amines

The mechanism of the appearance of sulfhydryl groups in alpha 2-macroglobulin in the reaction with amines was characterized by analyses of the kinetics with ammonia and methylamine. All reactions occurred under pseudo-first-order conditions in the range of pH (7.0-8.6) and amine concentration (10-600 mM) investigated. The logarithm of the pseudo-first-order rate constant increased linearly as a function of pH with a slope of unity, indicating that the unprotonated amine is the active species in the reaction. Plots of the observed pseudo-first-order rate constants vs. concentration of unprotonated amine at constant pH were also linear and gave second-order-rate constants of 0.32 and 13.8 M-1 s-1 for ammonia and methylamine, respectively, at pH 8.0; similar values were obtained at pH 8.6. Activation energies of 85 and 100 kJ mol-1 and activation entropies of 10 and 95 J K-1 mol-1 for ammonia and methylamine, respectively, were estimated from Arrhenius plots, suggesting that the higher reaction rate for methylamine is due primarily to a higher activation entropy. These results are consistent with the release of sulfhydryl groups being caused by a nucleophilic attack of the uncharged amine on a thio ester bond of alpha 2-macroglobulin in a bimolecular reaction occurring under pseudo-first-order conditions. The characteristics of the reaction suggest that the thio ester in each alpha 2-macroglobulin subunit reacts independently and equivalently with the amine and also that the thio ester bond cleavage initiates the reaction sequence leading to inactivation of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and characterization of polyamine-based biomimetic catalysts as artificial ribonuclease

Several polyamine derivatives (I-V) conjugated with or without an intercalative moiety were prepared as ribonuclease mimics. Although no DNA-cleaving activity was observed for all compounds tested, mimics I, III, and V bearing an intercalative moiety along with the primary amine and/or imidazole moieties exhibited potent RNA-cleaving activity at near physiological pH. The RNA-cleaving reactions of the compounds show characteristic bell-shaped pH dependency, and the optimal pH values for III and V were well correlated to the pKa values of their active sites, primary amine, and imidazole moieties.     

Removal of As(V) from water by pectin based active hydrogels following geochemical approach

Fe(II)-loaded pectin based hydrogels were used to remove As(V) from water. The hydrogels were synthesized by crosslinking pectin 'as such' or in the presence of 2-acrylamido-2-methylpropanesulphonic acid and three crosslinkers. The hydrogels crosslinked with 5% glutaraldehyde showed the maximum uptake of both Fe(II) and As(V). The sorption of As(V) was found to be sensitive to pH and temperature changes, as the maximum sorption was observed at 35 degrees C and 7.0-9.2 pH.     

Modeling the inactivation kinetics of bacillus spores by glutaraldehyde

Aims: Our goal was to develop a mathematical kinetic model to predict the sporicidal activity of glutaraldehyde, which is an active ingredient frequently used in commercial products employed for liquid disinfection and decontamination. Methods and Results: We used our previously published data on spore inactivation by glutaraldehyde to develop a predictive model obtained by calculating multiple independent modifying functions. The model was then validated by comparing model predicted values to new experimental data. For model validation, quality‐controlled spores of Bacillus athrophaeus (previously and generally known as Bacillus subtilis globigii) were exposed under conditions where several physicochemical variables were modified simultaneously, and the spore surviving fractions were measured by titration. Conclusions: The model predicted within one order of magnitude variations in sporicidal effectiveness due to changes in main parameters (glutaraldehyde concentration, temperature or time‐duration of the treatment). Other parameters such pH, salinity and the effect of serum concentration were also addressed, albeit with less accuracy. Significance and Impact of the study: The model should be useful to quantitatively estimate the effectiveness of glutaraldehyde‐based disinfectants, decontaminants, and germicides under the described conditions, particularly when limited data are available or when spore virulence (like that of Bacillus anthracis) precludes extensive experimentation. A similar approach could predict the effectiveness of a variety of decontaminant and disinfecting agents.     

Arginine-vasopressin modulates intracellular pH via V1 and V2 receptors in renal collecting duct cells.

Arginine-vasopressin (AVP) has been proposed to be involved in the modulation of acid-base transporters; however, the nature of the mechanisms underlying AVP direct action on intracellular pH (pH(i)) in the cortical collecting duct (CCD) is not yet clearly defined. The aim of the present study was to elucidate which are the proteins implicated in AVP modulation of pH(i), as well as the receptors involved in these responses using a CCD cell line (RCCD(1)); pH(i) was monitored with the fluorescent dye BCECF in basal conditions and after stimulation with basolateral 10(-8) M AVP. Specific V1- or V2-receptor antagonists were also used. RT-PCR studies demonstrated that RCCD(1) cells express V1a and V2 receptors. Functional studies showed that while V2-receptor activation induced a biphasic response (alkalinization-acidification), V1-receptor activation resulted in an intracellular acidification. The V2-mediated alkalinization phase involves the activation of basolateral NHE-1 isoform of the Na(+)/H(+) exchanger while in the acidification phase CFTR is probably implicated. On the other hand, V1-mediated acidification was due to activation of a Cl(-)/HCO(3)(-) exchanger. We conclude that in RCCD(1) cells AVP selectively activates, via a complex of V1 and V2 receptor-mediated actions, different ion transporters linked to pH(i) regulation which might have physiological implications.     

Investigation of the binding mechanism of glucoamylase to alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) has been coupled to several porous silica matrices by a new covalent process using alkylamine derivatives of titanium(IV)-activated supports. In order to investigate the interaction of the titanium element with the silanol groups of the inorganic matrices, activation was performed at different times, using titanium(IV) chloride, either pure or as a 15% w/v solution, in 15% w/v hydrochloric acid at 25, 45 and 80°C, followed by washing with sodium acetate buffer (0.02m, pH 4.5) or chloroform. Using pure TiCl₄, the highest activities of all preparations were obtained at 80°C and with acetate buffer washing, resulting from a higher content of titanium coating of the carrier. When activation was performed in aqueous TiCl₄ solution, followed by a drying step, the highest activity was obtained with preparations washed with chloroform, with or without amination. When reacting pure TiCl₄ with controlled pore glass (CPG) and with porous silica (Spherosil), colour formation was observed after reaction of glutaraldehyde with the aminated support. This did not happen when Celite was used as the support. As a criterion for comparison of the different immobilized enzyme preparations, the concept of an ‘instability factor’, which measures the percentage of immobilized enzyme activity due to release of enzyme into solution, is introduced. Instability factors of immobilized enzyme preparations on Celite were always higher than those obtained with the other matrices, confirming that there was no covalent coupling of the enzyme to Celite. However, when the activation was performed with aqueous TiCl₄ solution with drying, Schiff's base formation was observed in all preparations and very stable immobilized enzyme preparations were obtained. The results of the activation of controlled pore glass and porous silica with pure titanium(IV) chloride suggest the existence of a true reaction between the titanium element and the silanol groups of these carriers by formation of a bridge, Si-O-Ti, while with the titanium(IV) chloride solution in hydrochloric acid, a coating of hydrous titanium(IV) oxide is obtained.     

Intracellular distinction between peroxidase and catalase in exocrine cells of rat lacrimal gland: a biochemical and cytochemical study.

The lacrimal gland (Glandula orbitalis externa) of rat contains both peroxidase and catalase and was used as a model for biochemical and cytochemical distinction between peroxidase and catalase. Both enzymes were isolated by ammonium sulfate precipitation from tissue homogenates, and the effects of fixation with glutaraldehyde and various conditions of incubation were investigated colorimetrically using DAB as hydrogen donor. The lacrimal gland peroxidase is strongly inhibited by glutaraldehyde treatment. In contrast, for catalase the fixation with glutaraldehyde is the prerequistie for demonstration of its peroxidatic activity. The maximal peroxidatic activity was obtained after treatment of catalase with 3% glutaraldehyde, higher concentrations being inhibitory. For lacrimal gland peroxidase, the maximal rate of oxidation of DAB is at pH 6.5, whereas for catalase it is at pH 10.5. The optimal concentration of H2O2 for lacrimal gland peroxidase is at 10(-3)M and for peroxidatic activity of catalase at 10(-1)M. These optimal conditions obtained biochemically were applied to tissue sections of rat lacrimal gland. After the fixation of tissue with a low concentration of glutaraldehyde and incubation in the DAB medium at neutral pH containing 10(-3)M H2O2 (Peroxidase medium), the reaction product was localized in the cisternae of the rough endoplasmic reticulum, in elements of the Golgi apparatus, and in secretory granules. After the fixation of tissue with 3% glutaraldehyde and incubation in the DAB-medium containing 10(-1)M H2O2 and at pH 10.5 (catalase medium), the staining in the endoplasmic reticulum, the Golgi-apparatus and in secretory granules was completely inhibited and reaction product was localized exclusively in small (0.2-0.5 mu) particles similar to small peroxisomes described in various other cell-types.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds.

Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study; I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32-34 s, final temperature between 40 degrees C and 47 degrees C.     

Tresyl-mediated synthesis: kinetics of competing coupling and hydrolysis reactions as a function of pH, temperature, and steric factors

Kinetic parameters have been measured for coupled nucleophilic and solvolytic reactions of 2,2,2-trifluoroethanesulfonyl (tresyl)-modified poly(ethylene glycol) based on a system of coupled differential equations implied by recently proposed elementary reaction mechanisms. Fitted kinetic parameters were found to be strong functions of pH, temperature, and steric factors. To maximize the total yield of coupled amine as well as the fraction of secondary amine linkages, our model predicts that it is desirable to run tresyl coupling reactions at low temperatures at pH approximately 8.0, depending on the amine pKa for primary, unhindered amines. For branched primary amines, our data favor room temperature at a slightly higher pH.     

Structural Snapshots from the Oxidative Half-reaction of a Copper Amine Oxidase: IMPLICATIONS FOR O2 ACTIVATION*

The mechanism of molecular oxygen activation is the subject of controversy in the copper amine oxidase family. At their active sites, copper amine oxidases contain both a mononuclear copper ion and a protein-derived quinone cofactor. Proposals have been made for the activation of molecular oxygen via both a Cu(II)-aminoquinol catalytic intermediate and a Cu(I)-semiquinone intermediate. Using protein crystallographic freeze-trapping methods under low oxygen conditions combined with single-crystal microspectrophotometry, we have determined structures corresponding to the iminoquinone and semiquinone forms of the enzyme. Methylamine reduction at acidic or neutral pH has revealed protonated and deprotonated forms of the iminoquinone that are accompanied by a bound oxygen species that is likely hydrogen peroxide. However, methylamine reduction at pH 8.5 has revealed a copper-ligated cofactor proposed to be the semiquinone form. A copper-ligated orientation, be it the sole identity of the semiquinone or not, blocks the oxygen-binding site, suggesting that accessibility of Cu(I) may be the basis of partitioning O₂ activation between the aminoquinol and Cu(I).     

Studies of the mechanism of glutamine synthetase utilizing pH-dependent behavior in catalysis and binding

The pKa values of enzyme groups of Escherichia coli glutamine synthetase which affect catalysis and/or substrate binding were determined by measuring the pH dependence of Vmax and V/K. Analysis of these data revealed that two enzyme groups are required for catalysis with apparent pKa values of approximately 7.1 and 8.2. The binding of ATP is essentially independent of pH in the range studied while the substrate ammonia must be deprotonated for the catalytic reaction. Using methylamine and hydroxylamine in place of ammonia, the pKa value of the deprotonated amine substrate as expressed in the V/K profiles was shifted to a lower pKa value for hydroxylamine and a higher pKa value for methylamine. These data indicate that the amine substrate must be deprotonated for binding. Hydroxylamine is at least as good a substrate as ammonia judged by the kinetic parameters whereas methylamine is a poor substrate as expressed in both the V and V/K values. Glutamate binding was determined by monitoring fluorescence changes of the enzyme and the data indicate that a protonated residue (pKa = 8.3 +/- 0.2) is required for glutamate binding. Chemical modification by reductive methylation with HCHO indicated that the group involved in glutamate binding most likely is a lysine residue. In addition, the Ki value for the transition state analog, L-3-amino-3-carboxy-propanesulfonamide was measured as a function of pH and the results indicate that an enzyme residue must be protonated (pKa = 8.2 +/- 0.1) to assist in binding. A mechanism for the reaction catalyzed by glutamine synthetase is proposed from the kinetic data acquired herein. A salt bridge is formed between the gamma-phosphate group of ATP and an enzyme group prior to attack by the gamma-carboxyl of glutamate on ATP to form gamma-glutamyl phosphate. The amine substrate subsequently attacks gamma-glutamyl phosphate resulting in formation of the tetrahedral adduct before phosphate release. A base on the enzyme assists in the deprotonation of ammonia during its attack on gamma-glutamyl phosphate or after the protonated carbinol amine is formed. Based on the kinetic data with the three amine substrates, catalysis is not rate-limiting through the pH range 6-9.