Effect of gamma irradiation on immobilized trypsin

The effect of ionizing radiation of 0.05-10 Mrad on trypsin immobilized on dialdehyde cellulose was being studied. After irradiation the activity of native trypsin decreases by 25%, as compared with the initial, while the activity of immobilized trypsin remains constant. Before immobilization cellulose undergoes special pretreatment that leads to a decrease in the initial contamination. Some samples of modified cellulose were contaminated by staphylococcus culture (200,000 microbes per 0.2 g) and then exposed to irradiation of 0.05-0.4 Mrad. A distinct correlation between the irradiation dose (0.05-0.4 Mrad) and contamination of the object was registered.     

Effect of terrilytin on the activity of nonspecific resistance factors during immunization with staphylococcal anatoxin and staphylococcal infection

Terrilytin and immobilized terrilytin enhance the activity and intensity of phagocytosis and increase the concentration of lysozyme in nonimmunized animals. Both preparations increase the production of antibodies to staphylococcal alpha-hemolysin, the titers of beta-lysins, the activity and intensity of the phagocytosis of bacterial cells by peripheral blood leukocytes in animals immunized with staphylococcal toxoid and challenged with live staphylococcal culture. In healthy animals terrilytin and immobilized terrilytin induce an increase in total proteolytic activity and in the activity of alpha-1-antitrypsin and alpha-2-macroglobulin, decreased as the result of staphylococcal infection.     

Activity studies of immobilized subtilisin on functionalized pure cellulose-based membranes

The activity of immobilized subtilisin BPN' on pure cellulose-based membrane support was investigated using site-directed and random immobilization approaches. The catalytic activity of site-directed immobilized subtilisin on pure cellulose fiber-based materials was found to be 81% of that in homogeneous solution, while that of randomly immobilized subtilisin was 27%. Pure cellulose membrane supports provided large surface areas for high enzyme loading without diffusional limitations. The activity of immobilized subtilisin on pure cellulose support was more than twice that on a modified polyether sulfone (MPS) membrane, which was attributed to the higher hydrophilicity of cellulose. Immobilized subtilisin maintained its initial activity for 14 days at 4 degrees C and 7 days at 24 degrees C. The immobilized enzyme could resist higher temperature and operate over a wider range of pH without loss of activity. This study showed that pure cellulose fiber-based membranes are well suited for enzyme immobilization and biocatalysis.     

Immunocorrecting and protective effect of proteolytic enzymes on antibody formation in mice in staphylococcal infection and antibiotic treatment

The impact of proteolytic enzymes on the humoral immune response, survival rate and mean survival time of mice, infected with S. aureus culture and receiving antibiotics was studied. Infection with staphylococcal suppressed the formation of antibodies to sheep red blood cells. Ampicillin made this immunosuppression even more pronounced, while gentamicin produced practically no effect on the degree of immunosuppression in the infected animals. Proteolytic enzymes terrilytin and terridecase exhibited immunocorrecting properties when used in combination with antibiotics. Terridecase, the immobilized form of the enzyme proved to have the highest activity. In experimental generalized staphylococcal infection all preparations under study produced a protective effect. The maximum effect was noted after the use of ampicillin in combination with terridecase.     

Preparation of lysozyme immobilized on textile carriers and its use in treating suppurative wounds

Immobilized forms of lysozyme were prepared by its covalent binding on dialdehyde cellulose and polycaproamide fibres as woven and knitted fabrics respectively. The preparations were estimated by the content of protein and bacteriolytic activity. The lysozyme activity per 1 g of the carrier and the protein content on dialdehyde cellulose were several times higher than those on polycaproamide while the specific activity of lysozyme on the polycaproamide carrier was somewhat higher than that on dialdehyde cellulose. The effect of the immobilized lysozyme in treatment of purulent wounds was studied on albino rats. It was shown that the periods of the wound healing with the use of the immobilized lysozyme were shorter than those with the use of native lysozyme. Cytological and morphological investigation of the wound wall confirmed the higher efficacy of the lysozyme immobilized forms in treatment of purulent wounds as compared to the use of the native enzyme.     

Biologically active polymer supports based on cellulosic derivatives: synthesis and kinetic study

Bioactive cellulose derivatives have been synthesised by coupling enzymes/antibiotics on carboxymethyl cellulose acid chloride and cellulose carbonate. The effect of pH and temperature on the enzymatic activity of amyloglucosidase immobilised on cellulose carbonate was studied. Michaelis-Menten kinetics have been obeyed to the first degree of approximation despite the restricted mobility of the attached enzyme on the polymer support. Lineweaver-Burk plots for the amyloglucosidase immobilized on carboxymethyl cellulose acid chloride at ambient pH with cellulose carbonate at pH 8 have also been plotted. The Michaelis-Menten constant for the immobilized amyloglucosidase on cellulose carbonate at pH 8 was 9.1 mM, and the activation energy for starch hydrolysis was 21.8 kcals/mole.     

Characteristics of yeast invertase immobilized on porous cellulose beads.

Invertase from Candida utilis was immobilized on porous cellulose beads by an ionic-quanidino bond. The immobilized invertase showed optimum activity between pH 4.0 and 5.4, while the free enzyme had a sharp optimum at pH 4.1. Both temperature profiles were fairly similar up to 55 degrees C. However, above this temperature the immobilized enzyme was more stable than the free enzyme. From the temperature data, the activation energies were found to be 7,322 and 4,052 cal/g mol for the free and the immobilized enzyme, respectively. Candida invertase shows characteristics of substrate inhibition. Both the Km and Ki for the free and the immobilized enzymes were determined. The apparent Ki for the immobilized invertase was much higher than the Ki of the free enzyme, suggesting a diffusion effect. Immobilized invertase molecules deep in the pores only see sucrose concentrations much less than the bulk concentrations. Immobilization, thus, offers certain processing advantages in this regard.     

Studies on the immobilization of trypsin on sand

Trypsin was immobilized on sand using five different methods. Attempts were made to attach amino-functional groups onto sand using 3-aminopropyltriethoxysilane, hexamethylenetetramine, hexamethylenediamine, and melamine. Glutaraldehyde was used as a bifunctional agent in all the methods. Methods for the estimation of the proteolytic 1activity and protein content of immobilized trypsin were standardized. The maximum retained activity was observed for trypsin immobilized on sand via 3-aminopropytriethoxysilane and glutaraldehyde. Immobilized trypsin showed a shift in the pH optimum toward the acidic side over that of soluble trypsin in all five cases. The optimum temperature for both native and immobilized trypsin prepared by the silane-glutaraldehyde method was found to be 45°C. However, the pH and thermal stabilities of immobilized trypsin were observed to be better than that of the native enzyme.     

Immobilization of adenosine deaminase onto agarose and casein

In the present study adenosine deaminase (ADA) was immobilized onto two different polymeric materials, agarose and casein. The factors affecting the amount of enzyme attachment onto the polymeric supports such as incubation time were investigated. The maximum amount of enzyme immobilized onto different polymeric supports occurred at incubation pH value 7.5 and ADA concentration 42 units/g and the incubation time needed for the maximum amount of enzyme attachment to the polymeric supports was found to be 8 h. Some phsicochemical properties of the free and immobilized ADA such as operational stability, optimum temperature and thermal stability, pH optimum and stability, storage stability, and the effect of gamma-radiation were studied. The operational stability of the free and immobilized enzyme showed that the enzyme immobilized by a cross-linking technique using gultaric dialdehyde showed poor durability and the relative activity decreased sharply due to the leakage after repeated washing, while the enzymes immobilized by covalent bonds to the carriers showed a slight decrease in most cases in the relative activity (around 20%) after being used 10 times. Storage for 4-6 months, showed that the free enzyme lost its activity, while the immobilized enzyme showed the opposite behavior. Subjecting the immobilized enzyme to a dose of gamma radiation of 0.5-10 Mrad showed complete loss in the activity of the free enzyme at a dose of 5 Mrad, while the immobilized enzymes showed relatively high resistance to gamma radiation up to a dose of 5 Mrad.     

Experimental study of protease C--proteolytic enzyme of microbial origin]

The specific activity of protease C, a proteolytic enzyme isolated from Acremonium chrysogenum was studied under experimental conditions. Protease C was shown to lyse necrotic biological substrates (dry crusts of burn wounds) and blood clots. By the nature of the effect protease C was analogous to terrilytin and by the level of the effect it was superior in some experiments. Protease C was low toxic and had no mutagenic action.     

The isolation and characterization of phosphofructokinase from the epithelial cells of rat small intestine.

1. Only a single phosphofructokinase isoenzyme is present in the mucosa of rat small intestine. 2. Mucosal phosphofructokinase was purified to yield a homogeneous preparation of specific activity 175 units/mg of protein. 3. The native enzyme is a tetramer, with monomer Mr 84 500 +/- 5000. 4. The native enzyme may be degraded by the action of endogenous proteinases to give two products with the same specific activity as the native enzyme: degradation occurs in the order native enzyme leads to proteolytic product 1 leads to proteolytic product 2. 5. Proteolytic product 1 has a greater mobility in cellulose acetate electrophoresis at pH8 and binds more strongly to DEAE-cellulose than does native enzyme; the converse is true for proteolytic product 2. 6. Proteolytic product 1 is a tetramer with a monomer Mr about 74 300; proteolytic product 2 is also a tetramer. 7. Native enzyme can only be prepared in the presence of proteinase inhibitors; partial purifications based on simple fractionation of crude mucosal extracts in the absence of proteinases inhibitors contain proteolytic product 2 as the main component and proteolytic product 1 together with little native enzyme. 8. Purified native mucosal phosphofructokinase displayed little co-operativity with respect to fructose 6-phosphate at pH 7.0 and was only weakly inhibited by ATP.     

Stability improvement of immobilized lactoperoxidase using polyaniline polymer

Enzyme engineering via immobilization techniques is perfectly compatible against the other chemical or biological approximate to improve enzyme functions and stability. In this study lactoperoxidase was immobilized onto polyaniline polymer activated with glutaraldehyde as a bifunctional agent, to improve enzyme properties. Polyaniline polymer was used due its unique physical and chemical properties to immobilize lactoperoxidase (LPO). The optimum activity of immobilized LPO was observed at pH 6 and 55?°C, which has been increased about 10?°C for the immobilized enzyme. The immobilized enzyme maintained absolutely active for 60?days whereas the native enzyme lost 80?% of its initial activity within this period of time. Moreover, the immobilized enzyme can be reused for several times without loss of activity. The kinetic parameter studies showed slight differences between free and immobilized enzymes. The K_m and K_m.app were calculated to be 0.6 and 0.4; also V_max and V_max.app were 1.3 and 0.9 respectively.     

Site-specific immobilization of enzymes on magnetic nanoparticles and their use in organic synthesis

Magnetic nanoparticles (MNPs) are attractive materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field; this could facilitate the recycling of enzymes and broaden their applications in organic synthesis. Herein, we report the methods for the immobilization of water-soluble and membrane-bound enzymes, and the activity difference between free and immobilized enzymes is discussed. Sialyltransferase (PmST1, from Pasteurella multocida ) and cytidine monophosphate (CMP)-sialic acid synthetase (CSS, from Neisseria meningitides ) were chosen as water-soluble enzymes and expressed using an intein expression system. The enzymes were site-specifically and covalently immobilized on PEGylated-N-terminal cysteine MNPs through native chemical ligation (NCL). Increasing the length of the PEG linker between the enzyme and the MNP surface increased the activity of the immobilized enzymes relative to the free parent enzymes. In addition, the use of a fluorescent acceptor tag for PmST1 affected enzyme kinetics. In contrast, sialyltransferase from Neisseria gonorrheae (NgST, a membrane-bound enzyme) was modified with a biotin-labeled cysteine at the C-terminus using NCL, and the enzyme was then assembled on streptavidin-functionalized MNPs. Using a streptavidin-biotin interaction, it was possible to immobilize NgST on a solid support under mild ligation conditions, which prevented the enzyme from high-temperature decomposition and provided an approximately 2-fold increase in activity compared to other immobilization methods on MNPs. Finally, the ganglioside GM3-derivative (sialyl-lactose derivative) was synthesized in a one-pot system by combining the use of immobilized PmST1 and CSS. The enzymes retained 50% activity after being reused ten times. Furthermore, the results obtained using the one-pot two-immobilized-enzyme system demonstrated that it can be applied to large-scale reactions with acceptable yields and purity. These features make enzyme-immobilized MNPs applicable to organic synthesis.     

Lysozyme modification by the fenton reaction and gamma radiation

A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(II)/H2 O2) or hydroxyl radicals produced by y radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. Gamma radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr > Met = Cys > Lys > Ile + Leu > Gly > Pro = Phe > Thr + Ala > Trp = Ser > Arg > Asp + Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met His > Ile + Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by gamma radiation.     

Glucose oxidase immobilization on a novel cellulose acetate-polymethylmethacrylate membrane

Glucose oxidase (GOD) was immobilized on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane. The immobilized GOD showed better performance as compared to the free enzyme in terms of thermal stability retaining 46% of the original activity at 70 degrees C where the original activity corresponded to that obtained at 20 degrees C. FT-IR and SEM were employed to study the membrane morphology and structure after treatment at 70 degrees C. The pH profile of the immobilized and the free enzyme was found to be similar. A 2.4-fold increase in Km value was observed after immobilization whereas Vmax value was lower for the immobilized GOD. Immobilized glucose oxidase showed improved operational stability by maintaining 33% of the initial activity after 35 cycles of repeated use and was found to retain 94% of activity after 1 month storage period. Improved resistance against urea denaturation was achieved and the immobilized glucose oxidase retained 50% of the activity without urea in the presence of 5M urea whereas free enzyme retained only 8% activity.     

Preparations of immobilized lysozyme with reversibly soluble polymer for hydrolysis of microbial cells

Hen egg white lysozyme was immobilized by carbodiimide method to form amide bonds with a polymer (AS-L) showing reversibly soluble-insoluble characteristics with pH change. The immobilized enzyme (LY-AS) was soluble above pH 6 and precipitate below pH 4.5, offering advantages in that it can carry out hydrolysis of microbial cells in a soluble form yet be recovered after precipitation at low pH. The maximum specific activity of LY-AS was 66% of that of free lysozyme with M. lysodeikticus cells as substrate, which is much higher than the values reported in the literature using water-insoluble materials as carriers. The effects of pH and temperature on the activity of LY-AS were studied and compared with those of free lysozyme. With repeated pH cycles between 6.6 and 4.5, the operation half-life of immobilized enzyme activity was nine cycles. Repeated batch lysis of microbial cells could be carried out with intermittent enzyme precipitation and recovery steps. In such an operation the insoluble residual cells should be recovered together with the immobilized enzyme to minimize enzyme loss arising from adsorption to cells.     

Characterization of deoxyribonuclease I immobilized on magnetic hydrophilic polymer particles

Magnetic bead cellulose particles and magnetic poly(HEMA-co-EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA-co-EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg(2+), Ca(2+), Mn(2+) and Co(2+)) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co(2+) ions.     

Immunostimulating and protective effects of terrilytin preparations in staphylococcal infection

In mice infected with staphylococci there was observed less pronounced development of the immune response to sheep red blood cells (SRBC) than in intact animals subjected only to immunization. Administration of free terrilytin to the infected mice increased the immune response development induced by SRBC while the use of immobilized terrilytin normalized it. The supernatant liquid of the spleen cells (SLSC) from the mice treated with the free of immobilized terrilytins stimulated development of the immune response to SRBC in the infected animals and inhibited the function of the suppressor cells in the spleen. The immunomodulating and protective effects of the SLSC fractions isolated with column chromatography on Sephadex G-150 were investigated. Substances of the low molecular fraction of the SLSC proteins (molecular weight of 10-15 kD) from the mice treated with the terrilytins showed immunostimulating and protective properties. The factors inducing both the activity types included peptides and the ribonucleotide component playing a significant role in realization of the immunostimulating and protective effects of the free and immobilized proteases.     

Covalent immobilization of a glucoamylase to bagasse dialdehyde cellulose

Cellulose fibres from bagasse were oxidized by sodium periodate in sulphuric acid media at positions 2 and 3 of the anhydroglucose unit to produce dialdehyde cellulose. The aldehyde groups of the dialdehyde cellulose were able to react with amino groups of a glucoamylase to form covalent bonds and result in a dialdehyde cellulose immobilized enzyme. The optimum pH of this immobilized enzyme and free enzyme were in the range of 3.0–5.0 and 3.5–5.0, respectively. The optimum temperature for both the free and immobilized enzymes was 60–65 °C. The relative remaining activity of the immobilized enzyme was 36% and its stability was very good, since it could be reused for over 30 cycles. Its activity decreased from the first to the seventh reuse cycles, due to the slow detachment of non-covalently bound enzyme. However, activity tended to stabilize after the seventh cycle of reuse, indicating very stable covalent binding between the enzyme and dialdehyde cellulose.     

Immobilization of Papain on the Mesoporous Molecular Sieve MCM‐48

The immobilization of papain on the mesoporous molecular sieve MCM‐48 (with a pore size of 6.2 nm in diameter) with the aid of glutaraldehyde, and the characteristics of this immobilized papain are described. The optimum conditions for immobilization were as follows: 20 mg native free enzyme/g of the MCM‐48 and 0.75 % glutaraldehyde, 2 h at 10–20 °C and pH 7.0. Under these optimum conditions for immobilization, the activity yield [%] of the immobilized enzyme was around 70 %. The influence of the pH on the activity of the immobilized enzyme was much lower compared to the free enzyme. The thermostability of the immobilized enzyme, whose half‐life was more than 2500 min, was greatly improved and was found to be significantly higher than that of the free enzyme (about 80 min). The immobilized enzyme also showed good operational stability, and the activity of the immobilized enzyme continued to maintain 76.5 % of the initial activity even after a 12‐day continuous operation. Moreover, the immobilized enzyme still exhibited good storage stability. From these results, papain immobilized on the MCM‐48 with the aid of glutaraldehyde, can be used as a high‐performance biocatalyst in biotechnological processing, in particular in industrial and medical applications.