Immobilization of the enzyme E. coli L-asparaginase on a water-soluble copolymer of vinylpyrrolidone and acrolein

A method for immobilizingE. coli L-asparaginase on a copolymer of vinylpyrrolidone and acrolein has been developed and optimized. The influence on the nature of the modification of the number of acrolein residues in the copolymer has been established. The enzymatic and some physicochemical properties of the immobilized forms of the enzyme obtained have been studied.A. Kirkhenshtein Institute of Microbiology, Academy of Sciences of the Latvian SSR, Riga. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 562–565, July–August, 1988.     

Immobilization of the enzyme L-asparaginase from E. coli on polysaccharides I. Covalent binding with insoluble Sepharoses and CM-cellulose

Summary Immobilized L-asparaginase has been synthesized by various methods of covalent binding to insoluble Sepharoses and CM-cellulose.Some kinetic properties of the preparations obtained, the dependence of the activity on the pH of the medium, and their stability have been investigated.It has been established that the immobilized L-asparaginase possesses increased heat resistance, stability on storage, and stability to competing inhibitors.Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR, Riga. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 639–643, September–October, 1976.     

Immobilization of the enzyme L-asparaginase of E. coli on polysaccharides. V. Preparation and properties of polymeric conjugates based on water-soluble CM-cellulose differing by the quantitative amounts of polymer

A precedure has been developed for the chemical binding of L-asparaginase to soluble CM-cellulose which permits final immobilization products with different degrees of modification to be obtained. Some physicochemical and immunological properties of the samples obtained have been studied.     

Immobilization of the enzyme L-asparaginase of E. coli on polysaccharides. V. Preparation and properties of polymeric conjugates based on water-soluble CM-cellulose differing by the quantitative amounts of polymer

A precedure has been developed for the chemical binding of L-asparaginase to soluble CM-cellulose which permits final immobilization products with different degrees of modification to be obtained. Some physicochemical and immunological properties of the samples obtained have been studied.A. Kirkhenshtein Institute of Microbiology, Latvian SSR Academy of Sciences, Riga. All-Union Scientific-Research Technological Institute of Antibiotics and Enzymes for Medical Synthesis, Leningrad. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 581–587, July–August, 1987.     

Immobilization of the enzyme L-asparaginase fromE. coli on polysaccharides IV. Covalent binding with dialdehyde-dextran

The synthesis has been effected of immobilizedE. coli L-asparaginase on medical dextran — poliglyukin. The influence of the bound polymer on some physicochemical properties of the final products have been studied. An increased resistance to heat and stability on storage of the immobilized forms of L-asparagine in comparison with a native enzyme has been found. It has been shown that the polymer modification of L-asparaginase leads to a decrease in the antigenic affinity of the immobilized enzyme as compared with the native enzyme.     

Immobilization of the enzyme L-asparaginase fromE. coli on polysaccharides. II. Covalent binding with soluble CM-cellulose

The covalent binding of L-asparaginase to soluble CM-cellulose has been carried out by the azide method, and some physicochemical properties of the preparation obtained have been studied. It has been established that the modified L-asparaginase possesses a higher heat stability than the native enzyme and also a greater resistance to proteolytic enzymes.Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR, Riga. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 383–388, May–June, 1979.     

Chemical modification of E. coli L-asparaginase with polyethylene glycol grafted vinylpyrrolidone–maleic anhydride copolymer

A series of polyethylene glycol grafted vinylpyrrolidone–maleic anhydride copolymers [P(VMP)] was synthesized by radical copolymerization of vinylpyrrolidone, maleic anhydride and polyethylene glycol maleic acid monoester. Escherichia coli l -asparaginase was chemically modified with these copolymers. The modified l -asparaginase exhibited the complete loss of antigenicity towards anti-asparaginase serum from rabbit. The highest enzyme activity of the modified l -asparaginase without antigenicity was retained by 59% of the nonmodified one. The modified enzyme was also more resistant to trypsin in vitro. When tested in vivo, the native l -asparaginase was quickly cleared from the plasma of rabbits (half-life time: t_1/2=1.2 hr), whereas the modified enzyme showed prolonged clearance from plasma (t_1/2=53 hr). © 1997 John Wiley & Sons, Ltd.     

Immobilization of the enzyme L-asparaginase fromE. coli on polysaccharides IV. Covalent binding with dialdehyde-dextran

The synthesis has been effected of immobilizedE. coli L-asparaginase on medical dextran — poliglyukin. The influence of the bound polymer on some physicochemical properties of the final products have been studied. An increased resistance to heat and stability on storage of the immobilized forms of L-asparagine in comparison with a native enzyme has been found. It has been shown that the polymer modification of L-asparaginase leads to a decrease in the antigenic affinity of the immobilized enzyme as compared with the native enzyme.Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR, Riga. All-Union Scientific-Research Technological Institute of Antibiotics and Enzymes for Medical Purposes, Leningrad. A. Kirkhenshtein Institute of Microbiology, Academy of Sciences of the Latvian SSR, Riga. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 766–771, November–December, 1986.     

Chemical modification of the tryptophan residues of the L-asparaginase of E. coli with N-bromosuccinimide

Summary The role of the tryptophan residues in the L-asparaginase molecule has been studied by the method of chemical modification with N-bromosuccinimide, and it has been established that in an acid medium this reagent modifies all four tryptophan residues present in the molecule, completely suppressing the activity of the enzyme.The substrate — L-asparagine — and a competing inhibitor — S-benzyl-N-benzyloxycarbonyl-L-cysteine — protect the L-asparaginase from the action of N-bromosuccinimide, which shows the role of the tryptophan in the catalytic center of the L-asparaginase.Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 228–231, March–April, 1975.     

Immobilization of E. coli with autodisplayed Z-domains to a surface-modified microplate for immunoassay

Escherichia coli with autodisplayed Z-domains was reported to improve the sensitivity of immunoassays by the orientation control of antibodies. In this work, a sensitive microplate-based immunoassay is presented by immobilizing E. coli cells to a surface-modified microplate. The microplate was prepared by coating parylene-H film with formyl groups, and then covalently coupling poly-L-lysine to the parylene-H film. The E. coli cells were bound to the microplate by charge interactions between the negatively charged E. coli outer membrane and the positively charged microplate surface. In this work, the preparation of the microplate coated with poly-L-lysine is presented. The immobilization efficiency of E. coli to the modified surface was estimated to be far higher than non-specific interaction by fluorescence microscope and the optical transmittance of the modified microplate was measured to be feasible for immunoassay. The microplate-based immunoassay is demonstrated to be feasible for medical diagnosis of inflammatory diseases by using C-reactive protein as a target analyte for the medical diagnosis of inflammatory diseases.     

Effect of water-soluble polymers, polyethylene glycol and poly(vinylpyrrolidone), on the gelation of aqueous micellar solutions of Pluronic copolymer F127

The micellization of F127 (E(98)P(67)E(98)) in dilute aqueous solutions of polyethylene glycol (PEG6000 and PEG35000) and poly(vinylpyrrolidone) (PVP K30 and PVP K90) is studied. The average hydrodynamic radius (r(h,app)) obtained from the dynamic light scattering technique increased with increase in PEG concentration but decreased on addition of PVP, results which are consistent with interaction of the micelles with PEG and the formation of micelles clusters, but no such interaction occurs with PVP. Tube inversion was used to determine the onset of gelation. The critical concentration of F127 for gelation increased on addition of PEG and of PVP K30 but decreased on addition of PVP K90. Small-angle X-ray scattering (SAXS) was used to show that the 30 wt% F127 gel structure (fcc) was independent of polymer type and concentration, as was the d-spacing and so the micelle hard-sphere radius. The maximum elastic modulus (G(max)(')) of 30 wt% F127 decreased from its value for water alone as PEG was added, but was little changed by adding PVP. These results are consistent with the packed-micelles in the 30 wt% F127 gel being effectively isolated from the polymer solution on the microscale while, especially for the PEG, being mixed on the macroscale.     

Immobilization of E. coli bacteria in three-dimensional matrices for ISFET biosensor design

In recent years, cell-based biosensors (CBBs) have been very useful in biomedicine, food industry, environmental monitoring and pharmaceutical screening. They constitute an economical substitute for enzymatic biosensors, but cell immobilization remains a limitation in this technology. To investigate into the potential applications of cell-based biosensors, we describe an electrochemical system based on a microbial biosensor using an Escherichia coli K-12 derivative as a primary transducer to detect biologically active agents. pH variations were recorded by an ion-sensitive field effect transistor (ISFET) sensor on bacteria immobilized in agarose gels. The ISFET device was directly introduced in 100 ml of this mixture or in a miniaturized system using a dialysis membrane that contains 1 ml of the same mixture. The bacterial activity could be detected for several days. The extracellular acidification rate (ECAR) was analyzed with or without the addition of a culture medium or an antibiotic solution. At first, the microorganisms acidified their micro-environment and then they alkalinized it. These two phases were attributed to an apparent substrate preference of bacteria. Cell treatment with an inhibitor or an activator of their metabolism was then monitored and streptomycin effect was tested.     

壳聚糖微球固定化L-天门冬酰胺酶研究

以反相悬浮交联法合成了壳聚糖微球,并将其应用于固定化L-天门冬酰胺酶。探讨了不同合成条件对成球情况的影响,研究了固定化酶的性质。结果表明,壳聚糖载体可以较好地吸附L-天门冬酰胺酶。固定化酶的活力回收率较高,且稳定性得到提高。     

Immobilization of protein ligands with methyl vinyl ether-maleic anhydride copolymer.

Methyl vinyl ether-maleic anhydride copolymer (MMAC) is a water-insoluble polymer with an acid anhydride group which reacts with amino groups of ligands to form stable amide bonds. MMAC was used to immobilize protein ligands on two kinds of supports, the wells of plastic microtitre plates for enzyme-linked immunosorbent assay and related methods, and gels for affinity adsorbents. The wells were first coated with MMAC and then allowed to react with proteins. The immobilization of proteins by this method was efficient and occurred in a dose-dependent manner. Shodex Et123, a gel having amino groups, was incubated with MMAC, and then the activated Shodex was used to immobilize high concentrations of proteins. Concanavalin A-Shodex thus obtained had high affinities and was successfully used for the high-performance liquid affinity chromatography of sugar derivatives on a short column.