Preparation and properties of trypsin chemically attached to EEDQ activated styrene–methacrylic acid copolymers

Styrene–methacrylic acid copolymers of varying combinations crosslinked with p-DVB (1–2%) and porous structure were synthesized to be used as carriers in trypsin immobilization. The styrene–methacrylic acid copolymers containing free carboxy groups were activated by conversion into the mixed carbonic anhydride with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) at pH 4.0. The degree of activation of copolymers were determined from the amount of p-aminobenzoic acid each could bind. The activated copolymers were incubated with trypsin in phosphate buffer (pH 8.0) at 4°C for 24 h. The optimum conditions for enzymatic activity measurements determined and the activity tests were carried out in 1.5 × 10-2M CaCl2 solution (pH 8.0) at 0.05 ionic strength with a pH-stat instrument. The dependence of the activity of styrene–methacrylic acid (SMA)/trypsin derivatives to pH was investigated and it was observed that the optimum pH of the immobilized trypsin derivatives moved to the basic region compared to the native trypsin. It was found that as the ionic strength increased, the shift in the optimum pH decreased and the activity increased. The Michaelis constants for the SMA–trypsin derivatives were determined with aid of Lineweaver–Burk diagrams. The thermal, storage, and operational stabilities of SMA–trypsin derivatives were assessed. It was found that the above stabilities for all the immobilized trypsin derivatives were better than that for the native trypsin. Copyright © 1983 John Wiley & Sons, Inc

Preparation and properties of alpha-galactosidase chemically attached to activated chitin

WOS: 000184447900009PubMed ID: 12906314alpha-Galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) from watermelon was covalently immobilized on chitin. The immobilized alpha-galactosidase exhibited an activity of 0.61 U per g of carrier and an activity yield of 67%. The properties of free and immobilized a-galactosidase were also searched and compared. The results, showed that, optimum conditions for activity were not affected by immobilization. The optimum pH and temperature for free and immobilized enzyme found as pH 6.0 and 65degreesC, respectively. Compared with the free enzyme, the temperature and pH stabilities of the immobilized enzyme were similar. Both the enzymes were stable between pH 2-10 and below 50 C. The K-m values for free and immobilized enzyme were determined using p-nitrophenyl-alpha-D-galactopyranoside (PNPG) and raffinose as substrates. Operational stability of the immobilized enzyme was investigated by using both substrates. The operational half-life (t (1/2)) was calculated as 34 h for PNPG and 28 h for raffinose. The immobilized a-galactosidase was also utilized in the hydrolysis of raffinose. The immobilization procedure on chitin was cheap and also easy to carry out, and the immobilized enzyme had good properties that the potential for practical application is considerable

Diffusion characteristics of chitosan-entrapped microsomal UDP-glucuronyl transferase gel beads

PubMed ID: 7599146Rabbit hepatic microsomal UDP-glucuronyl transferase (EC 2.4.1.17) was immobilized by entrapment in chitosan which is an ionotropic gelation agent and the resulting preparation was used as a biocatalyst for the glucuronidation of 1-naphthol in order to study the drug metabolism in vitro and obtaining artificial liver support (detoxification). In this study, depending on the porous structure of the chitosan/UDPGT gel beads, mainly the diffusion characteristics of 1-naphthol from chitosan gel beads in well stirred solutions were investigated, and the values of the effective diffusion coefficients (De) indicate that UDP-glucuronyl transferase in chitosan beads has no additional diffusion barrier. Using the data, the capacity of the chitosan-gel as an immobilization matrix for microsomal UDP-glucuronyl transferase is discussed from the point of view of diffusion characteristics. © 1995

Effect of Enzyme Ratio on the Properties of Glucose-Oxidase and Catalase Immobilized on Modified Perlite

WOS: A1992HF48900002Processed perlite modified by silanization and treatment with glutaraldehyde was used as a support for the co-immobilization of glucose oxidase and catalase. Immobilization was carried out with several enzyme ratios and activity investigated with respect to pH and temperature. The deactivation rates of the preparations were investigated in recycling fluid bed reactors

Enzyme electrode based on oxalate oxidase immobilized in gelatin for specific determination of oxalate.

PubMed ID: 8144172A biosensor for the specific determination of oxalate was developed using oxalate oxidase (EC 1.2.3.4) from barley (Hordeum vulgare) seedling roots in combination with a dissolved oxygen probe. Oxalate oxidase immobilized with gelatin using glutaraldehyde and fixed on pretreated teflon membrane served as an enzyme electrode. The electrode response was maximum when 50 mM succinate buffer was used at pH 3.2 and 35 degrees C. The biosensor response depends linearly on oxalate concentration between 5 x 10(-6)-2 x 10(-4) M with response time 30 sec and substrate specificity of the oxalate oxidase electrode of 100%. The sensor is stable for more than 3 months during which time more than 400 assays can be performed