Enrico Boccu

Surface modification of proteins activation of monomethoxy-polyethylene glycols by phenylchloroformates and modification of ribonuclease and superoxide dismutase

A single-step method of activation of monomethoxy-polyethylene glycols suitable for its binding to polypeptides and proteins is proposed. Based on the reaction with 2,4,5-trichloro-phenylchloroformate orp-nitrophenylchloroformate, it gives reactive PEG-phenylcarbonate derivatives. The PEG intermediate is stable on storage, the activating group is easily quantified, and the reaction with amino acid and proteins proceeds rapidly at pH near neutrality. The PEG derivatization of enzymes with this procedure is less inactivating than those previously reported. Ribonuclease and super-oxide dismutase were modified and the effect of (a) bound polymer on clearance time in rats, (b) antibody recognition, and (c) on the enzymatic activity toward low and high molecular weight substrates were studied.

Glutamate dehydrogenase from Escherichia coli: induction, purification and properties of the enzyme.

When Escherichia coli was grown in a minimum medium with glucose as sole carbon source and a proper level of ammonia, NADP+ specific glutamate dehydrogenase (L-glutamate: NADP+ oxidoreductase (deaminating), ED 1.4.1.4) was induced. The enzyme was solubilized by French press treatment and purified to homogeneity by (NH4)2SO4 fractionation, heat treatment followed by DEAE-cellulose, hydroxylapatite and Bio-Gel chromatography with an overall yield of 30%. The enzyme proved to be heat stable and relatively resistant to protein denaturants. The optimum of enzymic activity for the reductive amination is at pH 8 and at pH 9 for the oxidative deamination. The activity is affected by adenine nucleotides. The molecular weight (about 250 000 for the native form and 46 000 for the inactive subunit) and amino acid composition, suggest strict similarities with the NADP+ enzyme from fungal origin.

Reaction of sulfenyl halides with cytochrome c. A novel method for heme cleavage

The h.eme group of ¢y~oc~rome c is linked ~o .the polypep~ide ehAn by thieeth,er b.rAges 11 ]. Me~ods presently used for home cleavage involve the use of heawj metals ~uth as silver ,and m.ercury I2, 3], performic acid [4], or, as recently reported, iodine mad cyanogen bromide I5]. Attempting to selectively modify No single ~ryp~ophan residue of horse heart ¢ytochlome c by sulfeny] halide~ . ~ ] , we found tha~ ~h~, ~-eagen~ alS~ a]lOv~ a rapid and quanIi~a.tiv-e cleavage of ~ e tlaioethe~ bonds linking tlae h.eme to ~,e p~o!ein. Sulfeny] halide~ a~e known to react w~th unsyrnmearico] Nioetla.ers gSv~g ~ise to disu~fi,des among other produc~ depending upon Ne stmctme of the Nioelhers I7, NThese r.eagen~s have found an %vplicadon in the removal of S-protecting g,oups in peptide syn-~es~s I9]. ~Ilae results herewith reported indi~zie thai 2-nit.mffnewl~lfenyl ¢Mofide ,(NPS-Ct) ~ea,ets :(see scheme) s

Isolation and some properties of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus☆

Abstract A purification procedure is described for 6-phosphogluconate dehdyrogenase (6-phospho- d -gluconate:NADP oxidoreductase (decarboxylating), EC 1.1.1.44) of Bacillus stearothermophilus (NCA 1503). A 1200-fold purification was obtained with a 15% yield. The purified enzyme has an approximate mol. wt of 101 000 as estimated by sucrose density gradient centrifugation and consists of subunits of a mol. wt of 51 000. Sulfhydryl group(s) are essential for enzymatic activity as indicated by p -chloromercuribenzoate and Ellmans reagent inactivation. Mg 2+ -activate the enzyme at a low concentration (0.01–0.04 M), whereas they inhibit at a higher concentration. The optimum of the activity was found at about pH 8, with a K m value at 43 °C of 2.5·10 −5 M for NADP and 2.0·10 −5 M for 6-phosphogluconic acid. The enzyme is stable at 60 °C, whereas it is inactivated at higher temperatures, with a denaturation half-time of 80 min at 70 °C and 3 min at 80 °C. The enzyme shows a broken Arrhennius plot, with two straight lines meeting at about 50 °C.

Radiation-induced polymerization for the immobilization of penicillin acylase

The immobilization ofEscherichia coli penicillin acylase (EC 3.5.1.11) was investigated by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperature. A leak-proof composite that does not swell in water was obtained by adding the crosslinking agent trimethylolpropane trimethacrylate to the monomer-aqueous enzyme mixture. Penicillin acylase, which was immobilized with greater than 70% yield, possessed a higherKm value toward the substrate 6-nitro-3-phenylacetamidobenzoic acid than the free enzyme form (Km = 1.7 × 10−5 and 1 × 10−5M, respectively). The structural stability of immobilized penicillin acylase, as assessed by heat, guanidinium chloride, and pH denaturation profiles, was very similar to that of the free-enzyme form, thus suggesting that penicillin acylase was entrapped in its native state into aqueous free spaces of the polymer matrix.

Coupling of monomethoxypolyethyleneglycols to proteins via active esters.

Two alternative methods for the attachment of monomethoxypolyethyleneglycols (PEG) to proteins are proposed; they are based upon the replacement of the hydroxy terminal function of PEG to carboxylate followed by its activation with dicyclohexylcarbodiimide and N-hydroxy-succinimide. The methods, which give more homogeneous product than that employing trichloro-s-triazine as coupling reagent, may also be used for the modification of essential -SH containing enzymes. The attachment of PEG activated via esters was tested with several model proteins and the influence of the extent of modification i. on the biological activity of various enzymes, ii. on the binding capacity for albumin and iii. on the clearance time in rats using superoxide dismutase as model tracer was evaluated. It was also demonstrated that the extent of PEG attachment varies greatly according to the different proteins used.

Effect of Edta on the Conformational Stability of Thermolysin

Thermolysin is a remarkably thermostable metallo endopeptidase produced by Bacillus Thermoproteolyticus Rokko[l]. The enzyme contains one catalytically essential zinc as well as four calcium atoms per molecule[2l. Calcium ions are not directly involved with the catalytic activity, but the nature of interactions in which they partecipate, as obtained by X-ray analysis,u gest that these ions may contribute to the thermostability of the molecule [3,4]. The pre sent communication describes the effect of the chelating agent EDTA which is an effective inhibitor of the enzyme [5,6], on the stability against heat and protein denaturants of the secondary structure of thermolysin, as assessed by circular dichroism and emission fluorescence measurements.

Immobilization of Purified Penicillin Acylase in a Polarized Ultrafiltration Membrane Reactor

Penicillin acylase from E. coli is widely used in the pharmaceutical industry for the mild hydrolysis of penicillin G, obtained by fermentation. The product, 6-aminopenicillanic acid (6-APA), is the starting material in the production of new, semisynthetic penicillins.’ Usually, crude penicillin acylase preparations are employed (either bound to insoluble matrices, or as soluble enzyme). This is the reason why few purification to homogeneity procedures have been described so far, and why little is known about acylase physico-chemical characteristics. In this paper, three items are briefly discussed: ( I ) a purification technique with a final affinity chromatography step; (2) an analysis of the enzyme thermal deactivation kinetics; and (3) the results of preliminary acylase stabilization tests, performed in a polarized ultrafiltration membrane reactor, where the enzyme is injected together with linear-chain polyelectrolytes. The procedure for penicillin acylase purification, usually employed in our laboratories,* involves the following steps: ( 1) enzyme extraction by washed-cell paste freezing and subsequent thawing in distilled water; (2) batch adsorption of acidic components, from the cell-free extract by DEAE-cellulose; (3) batch acylase adsorption by CM-cellulose at pH 5, followed by elution with 0.1 M acetate buffer; (4) ammonium sulfate cut between 50% and 70% saturation; and (5) adsorption on a hydroxylapatite column and elution with a linear phosphate buffer gradient. With some stocks of E. coli cells, however, this method did not yield a homogeneous enzyme. An affinity resin was therefore prepared, which employs 6-APA as a ligand and Sepharose as a support. The activation agent is tosyl chloride.’ The procedures for resin preparation are the following: ( 1 ) dry Sepharose (corresponding to I30 g of wet resin) is added to a solution of tosyl chloride (18.5 g in 37 ml acetone); (2) the mixture is agitated while 18 ml of pyridine are added dropwise; (3) after I hour at room temperature, the resin is washed with 0.1 M borate buffer, pH

[49] 6-Phosphogluconate dehydrogenase from Bacillus stearothermophilus

Publisher Summary The enzyme 6-phosphogluconate dehydrogenase plays a key role in the pentose phosphate cycle because it catalyzes the reversible oxidative decarboxylation of 6-phospho-D-gluconate to yield D-ribulose 5-phosphate and CO 2 , with nicotinamide adenine dinucleotide phosphate (NADP) reduced to nicotinamide adenine dinucleotide phosphate dehydrogenase (NADPH). This chapter describes a purification procedure for 6-PGDH from the thermophilic microorganism, B. stearotherrnophilus , which shows an optimum growth temperature near 60°C. The method of the determination of enzymic activity is based on the spectrophotometric measurement of NADPH formed during the oxidative decarboxylation of the substrate. The reaction is initiated by the addition of the enzyme and followed by the rate of NADPH formation at 340 nm using a thermostatted spectrophotometer. The steps involved in the purification procedure are: (1) ammonium sulfate fractionation, (2) diethylaminoethyl (DEAE)-cellulose column chromatography, (3) hydroxyapatite column chromatography, (4) phosphocellulose column chromatography, and (5) gel filtration with BioGel A-0.5m. The purified enzyme is foundto be homogeneous on alkaline polyacrylamide gels at pH 8.5.

Isolation and some Properties of Enolase from Bacillus Stearothermophilus

Several enzymes from the obligate thermophile B.stearothermophilus are under active investigation in several laboratories, in order to ascertain whether a general explanation can be given of the enhanced stability toward heat of thermophilic enzymes. We are currently involved in physico-chemical analyses of several enzymes from B.stearothermophilus and, in our multi-enzyme separation procedure from this bacterial source, we have also isolated enolase (EC 4.2,1.11). It seemed to us of interest to verify whether the enzyme from this moderately thermostable microorganism shows a dimeric structure similar to enolase from all mesophilic sources, from microorganisms to vertebrates (1), or an octameric structure as shown for the enzyme from two extremely thermophilic microorganisms Thermus X-1 and Thermus aquaticus YT-1 recently isolated by Stellwagen and coworkers (2,3).