The plot of kcat/Km against pH for the Bacillus cereus 569/H beta-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pKa 5.6+/-0.2 and one of pKa 9.5+/-0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pKa of 5.6+/-0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a Ki of 70 microM. The pH-dependence of Ki shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of Ki between pH 6 and 9 indicates that the pKa of zinc(II)-bound water must be 5.6 and not the higher pKa of 9.5. The kinetic solvent isotope effect on kcat/Km is 1.3+/-0.5 and that on kcat is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B beta-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.

Experimental evidence for a [2 + 2] mechanism in the Lewis acid-promoted formation of a,b-unsaturated esters from ethoxyacetylene and aldehydes. Synthesis and characterisation of 4-ethoxyoxetes.

No abstract

The potential of blackcurrant pomace as a raw material for the extraction of dietary fibre was evaluated using two pomaces one sourced from the UK and one from Poland.A fractionation protocol was designed to isolate and subsequently quantify the soluble and insoluble dietary fibre fractions. Blackcurrant pomace and isolated pectins, hemicelluloses and celluloses were assessed by means of sugar compositional analysis, spectroscopy, size exclusion chromatography and dilute solution viscometry. The blackcurrant pomaces presented considerable amounts of dietary fibre with soluble fibre ranging from 25-30% w/w and insoluble dietary fibre accounting for about 47% w/w for both pomaces. Blackcurrant pomaces differed in the amount of extracted pectins with an almost two times higher pectin yield obtained from blackcurrant pomace sourced from Poland. The hemicellulosic polysaccharide content was 15% w/w whereas the amount of cellulosic fraction varied from 14-17% w/w. Pectins isolated from both blackcurrant pomaces were LM pectins with a degree of esterification in the range of 11-38%. The work has identified that dietary fibres obtained from blackcurrant pomace had desirable ratio of insoluble to soluble fibre and are a potential new source of dietary fibre.

48 49Pectin was isolated by aqueous extraction at pH 6.0 or 2.0 from okra 50 (Abelmoschus esculentus L.) pods. An isolation protocol was designed to extract 51 pectin and to study the influence of the extraction pH on their composition and 52 physicochemical properties. The extracted pectin was assessed using sugar 53 compositional analysis (neutral sugars, galacturonic acid, acetyl and methyl contents). 54FT-IR and NMR spectroscopy, size exclusion chromatography (SEC) and dilute 55 solution viscometry were also used to determine the macromolecular characteristics of 56 isolated pectin. The extraction protocols resulted in the isolation of pectin of high 57 purity as evidenced by their high total carbohydrate (70.0 -81.8%) and low protein 58 (4.3 -6.3%) contents. Samples contained between 46-56% galacturonic acid, had 59 broad molecular weight distributions, a low degree of methylation (40.0 and 24.6 %) 60 and high degree of acetylation (52.2 and 37.6 %). Neutral sugar analysis showed that 61 the pectin extracted at pH 6.0 contained more neutral sugars, particularly, galactose 62 (21.7 -25.7 mol%), rhamnose (10.1 -13.2 mol%) and arabinose (7.1 -7.3 mol%) 63 than that extracted at pH 2.0 indicating variations in fine structure. In addition, 64 molecular parameters of the isolated pectins, such as intrinsic viscosity (2.8 -4.4 dL 65 g -1 ), critical concentration (0.15 -0.45 dL g -1 ) and coil overlap parameter (0.66 -66 1.51), showed that extraction conditions resulted in pectin with different chain 67 morphology. The yield and physico-chemical characteristics of the extracted pectin 68 from okra pods were influenced by the extraction conditions. 69 70 71