Preparation of autoxidized cholesterol mixtures from [4-14C] cholesterol and cholesteryl fatty acid esters

Autoxidation of non-esterified cholesterol, in the solid state, at 100 degrees C, is known to be a relatively slow reaction. The presence of carefully chosen cholesteryl esters considerably increases the ratio of autoxidation. Using this method, mixtures of autoxidized free cholesterol (oxycholesterol) labelled on carbon 4 can be obtained almost quantitatively, in the presence of benzoyl peroxide. The ratio of [4-14C] ester produced by transesterification at the end of the reaction is about 10%.     

Preparation of L-tyrosine-ring- 14 C, L-DOPA-ring- 14 C and related metabolites

The reversibility of the tyrosine phenol-lyase reaction has been utilized to develop a simple system in which phenol-¹⁴C is incorporated into l-tyrosine in high yield. By use of mushroom tyrosinase, catechol-¹⁴C can be prepared from phenol-¹⁴C and l-DOPA-¹⁴C from l-tyrosine-¹⁴C. Catechol-¹⁴C can also be incorporated into l-DOPA-¹⁴C by use of tyrosine phenol-lyase, giving the possibility of preparing DOPA with two labeling patterns in the ring when starting with phenol-¹⁴C. Two further tyrosine metabolites, para-coumaric acid and homogentisic acid, have also been enzymatically prepared with ¹⁴C in the ring.     

Preparation and some properties of cholesterol oxidase from Rhodococcus sp. R14-2

Rhodococcus sp. R_14-2, isolated from Chinese Jin-hua ham, produces a novel extracellular cholesterol oxidase (COX). The enzyme was extracted from fermentation broth and purified 53.1-fold based on specific activity. The purified enzyme shows a single polypeptide band on SDS-PAGE with an estimated molecular weight of about 60 kDa, and has a pI of 8.5. The first 10 amino acid residues of the NH₂-terminal sequence of the enzyme are A-P-P-V-A-S-C-R-Y-C, which differs from other known COXs. The enzyme is stable over a rather wide pH range of 4.0–10.0. The optimum pH and temperature of the COX are pH 7.0 and 50°C, respectively. The COX rapidly oxidizes 3β-hydroxysteroids such as cholesterol and phytosterols, but is inert toward 3α-hydroxysteroids. Thus, the presence of a 3β-hydroxyl group appears to be essential for substrate activity. The Michaelis constant (Km) for cholesterol is estimated at 55 μM; the COX activity was markedly inhibited by metal ions such as Hg²⁺ and Fe³⁺ and inhibitors such as p-chloromercuric benzoate, mercaptoethanol and fenpropimorph. Inhibition caused by p-chloromercuric benzoate, mercuric chloride, or silver nitrate was almost completely prevented by the addition of glutathione. These suggests that -SH groups may be involved in the catalytic activity of the present COX.     

Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl–sepharose chromatography. The estimated molecular mass (SDS-PAGE) and K_m of the enzyme were ∼55.0 kDa and 151 μM, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45 °C, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe²⁺ and Pb²⁺ at 0.1 mM of each acted as inhibitors, while Ag⁺, Ca²⁺, Ni²⁺ and Zn²⁺ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.     

The chemical synthesis and biological oxidation of 7α-hydroxy[26-14C]cholesterol, 7-dehydro[26-14C]cholesterol and 26-hydroxy[26-14C]cholesterol

1. 26-Hydroxycholesterol was obtained by reducing the methyl ester of (±)-3β-hydroxycholest-5-en-26-oic acid, which was synthesized from 25-oxonorcholesterol. 2. Methods for preparing 7α-hydroxycholesterol and 7-dehydrocholesterol were modified to allow the micro-scale preparation of these [¹⁴C]sterols from [26-¹⁴C]-cholesterol. 3. 26-Hydroxycholesterol was oxidized more readily than 7α-hydroxycholesterol, 7-dehydrocholesterol or cholesterol by mitochondrial preparations from livers of mice, rats, guinea pigs, common toads (Bufo vulgaris) and Caiman crocodylus. 4. (±)-3β-Hydroxy[26-¹⁴C]cholest-5-en-26-oic acid was oxidized very rapidly to ¹⁴CO₂ by mouse and guinea-pig mitochondria without evident discrimination between the two optical isomers. 5. An enzyme system that oxidizes 26-hydroxycholesterol to 3β-hydroxycholest-5-en-26-oic acid was identified in the soluble extract of rat-liver mitochondria. This enzyme could use NADP in place of NAD but was not identical with liver alcohol dehydrogenase (EC 1.1.1.1). 6. [26-¹⁴C]Cholesteryl 3β-sulphate was not oxidized by fortified mouse-liver preparations that oxidized [26-¹⁴C]cholesterol to ¹⁴CO₂.     

Preparation of Radioactive Starch, Glucose and Fructose from C14O2

Radioactive starch, glucose and fructose have been preparedfrom tobacco leaves after assimilation of C¹⁴O₂. The apparatusused for photosynthesis consisted of a shallow Perspex leafchamber connected to a closed gas system, in which C¹⁴O₂ wasgenerated from BaC¹⁴O₂. Six leaves, area 14 to 18 sq. dm. whenexposed to bright sunlight with an initial CO₂ concentrationof 8 to 10 per cent., assimilated 3.35 g. of C¹⁴O₂ in 8 to 10hours. At least 80 per cent. of the C¹⁴O₂ supplied appearedin the leaves as starch and sugar and over 80 per cent. of theradioactivity was accounted for in these carbohydrates. Thespecific activity per m. atom of carbon of the isolated productswas 85 to 90 per cent. of that of the C¹⁴O₂. Small amounts ofradioactive carbon were also incorporated in the leaf proteinand in the celluose, hemicellulose and polyuronides.