Purification, characterization, and biological cytotoxic activity of the extracellular cholesterol oxidase produced by Castellaniella sp. COX.

A new bacterial strain producing extracellular cholesterol oxidase (ChOx) was isolated and identified as Castellaniella sp. COX. The ChOx was purified by salting-out and ion-exchange chromatography up to 10.4-fold, with a specific activity of 15 U/mg with a molecular mass of 59 kDa. The purified ChOx exhibited pH 8.0 and temperature 40°C for its optimum activity. The enzyme showed stability over a wide pH range and was most stable at pH value 7.0, and at pH 8.0, it retained almost 86% of its initial activity after 3 h of incubation at 37°C. The enzyme possessed a half-life of 8 h at 37°C, 7 h at 40°C, and 3 h at 50°C. A Lineweaver-Burk plot was calibrated to determine its Km (0.16 mM) and Vmax (18.7 µmol·mg-1 ·min-1 ). The ChOx activity was enhanced with Ca2+ , Mg2+ , and Mn2+ while it was inhibited by Hg2+ , Ba2+ , Fe2+ , Cu2+ , and Zn2+ ions. Organic solvents like acetone, n-butanol, toluene, dimethyl sulfoxide, chloroform, benzene, and methanol were well tolerated by the enzyme while iso-propanol and ethanol were found to enhance the activity of purified ChOx. ChOx induced cytotoxicity with an IC50 value of 1.78 and 1.88 U/ml against human RD and U87MG established cell lines, respectively, while broadly sparing the normal human cells.

Purification, characterization and amino acid content of cholesterol oxidase produced by Streptomyces aegyptia NEAE 102.

BACKGROUND: There is an increasing demand on cholesterol oxidase for its various industrial and clinical applications. The current research was focused on extracellular cholesterol oxidase production under submerged fermentation by a local isolate previously identified as Streptomyces aegyptia NEAE 102. The crude enzyme extract was purified by two purification steps, protein precipitation using ammonium sulfate followed by ion exchange chromatography using DEAE Sepharose CL-6B. The kinetic parameters of purified cholesterol oxidase from Streptomyces aegyptia NEAE 102 were studied. RESULTS: The best conditions for maximum cholesterol oxidase activity were found to be 105 min of incubation time, an initial pH of 7 and temperature of 37 °C. The optimum substrate concentration was found to be 0.4 mM. The higher thermal stability behavior of cholesterol oxidase was at 50 °C. Around 63.86% of the initial activity was retained by the enzyme after 20 min of incubation at 50 °C. The apparent molecular weight of the purified enzyme as sized by sodium dodecyl sulphate-polyacryalamide gel electrophoresis was approximately 46 KDa. On DEAE Sepharose CL-6B column cholesterol oxidase was purified to homogeneity with final specific activity of 16.08 U/mg protein and 3.14-fold enhancement. The amino acid analysis of the purified enzyme produced by Streptomyces aegyptia NEAE 102 illustrated that, cholesterol oxidase is composed of 361 residues with glutamic acid as the most represented amino acid with concentration of 11.49 µg/mL. CONCLUSIONS: Taking into account the extracellular production, wide pH tolerance, thermal stability and shelf life, cholesterol oxidase produced by Streptomyces aegyptia NEAE 102 suggested that the enzyme could be industrially useful.

Purification and characterisation of the extracellular cholesterol oxidase enzyme from Enterococcus hirae.

BACKGROUND: Recently many efforts are being carried out to reduce cholesterol in foods. Out of the 50 selected isolates that were tested using the agar well diffusion method to assess their ability to decompose cholesterol, 24 bacterial isolates were screened based on their cholesterol-decomposition ability in liquid media. RESULTS: The bacterial isolate that displayed the highest cholesterol oxidase activity was identified as Enterococcus hirae. The maximal growth and cholesterol decomposition were achieved with a 1-day incubation under static conditions at 37 °C in cholesterol basal medium adjusted to pH 7 supplemented with 1 g/l cholesterol as the substrate, no additional carbon or nitrogen sources and 0.5 % CaSO4. The cholesterol oxidase enzyme (ChoX) produced by E. hirae was extracted at an (NH4)2SO4 saturation level of 80 % and purified with 79 % yield, resulting in 2.3-fold purification. The molecular weight of (ChoX) was 60 kDa. The optimal conditions required for the maximal activity of the purified COD enzyme produced by E. hirae were 30 min, 40 °C, pH 7.8, substrate concentration of 1 g/l and 200 ppm of MgCl2. The enzyme maintained approximately 36 % and 58.5 % of its activity after 18 days of storage at 4-8 °C. Also, the enzyme loss its activity by gradual thermal treatment, but it maintained 58.5 % of its activity at 95 °C for 2 hr. CONCLUSIONS: E. hirae Mil-31 isolated from milk had a great capacity to decompose cholesterol in basal medium supplemented with cholesterol under its optimal growth conditions. Decomposition process of cholesterol by this strain results from its production of cholesterol oxidase enzyme (ChoX). The highest specific enzyme activity and highest purification fold of purified enzyme were achieved after using Sephadex G-100.

Assessment of alkaline cholesterol oxidase purified from Rhodococcus sp. PKPD-CL for its halo tolerance, detergent and organic solvent stability.

The novel bacterium, Rhodococcus sp. PKPD-CL was isolated and identified from the 'Chilika Lake' located at Odisha state of India, which is a largest brackish water habitat in Asia. Rhodococcus sp. PKPD-CL produces extracellular halo tolerant, detergent and organic solvent stable alkaline cholesterol oxidase. It has apparent molecular weight of 60 kDa and was purified 59 fold by using 60% saturated ammonium sulfate fractionation, anion exchange followed by size exclusion chromatographic techniques with 37% recovery. It showed substrate specificity for 3ß-hydroxysteroids with Km of 1.1 × 10(-4)M for cholesterol. The pH, 8.0 and the temperature, 37 °C were required for its optimum activity. Enzyme is considerably stable at pH 6.0-8.5 and temperature up to 50 °C. At 4 and 30 °C it maintained its 100% activity up to 60 days. The isoelectric point of the enzyme was 9.5. It showed 80% residual activity with 20% NaCl (3.42 M) and 83% relative activity with 12% NaCl (2.05 M) concentration. The metal ions like Zn(2+), Cu(2+), Ag+, Fe(3+), Ba(2+) inhibited the enzyme activity >60% while Hg(2+) served a potent inhibitor whereas Mg(2+) found to be a good enhancer for it. The enzyme was stable in presence of chemical reagents (NaN3, EDTA), detergents (Tween-80, Tween-20, Triton X-100, sodium cholate) and various organic solvents (isopropanol, ethanol, benzene, chloroform, methanol, toluene, ethyl acetate, butanol and dimethylsulfoxide). Such a multi stress tolerant and versatile enzyme produced by Rhodococcus sp. PKPD-CL may serve as a good choice for industrial applications.

Production, purification and characterization of cholesterol oxidase from a newly isolated Streptomyces sp.

Cholesterol oxidase production (COD) by a new isolate characterized as Streptomyces sp. was studied in different production media and fermentation conditions. Individual supplementation of 1 % maltose, lactose, sucrose, peptone, soybean meal and yeast extract enhanced COD production by 80-110 % in comparison to the basal production medium (2.4 U/ml). Supplementation of 0.05 % cholesterol (inducer) enhanced COD production by 150 %. COD was purified 14.3-fold and its molecular weight was found to be 62 kDa. Vmax (21.93 µM/min mg) and substrate affinity Km (101.3 µM) suggested high affinity of the COD for cholesterol. In presence of Ba(2+) and Hg(2+) the enzyme activity was inhibited while Cu(2+) enhanced the activity nearly threefold. Relative activity of the enzyme was found maximum in triton X-100 whereas sodium dodecyl sulfate inactivated the enzyme. The enzyme activity was also inhibited by the thiol-reducing reagents like Dithiothreitol and ß-mercaptoethanol. The COD showed moderate stability towards all organic solvents except acetone, benzene and chloroform. The activity increased in presence of isopropanol and ethanol. The enzyme was most active at pH 7 and 37 °C temperature. This organism is not reported to produce COD.

Cholesterol degradation by Gordonia cholesterolivorans.

This paper reports physiological and genetic data about the type strain Gordonia cholesterolivorans, a strain that is able to degrade steroid compounds containing a long carbon side chain such as cholesterol (C(27)), cholestenone (C(27)), ergosterol (C(28)), and stigmasterol (C(29)). The length of the carbon side chain appears to be of great importance for this bacterium, as the strain is unable to grow using steroids with a shorter or nonaliphatic carbon side chain such as cholic acid (C(24)), progesterone (C(21)), testosterone, androsterone, 4-androstene-3,17-dione (all C(19)), and further steroids. This study also demonstrates that the degradation of cholesterol is a quite common feature of the genus Gordonia by comparing Gordonia cholesterolivorans with some other species of this genus (e.g., G. sihwensis, G. hydrophobica, G. australis, and G. neofelifaecis). Pyrosequencing of the genome of G. cholesterolivorans led to the identification of two conventional cholesterol oxidase genes on an 8-kb and a 12.8-kb genomic fragment with genetic organizations that are quite unique as compared to the genomes of other cholesterol-degrading bacteria sequenced so far. The identified two putative cholesterol oxidases of G. cholesterolivorans are both intracellularly acting enzymes of the class I type. Whereas one of these two cholesterol oxidases (ChoOx-1) shows high identity with an oxidoreductase of the opportunistic pathogen G. bronchialis and is not transcribed during growth with cholesterol, the other one (ChoOx-2) appears phylogenetically closer to cholesterol oxidases from members of the genus Rhodococcus and is transcribed constitutively. By using targeted gene disruption, a G. cholesterolivorans ChoOx-2 gene mutant strain that was unable to grow with steroids was obtained.

Preparation and characterization of affinity sorbents based on isoalloxazine-like ligands for separation of flavoenzymes.

Affinity ligands for flavoenzymes were synthesized based on the natural structure of flavo-coenzymes. Two typical flavoenzymes, cholesterol oxidase from Brevibacterium sp. and xanthine oxidase from bovine milk, were employed as standard enzymes. Fluorescent probes were synthesized from eight isoalloxazine-like chemicals and 5-aminofluorescein. Probe-enzyme interactions were analyzed via fluorescence spectra. Chemicals with high binding abilities to flavoenzymes were coupled with Sepharose through spacers composed of epichlorohydrin, ethylenediamine, 1,3-diaminopropane, 2-hydroxy-1,3-diaminopropane, and 1,4-diaminobutane, and subjected to adsorption analysis with flavoenzymes. The results indicated that ligands synthesized from 2,4-dioxohexahydropyrimidine-5-carboxylic acid, cytosine, 7-chloroalloxazine, and 8-chloroalloxazine had high binding abilities to the flavoenzymes. The affinity sorbent based on these ligands revealed a high theoretical maximum adsorption (Q(max)). Protein and bioactivity recoveries were tested after one step of affinity binding via chromatographic analysis on small columns. Results showed that ligands linked with sorbents through long hydrophilic spacers had higher activity recoveries.

Production of recombinant cholesterol oxidase containing covalently bound FAD in Escherichia coli.

BACKGROUND: Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO). RESULTS: Design of the plasmid construct encoding the mature BCO, optimization of medium composition and identification of the best cultivation/induction conditions for growing and expressing the active protein in recombinant E. coli cells, concurred to achieve a valuable improvement: BCO volumetric productivity was increased from approximately 500 up to approximately 25000 U/L and its crude extract specific activity from 0.5 up to 7.0 U/mg protein. Interestingly, under optimal expression conditions, nearly 55% of the soluble recombinant BCO is produced as covalently FAD bound form, whereas the protein containing non-covalently bound FAD is preferentially accumulated in insoluble inclusion bodies. CONCLUSIONS: Comparison of our results with those published on non-covalent (type I) COs expressed in recombinant form (either in E. coli or Streptomyces spp.), shows that the fully active type II BCO can be produced in E. coli at valuable expression levels. The improved over-production of the FAD-bound cholesterol oxidase will support its development as a novel biotool to be exploited in biotechnological applications.

Cholesterol oxidase from Rhodococcus erythropolis with high specificity toward ß-cholestanol and pytosterols.

Two genes (choRI and choRII) encoding cholesterol oxidases belonging to the vanillyl-alcohol oxidase (VAO) family were cloned on the basis of putative cholesterol oxidase gene sequences in the genome sequence data of Rhodococcus erythropolis PR4. The genes corresponding to the mature enzymes were cloned in a pET vector and expressed in Escherichia coli. The two cholesterol oxidases produced from the recombinant E. coli were purified to examine their properties. The amino acid sequence of ChoRI showed significant similarity (57%) to that of ChoRII. ChoRII was more stable than ChoRI in terms of pH and thermal stability. The substrate specificities of these enzymes differed distinctively from one another. Interestingly, the activities of ChoRII toward ß-cholestanol, ß-sitosterol, and stigmasterol were 2.4-, 2.1-, and 1.7-fold higher, respectively, than those of cholesterol. No cholesterol oxidases with high activity toward these sterols have been reported so far. The cholesterol oxidation products from these two enzymes also differed. ChoRI and ChoRII oxidized cholesterol to form cholest-4-en-3-one and 6ß-hydroperoxycholest-4-en-3-one, respectively.

Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase.

Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53-62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. K(m) and V(max) values of the cholesterol oxidase were estimated from Lineweaver-Burk plots. The V(max)/K(m) ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures.

Expression optimization, purification, and functional characterization of cholesterol oxidase from Chromobacterium sp. DS1.

Cholesterol oxidase is a bifunctional bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This valuable enzyme has attracted a great deal of attention because of its wide application in the clinical laboratory, synthesis of steroid derived drugs, food industries, and its potentially insecticidal activity. Therefore, development of an efficient protocol for overproduction of cholesterol oxidase could be valuable and beneficial in this regard. The present study examined the role of various parameters (host strain, culture media, induction time, isopropyl ß-D-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature) on over-expression of cholesterol oxidase from Chromobacterium sp. DS1. Applying the optimized protocol, the yield of recombinant cholesterol oxidase significantly increased from 92 U/L to 2115 U/L. Under the optimized conditions, the enzyme was produced on a large-scale, and overexpressed cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax values of the purified enzyme for cholesterol were estimated using Lineweaver-Burk plot. Further, the optimum pH and optimum temperature for the enzyme activity were determined. This study reports a straightforward protocol for cholesterol oxidase production which can be performed in any laboratory.

The binding and release of oxygen and hydrogen peroxide are directed by a hydrophobic tunnel in cholesterol oxidase.

The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, determined at sub-angstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To elucidate the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or tunnel residue Phe359 or Gly347 to Trp or Asn reduces the catalytic efficiency of oxidation. The K mO 2 increases from 300 +/- 35 microM for the wild-type enzyme to 617 +/- 15 microM for the F359W mutant. The k cat for the F359W mutant-catalyzed reaction decreases 13-fold relative to that of the wild-type-catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Transfer of hydride from the sterol to the flavin prosthetic group is no longer rate-limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively, is consistent with the cooperative kinetics. In the atomic-resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is observed in only a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.

Heterologous expression of cholesterol oxidase in Bifidobacterium longum under the control of 16S rRNA gene promoter of bifidobacteria.

We have constructed a constitutive high-level-expression vector for the genus Bifidobacterium and used it to express cholesterol oxidase from Streptomyces coelicola. The promoter region of the 16S rRNA gene was amplified by inverse PCR and used for the construction of pBES16PR. The optimal ribosome-binding site (RBS) for Bifidobacterium was incorporated in pBES16PR. In order to test the efficacy of this expression vector, we constructed pBES16PR-CHOL with the structural gene for cholesterol oxidase under the control of the 16S rRNA promoter, and used it to transform Bifidobacterium longum. The gene was successfully expressed and high level of cholesterol oxidase activity was obtained in B. longum. This is the first report of an expression vector for the genus Bifidobacterium using a 16S rRNA gene promoter and successful expression of cholesterol oxidase.

Response Surface Methodology-Genetic Algorithm Based Medium Optimization, Purification, and Characterization of Cholesterol Oxidase from Streptomyces rimosus.

The applicability of the statistical tools coupled with artificial intelligence techniques was tested to optimize the critical medium components for the production of extracellular cholesterol oxidase (COD; an enzyme of commercial interest) from Streptomyces rimosus MTCC 10792. The initial medium component screening was performed using Placket-Burman design with yeast extract, dextrose, starch and ammonium carbonate as significant factors. Response surface methodology (RSM) was attempted to develop a statistical model with a significant coefficient of determination (R2 = 0.89847), followed by model optimization using Genetic Algorithm (GA). RSM-GA based optimization approach predicted that the combination of yeast extract, dextrose, starch and ammonium carbonate at concentrations 0.99, 0.8, 0.1, and 0.05 g/100 ml respectively, has resulted in 3.6 folds increase in COD production (5.41 U/ml) in comparison with the un-optimized medium (1.5 U/ml). COD was purified 10.34 folds having specific activity of 12.37 U/mg with molecular mass of 54 kDa. The enzyme was stable at pH 7.0 and 40 °C temperature. The apparent Michaelis constant (Km) and Vmax values of COD were 0.043 mM and 2.21 µmol/min/mg, respectively. This is the first communication reporting RSM-GA based medium optimization, purification and characterization of COD by S. rimosus isolated from the forest soil of eastern India.

Coenzyme-like ligands for affinity isolation of cholesterol oxidase.

Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)µM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%.

Cholesterol oxidase with high catalytic activity from Pseudomonas aeruginosa: Screening, molecular genetic analysis, expression and characterization.

An extracellular cholesterol oxidase producer, Pseudomonas aeruginosa strain PA157, was isolated by a screening method to detect 6ß-hydroperoxycholest-4-en-3-one-forming cholesterol oxidase. On the basis of a putative cholesterol oxidase gene sequence in the genome sequence data of P. aeruginosa strain PAO1, the cholesterol oxidase gene from strain PA157 was cloned. The mature form of the enzyme was overexpressed in Escherichia coli cells. The overexpressed enzyme formed inclusion bodies in recombinant E. coli cells grown at 20 °C and 30 °C. A soluble and active PA157 enzyme was obtained when the recombinant cells were grown at 10 °C. The purified enzyme was stable at pH 5.5 to 10 and was most active at pH 7.5-8.0, showing optimal activity at pH 7.0 and 70 °C. The enzyme retained about 90% of its activity after incubation for 30 min at 70 °C. The enzyme oxidized 3ß-hydroxysteroids such as cholesterol, ß-cholestanol, and ß-sitosterol at high rates. The Km value and Vmax value for the cholesterol were 92.6 µM and 15.9 µmol/min/mg of protein, respectively. The Vmax value of the enzyme was higher than those of commercially available cholesterol oxidases. This is the first report to characterize a cholesterol oxidase from P. aeruginosa.

Rhodococcus erythropolis ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis, purification and concentration.

BACKGROUND: The suitability of the strain Rhodococcus erythropolis ATCC 25544 grown in a two-liter fermentor as a source of cholesterol oxidase has been investigated. The strain produces both cell-linked and extracellular cholesterol oxidase in a high amount, that can be extracted, purified and concentrated by using the detergent Triton X-114. RESULTS: A spray-dry method of preparation of the enzyme inducer cholesterol in Tween 20 was found to be superior in both convenience and enzyme synthesis yield to one of heat-mixing. Both were similar as far as biomass yield is concerned. Cell-linked cholesterol oxidase was extracted with Triton X-114, and this detergent was also used for purification and concentration, following temperature-induced detergent phase separation. Triton X-114 was utilized to purify and to concentrate the cell-linked and the extracellular enzyme. Cholesterol oxidase was found mainly in the resulting detergent-rich phase. When Triton X-114 concentration was set to 6% w/v the extracellular, but not the cell-extracted enzyme, underwent a 3.4-fold activation after the phase separation process. This result is interpreted in the light of interconvertible forms of the enzyme that do not seem to be in equilibrium. Fermentation yielded 360 U/ml (672 U/ml after activation), 36% of which was extracellular (65% after activation). The Triton X-114 phase separation step yielded 11.6-fold purification and 20.3-fold concentration. CONCLUSIONS: The results of this work may make attractive and cost-effective the implementation of this bacterial strain and this detergent in a purification-based industrial production scheme of commercial cholesterol oxidase.

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.

Hydrophobic-ionic chromatography: its application to microbial glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase.

Glucose oxidase from Aspergillus niger, hyaluronidase from Streptomyces hyalurolyticus, and cholesterol oxidase and cholesterol esterase from Pseudomonas fluorescens were effectively adsorbed on an Amberlite CG-50 column, when the cell-free cultured medium or the cultured medium with cell extract and without cell debris was applied without desalting but at pH less than or equal to 4.5. At the acidic pH, all the ion-exchange groups (-COOH) exist in the protonated form; the adsorption is not due to electrostatic attraction, but to hydrophobic interaction. The enzymes thus adsorbed were effectively eluted by increasing pH, at which the ion-exchange groups became dissociated. This type of adsorption-elution is called hydrophobic-ionic chromatography. By a single run of chromatography, glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase were purified 30-fold, 12-fold, 45-fold, and 20-fold with yields of 82%, 83%, 80%, and 90%, respectively. This indicates that hydrophobic-ionic chromatography on an Amberlite CG-50 column is effective for the purification of various enzymes, provided that they are stable at the acidic pH.

Cholesterol oxidase: sources, physical properties and analytical applications.

Since Flegg (H.M. Flegg, An investigation of the determination of serum cholesterol by an enzymatic method, Ann. Clin. Biochem. 10 (1973) 79-84) and Richmond (W. Richmond, The development of an enzymatic technique for the assay of cholesterol in biological fluids, Scand. J. clin. Lab. Invest. 29 (1972) 25; W. Richmond, Preparation and properties of a bacterial cholesterol oxidase from Nocardia sp. and its application to enzyme assay of total cholesterol in serum, Clinical Chemistry 19 (1973) 1350-1356) first illustrated the suitability of cholesterol oxidase (COD) for the analysis of serum cholesterol, COD has risen to become the most widely used enzyme in clinical laboratories with the exception of glucose oxidase (GOD). The use is widespread because assays incorporating the enzyme are extremely simple, specific, and highly sensitive and thus offer distinct advantages over the Liebermann-Burchard analytical methodologies which employ corrosive reagents and can be prone to unreliable results due to interfering substances such as bilirubin. Individuals can now readily determine their own serum cholesterol levels with a simple disposable test kit. This review discusses COD in some detail and includes the topics: (1) The variety of bacterial sources available; (2) The various extraction/purification protocols utilised in order to obtain protein of sufficient clarification (purity) for use in food/clinical analysis; (3) Significant differences in the properties of the individual enzymes; (4) Substrate specificities of the various enzymes; (5) Examples of biological assays which have employed cholesterol oxidase as an integral part of the analysis, and the various assay protocols; (6) New steroidal products of COD. This review is not a comprehensive description of published work, but is intended to provide an account of recent and current research, and should promote further interest in the application of enzymes to analytical selectivity.