Efficient production of active recombinant cholesterol oxidase from Rhodococcus erythropolis in Escherichia coli via his-tag assisted refolding strategy for cholesterol oxidation.

Classical (one-variable-at-a-time) and statistical methods (Plackett-Burman and Central composite design) were used to optimise growth medium for the production of cholesterol oxidase (COX) from Rhodococcus sp. NCIM 2891. COX activities from the classically and statistically optimised media were 0.75 and 3.25 U/ml, respectively. The statistically optimised medium had 4.33- and 9.7-fold higher enzymatic activity than the classically optimised and un-optimised basal medium, respectively. The ratio of enzyme production to cell growth rate was 29-fold higher in our statistically optimised medium than in the basal medium, indicating that the enzyme production could be classified as mixed type of growth. Cell-bound COX accounted for 90.68 ± 2 % of the total enzymatic activity of the growth medium. Interactions between the COX-inducing substrate cholesterol and medium growth substrates yeast extract and (NH4)2HPO4 significantly enhanced the production of cell-bound COX. Our results validate the statistical approach as a potential technique for achieving the large-scale production of cell-bound COX from Rhodococcus sp. NCIM 2891.

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 β-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 oxidase (CO) was successfully produced from Streptomyces sp. via the submerged fermentation method, and 69 U/mL enzyme activity was obtained. This study aimed to determine cholesterol oxidation kinetics and the production of CO as a catalyst. The enzyme was diluted to 0.15, 0.075, and 0.00375 U/mL for the oxidation reaction. The substrate was also prepared in three concentrations: 3.23, 6.46, and 12.93 mM. The optimization of conditions for enzymatic cholesterol oxidation was investigated through measurement of the effect of initial cholesterol and enzyme concentrations. Cholesterol concentration was rapidly measured via high-performance liquid chromatography (HPLC). The kinetics of CO were modeled using the first-order irreversible reaction. An enzymatic kinetic model was derived, and it was verified using experimental data and sensitivity analysis. Based on the experiment, the highest enzyme concentrations of crude and commercial CO can oxidize the substrate up to 84% within 240 min. However, the oxidation reaction showed a slightly different behavior in the early 60 min, and crude CO exhibited a slower substrate oxidation. The kinetic rate constant obtained by Euler’s method reached 1.0 x 10−3/min and 1.41 x 10−3/min for 0.15 U/mL crude and commercial CO, respectively.

BackgroundCholesterol oxidase biosensors have been used to determine the level of cholesterol in different serum and food samples. Due to a wide range of industrial and clinical applications of microbial cholesterol oxidase, isolation and identification of a new microbial source (s) of cholesterol oxidase are very important.ResultsThe local isolate Streptomyces sp. strain NEAE-94 is a promising source of cholesterol oxidase. It was identified based on cultural, morphological and physiological characteristics; in addition to the 16S rRNA sequence. The sequencing product had been deposited in the GenBank database under the accession number KC354803. Cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 in shake flasks was optimized using surface response methodology. The different process parameters were first screened using a Plackett-Burman design and the parameters with significant effects on the production of cholesterol oxidase were identified. Out of the 15 factors screened, agitation speed, cholesterol and yeast extract concentrations had the most significant positive effects on the production of cholesterol oxidase. The optimal levels of these variables and the effects of their mutual interactions on cholesterol oxidase production were determined using Box-Behnken design. Cholesterol oxidase production by Streptomyces anulatus strain NEAE-94 was 11.03, 27.31 U/mL after Plackett-Burman Design and Box-Behnken design; respectively, with a fold of increase of 6.06 times compared to the production before applying the Plackett-Burman design (4.51 U/mL).ConclusionsMaximum cholesterol oxidase activity was obtained at the following fermentation conditions: g/L (cholesterol 4, yeast extract 5, NaCl 0.5, K2HPO4 1, FeSO4.7H2O 0.01, MgSO4.7H2O 0.5), pH 7, inoculum size 4% (v/v), temperature 37°C, agitation speed of 150 rpm, medium volume 50 mL and incubation time 5 days.

Effects of enzyme concentration, substrate concentration and residence time on substrate conversion, reactor capacity and productivity were evaluated. As enzyme concentration increased from 1 to 6 g/L, substrate conversion also increased. Substrate conversion was essentially unchanged at enzyme concentrations greater than 6 g/L. Reactor capacity was significantly affected by both substrate and enzyme concentration. As residence time increased, substrate conversion increased 15–22%. Productivities of the continuous reactor were 10 to 20 times greater than those obtained in a batch reactor. The continuous membrane reactor was capable of producing a highly pure glucose syrup at low residence times. Due to the high levels of substrate conversion, minimal operational problems were experienced.

Rhodococcus equi No. 23 was grown in a batch fermenter. The effects of cultivation temperature, pH of the culture medium, aeration rate and agitation speed on the production of cholesterol oxidase (CholOx) by the test organism were examined. Results revealed that the cultivation temperature, the pH of the medium, the aeration rate and the agitation speed all affected the production of CholOx by R. equi No. 23. Adjusting the operation variables during the cultivation period increased the production of CholOx effectively and prevented the occurrence of overflow of foam during the fermentation period. A maximum CholOx activity of 0.34 unit/ml with a volumetric production rate of 0.011 unit/h per ml could be achieved in 30 h of cultivation at an aeration rate of 5.0 l/min, if the pH of the culture medium, the cultivation temperature and the agitation speed were controlled at 6.5, 39 degrees C and 200 rev. /min respectively during the first 24 h of cultivation, then shifted to 7.5, 37 degrees C and 300 rev./min respectively.

Cholesterol oxidase is a bacterial flavoenzyme which catalyzes oxidation and isomerization of cholesterol. This enzyme has a great commercial value because of its wide applications in cholesterol analysis of clinical samples, synthesis of steroid-derived drugs, food industries, and potentially insecticidal activity. Accordingly, development of an efficient protocol for overexpression of cholesterol oxidase can be very valuable and beneficial. In this study, expression optimization of cholesterol oxidase from Streptomyces sp. SA-COO was investigated in Escherichia coli host strains. Various parameters that may influence the yield of a recombinant enzyme were evaluated individually. The optimal host strain, culture media, induction time, Isopropyl ß-d-1-thiogalactopyranoside concentration, as well as post-induction incubation time and temperature were determined in a shaking flask mode. Applying the optimized protocol, the production of recombinant cholesterol oxidase was significantly enhanced from 3.2 to 158 U/L. Under the optimized condition, the enzyme was produced on a large-scale, and highly expressed 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 also determined. We report a straightforward and easy protocol for cholesterol oxidase production which can be performed in any laboratory.

Production of cholesterol oxidase (COD) under batch conditions through Ca-alginate immobilized cells of Streptomyces sp. was investigated. The process was studied for optimal immobilization conditions, beads operational stability and comparisons were made with the COD production via free cells. Influence of Na-alginate concentration (1-5 g L(-1) ) and initial biomass loading on enzyme production were studied. Effects of initial pH of the production medium, temperature, shaker speed, as well as reuse of beads on the COD production were also investigated. It was observed that COD production with immobilized cells (5.6 U ml(-1) ) was higher in comparison to free cells (4.5 U ml(-1) ) under optimized conditions. The maximum COD production by free cells was observed with initial pH 7.0, rpm 200 after 96 h of incubation while immobilized cells sustain a broad pH range 6.0-9.0, rpm 300 for maximum production after 72 h. The immobilized and free cells produced maximum COD in the culture incubated at 37 and 30 °C, respectively. Other parameters bead size and Na-alginate concentration found to be optimum with 1.5 mm and 4% w/v, respectively. Scanning electron microscope study of the immobilized cells indicated that the cells in Ca-alginate beads remained in normal shape with no alterations in the morphology.

Due to broad range of clinical and industrial applications of cholesterol oxidase, isolation and screening of bacterial strains producing extracellular form of cholesterol oxidase is of great importance. One hundred and thirty actinomycete isolates were screened for their cholesterol oxidase activity. Among them, a potential culture, strain NEAE-42 is displayed the highest extracellular cholesterol oxidase activity. It was selected and identified as Streptomyces cavourensis strain NEAE-42. The optimization of different process parameters for cholesterol oxidase production by Streptomyces cavourensis strain NEAE-42 using Plackett–Burman experimental design and response surface methodology was carried out. Fifteen variables were screened using Plackett–Burman experimental design. Cholesterol, initial pH and (NH4)2SO4 were the most significant positive independent variables affecting cholesterol oxidase production. Central composite design was chosen to elucidate the optimal concentrations of the selected process variables on cholesterol oxidase production. It was found that, cholesterol oxidase production by Streptomyces cavourensis strain NEAE-42 after optimization process was 20.521U/mL which is higher than result obtained from the basal medium before screening process using Plackett-Burman (3.31 U/mL) with a fold of increase 6.19. The cholesterol oxidase level production obtained in this study (20.521U/mL) by the statistical method is higher than many of the reported values.

BackgroundDue to broad range of clinical and industrial applications of cholesterol oxidase, isolation and screening of bacterial strains producing extracellular form of cholesterol oxidase is of great importance.ResultsOne hundred and thirty actinomycete isolates were screened for their cholesterol oxidase activity. Among them, a potential culture, strain NEAE-42 is displayed the highest extracellular cholesterol oxidase activity. It was selected and identified as Streptomyces cavourensis strain NEAE-42. The optimization of different process parameters for cholesterol oxidase production by Streptomyces cavourensis strain NEAE-42 using Plackett–Burman experimental design and response surface methodology was carried out. Fifteen variables were screened using Plackett–Burman experimental design. Cholesterol, initial pH and (NH4)2SO4 were the most significant positive independent variables affecting cholesterol oxidase production. Central composite design was chosen to elucidate the optimal concentrations of the selected process variables on cholesterol oxidase production. It was found that, cholesterol oxidase production by Streptomyces cavourensis strain NEAE-42 after optimization process was 20.521U/mL which is higher than result obtained from the basal medium before screening process using Plackett-Burman (3.31 U/mL) with a fold of increase 6.19.ConclusionsThe cholesterol oxidase level production obtained in this study (20.521U/mL) by the statistical method is higher than many of the reported values.

Formation of mixed crystals in microbial conversion of sterols and steroids

This paper deals with the optimization of culture conditions for the production of cholesterol oxidase (COD) by Streptomyces lavendulae NCIM 2499 using the one-factor-at-a-time method, orthogonal array method and response surface methodology (RSM) approaches. The one-factor-at-a-time method was adopted to investigate the effects of medium components (i.e. carbon and nitrogen) and environmental factors (i.e. initial pH) on biomass growth and COD production. Subsequently, an L12 orthogonal matrix was used to evaluate the significance of glycerol, soyabean meal, malt extract, K2HPO4, MgSO4 and NaCl. The effects of media components were ranked according to their effects on the production of COD as malt extract > soyabean meal > K2HPO4 > NaCl > MgSO4 > glycerol. The subsequent optimization of the four most significant factors viz. malt extract, soyabean meal, K2HPO4 and NaCl, was carried out by employing a central composite rotatable design (CCRD) of RSM. There was a 2.48-fold increase in productivity of COD as compared to the unoptimized media by using these statistical approaches.

Inferring the evolutionary relationship of 23 Malaysian Rhodococcus isolates with potential as cholesterol degrading bacteria

Overexpression of recombinant proteins in bacterial systems (such as E. coli) often leads to formation of inactive and insoluble ' inclusion bodies' . Protein refolding refers to folding back the proteins after solubilizing/unfolding the misfolded proteins of the inclusion bodies. Protein aggregation, a concentration dependent phenomenon, competes with refolding pathway. The refolding strategies largely aim at reducing aggregation and/or promoting correct folding. This review focuses on non-chromatographic strategies for refolding like dilution, precipitation, three phase partitioning and macro-(affinity ligand) facilitated three phase partitioning. The nanomaterials which disperse well in aqueous buffers are also discussed in the context of facilitating protein refolding. Apart from general results with these methods, the review also covers the use of non-chromatographic methods in protein refolding in the patented literature beyond 2000. The patented literature generally describes use of cocktail of additives which results in increase in refolding yield. Such additives include low concentration of chaotropic agents, redox systems, ions like SO4(2-) and Cl-, amines, carboxylic acids and surfactants. Some novel approaches like use of a "pressure window" or ionic liquids for refolding and immobilized diselenide compounds for ensuring correct -S-S- bonds pairing have also been discussed in various patents. In most of the patented literature, focus naturally has been on refolding in case of pharmaceutical proteins.

BackgroundProduction of recombinant proteins in bacterial hosts often produces insoluble intracellular particles called inclusion bodies. Recovery of active protein from inclusion bodies generally requires their solubilization in chemical denaturants followed by a refolding strategy. The solubilization is carried out with shaking/stirring and takes several hours.ResultsUsing inclusion bodies of seven diverse kinds of recombinant proteins [mutants of controller of cell division or death protein B (CcdB), human CD4D12, thioredoxin fusion protein (malETrx), mutants of maltose binding protein (MBP), single chain variable fragment (ScFv) b12 and single chain antigen binding fragment (ScFab) b12 (anti-HIV-1)], it is shown that exposure to microwave irradiation (200 W) for 2 min, solubilized these inclusion bodies completely. This was confirmed by data based upon turbidity measurements at 400 nm and dynamic light scattering studies. These solubilized inclusion bodies could be refolded correctly in all the cases by known methods. The refolding was confirmed by fluorescence emission spectra and biological activity studies.ConclusionSolubilization of the inclusion bodies before refolding is a part of protein production processes for several recombinant proteins which are overexpressed in the bacterial host systems. Our results show that microwave assistance can considerably shorten the process time.