Cholesterol Oxidase Production Research Articles

Improved production of Bacillus subtilis cholesterol oxidase by optimization of process parameters using response surface methodology

BackgroundCholesterol oxidase has numerous biomedical and industrial applications. In the current study, a new bacterial strain was isolated from sewage and was selected for its high potency for cholesterol degradation (%) and production of high cholesterol oxidase activity (U/OD600). ResultsBased on the sequence of 16S rRNA gene, the bacterium was identified as Bacillus subtilis. The fermentation conditions affecting cholesterol degradation (%) and the activity of cholesterol oxidase (U/OD600) of B. subtilis were optimized through fractional factorial design (FFD) and response surface methodology (RSM). According to this sequential optimization approach, 80.152% cholesterol degradation was achieved by setting the concentrations of cholesterol, inoculum size, and magnesium sulphate at 0.05 g/l, 6%, and 0.05 g/l, respectively. Moreover, 85.461 U of cholesterol oxidase/OD600 were attained by adjusting the fermentation conditions at initial pH, 6; volume of the fermentation medium, 15 ml/flask; and concentration of cholesterol, 0.05 g/l. The optimization process improved cholesterol degradation (%) and the activity of cholesterol oxidase (U/OD600) by 139% and 154%, respectively. No cholesterol was detected in the spectroscopic analysis of the optimized fermented medium via gas chromatography-mass spectroscopy (GC–MS). ConclusionThe current study provides principal information for the development of efficient production of cholesterol oxidase by B. subtilis that could be used in various applications.

Production and statistical optimization of cholesterol-oxidase generated by Streptomyces sp. AN strain

BackgroundCholesterol oxidases (CHOs) have attracted enormous attention because of their wide biotechnological potential. The present study explores the production of CHOs by Streptomyces sp. AN. Evaluation of culture conditions affecting enzyme production, medium optimization and released metabolite characteristics were also investigated. ResultsThe current work reports the isolation of 37 colonies (bacteria/actinobacteria) with different morphotypes from different soil/water samples. The isolate-coded AN was selected for its high potency for CHO production. Morphological characteristics and the obtained partial sequence of 16srRNA of AN showed 99.38% identity to Streptomyces sp. strain P12–37. Factors affecting CHO production were evaluated using Plackett-Burman (PB) and Box-Behnken (BB) statistical designs to find out the optimum level of the most effective variables, namely, pH, starch, NH4NO3 and FeSO4.7H2O with a predicted activity of 6.56 U/mL. According to this optimization, the following medium composition was considered to be optimum (g/L): cholesterol 1, starch 6, MgSO4.7H2O 0.1, CaCl2 0.01, FeSO4.7H2O 0.1, NH4NO3 23.97, yeast extract (YE) 0.2, K2HPO4 0.01, KH2PO4 0.1, NaCl 0.01, Tween 20 0.01, pH 6.36 and incubation temperature (30 °C) for 9 days. Spectophotometric analysis for released metabolites against cholesterol (standard) via Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) was carried out. FTIR spectrum showed the appearance of new absorption peaks at 1644 and 1725cm−1; this confirmed the presence of the Keto group (C=O) stretch bond. Besides, fermentation caused changes in thermal properties such as melting temperature peak (99.26; 148.77 °C), heat flow (− 8; − 3.6 Mw/mg), capacity (− 924.69; − 209.77 mJ) and heat enthalpy (− 385.29; 69.83 J/g) by comparison to the standard cholesterol as recognized through DSC thermogram. These changes are attributed to the action of the CHO enzyme and the release of keto derivatives of cholesterol with different properties. ConclusionStreptomyces sp. AN was endowed with the capability to produce CHO. Enzyme maximization was followed using a statistical experimental approach, leading to a 2.6-fold increase in the overall activity compared to the basal condition. CHO catalyzed the oxidation of cholesterol; this was verified by the appearance of a new keto group (C=O) peak at 1644 and 1725 cm−1 observed by FTIR spectroscopic analysis. Also, DSC thermogram demonstrates the alteration of cholesterol triggered by CHO.

Enzymatic production of 4-cholesten-3-one using cholesterol oxidase from Bacillus cereus strain KAVK5 and its potentiality toward the inhibition of disease-associated proteins an in silico approach

Optimization of cholesterol oxidase production and 16S rRNA partial sequence of Bacillus cereus strain KAVK4 isolated from butterAmutha Kuppusamy, Kokila Vasantha Kumar

RESPONSE SURFACE METHODOLOGY BASED MEDIUM OPTIMIZATION FOR IMPROVEMENT OF CHOLESTEROL OXIDASE ENZYME PRODUCTION BY NEWLY ISOLATED STREPTOMYCES SP.

Cholesterol oxidase is an important enzyme with great commercial value in many clinical and industrial processes, such as determination of cholesterol levels in food, serum and other clinical samples as well as its potential application, as a biocatalyst for bioconversion of sterol compounds and as an insecticide. A Preliminary comparative study of cholesterol oxidase enzyme production from Streptomyces sp. strain NHIA_CH5 using six different media containing cholesterol in submerged fermentation was investigated. An increase in the production was detected after two days in all of the studied media with a maximum production of 23.7 U/mL, noticed in medium containing soluble starch, glucose, peptone, beef extract, MgSO4.7H2O, and NaCl. A classical (one-variable-at-a-time) method revealed that the elimination of MgSO4.7H2O and NaCl favored cholesterol oxidase enzyme production. Statistical optimization of the other components was studied using the screening method Plackett-Burman design followed by the modeling method of the central composite design (CCD). The results showed that beef extract and glucose are the variables that significantly influenced and considered as the most important factors for cholesterol oxidase production. Experiments of CCD based on response surface methodology determine the optimum concentrations of these significant medium components and revealed maximum cholesterol oxidase enzyme production (50.5 U/mL) at beef extract 55 g/L and glucose 40 g/L which represented 2- fold higher titers than acquired from the non-optimized medium.

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

Abstract Cholesterol is an integral molecule which is used as a building block to numerous biological structures. However, excessive amount of cholesterol in the body causes hypercholesterolemia. Microbial degradation of cholesterol has become a great interest and preferred method to degrade the compound because it is effective and environmental friendly. The genus of Rhodococcus is one of the microorganisms reported to be able to degrade cholesterol by its ability to produce cholesterol oxidase (CHO). Nevertheless, no documentation found so far on the cholesterol degradation by local Rhodococcus species. Thus, this study aims to evaluate the ability of local Rhodococcus isolates in degrading cholesterol. Twenty-three Malaysian Rhodococcus isolates were screened for cholesterol degradation activity using MSC agar which has revealed the ability of all Rhodococcus strains in degrading cholesterol with Rhodococus strain UCC0021 was observed as the best cholesterol degrader. The enzyme activity for CHO was measured and the highest enzyme activity by this bacteria strain was 1.180 U/mL at 72 h, which is 48% higher when compared with other actinobacteria. 16S rRNA sequencing was performed for species identification and phylogenic analysis. The finding in this study has proven the interesting ability of Malaysian Rhodococcus strains to degrade cholesterol and could be used as an alternative tool for cholesterol degradation through the production of cholesterol oxidase. Therefore, future work will be concentrated to enhance the production of enzyme activity through optimization of growth parameters and nutrient conditions by this Rhodococcus strain.

Identification of cholesterol-assimilating actinomycetes strain and application of statistical modeling approaches for improvement of cholesterol oxidase production by Streptomyces anulatus strain NEAE-94

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.

Medium Optimization by Response Surface Methodology for Improved Cholesterol Oxidase Production by a Newly Isolated Streptomyces rochei NAM-19 Strain.

Cholesterol oxidase is an alcohol oxidoreductase flavoprotein with wide biotechnological applications. The current work describes the isolation of a potential cholesterol oxidase producing streptomycete from Egyptian soil. The isolated strain produced cholesterol oxidase in submerged culture using a medium containing glucose, yeast extract, malt extract, and CaCO3 with the addition of cholesterol as an inducer. The isolated strain was identified as Streptomyces rochei NAM-19 based on 16S rRNA sequencing and phylogeny. Optimization of cholesterol oxidase production has been carried out using response surface methodology. The Plackett-Burman design method was used to evaluate the significant components of the production medium followed by Box-Behnken experimental design to locate the true optimal concentrations, which are significantly affecting enzyme production. Results showed that the predicted enzyme response could be closely correlated with the experimentally obtained production. Furthermore, the applied optimization strategy increased volumetric enzyme production by 2.55 times (65.1 U/mL) the initial production obtained before medium optimization (25.5 U/mL).

Characterization of cholesterol oxidase from a marine Streptomyces sp. and its cytotoxicity

Abstract A marine actinobacterial strain (designated as AKHSS) capable of producing cholesterol oxidase on the enzyme indicator plates was identified as Streptomyces sp. The cell-free lysate of the strain was used for monitoring the production of cholesterol oxidase and the maximal enzyme yields were recorded at 72 h post inoculation. The cholesterol oxidase was purified using polyethylene glycol 4000 precipitation, diethylaminoethyl Sephacel anionic column chromatography and Superdex-200 gel filtration to near homogeneity. Through electron-spray ionization mass spectrometry, molecular mass of the purified enzyme was recorded as 42.84 kDa. The optimum pH of the enzyme was found to be 9 and it was stable up to 60 °C. Metal salts like MgSO4 and ZnSO4 stimulated the enzyme activity. The Vmax and Km of the purified enzyme with cholesterol as substrate were found to be 1.22 μmoles/min/mL and 0.54 mM respectively. The enzyme showed significant cytotoxicity on breast (MCF-7), nasopharyngeal (KB) and ovarian (OVCAR) cancer cell lines at very low concentrations ranging from 0.093 to 0.14 μM, as evident from MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell viability assay. Besides, the enzyme exhibited relatively less cytotoxicity on primary mouse embryonic fibroblast (3T3) cells. Thus, cholesterol oxidase of Streptomyces sp. AKHSS could be a potential anticancer agent.

Optimization of Process Parameters for Cholesterol Oxidase Production by Streptomyces Olivaceus MTCC 6820

Background:Streptomyces olivaceusMTCC 6820 is a potent microorganism for cholesterol oxidase (ChOx) production through the submerged fermentation process. Statistical optimization of the process parameters for submerged fermentation enhances the production of enzymes.Objective:This work is aimed to optimize the culture conditions for the fermentative production of cholesterol oxidase byStreptomyces olivaceusMTCC 6820 using combined Response Surface Methodology (RSM) and Artificial Neural Network (ANN) techniques.Methods:The ChOx production (U/ml) was modeled and optimized as a function of six independent variables (culture conditions) using RSM and ANN.Results:ChOx production enhanced 2.2 fold,i.e1.9 ± 0.21 U/ml under unoptimized conditions to 4.2 ± 0.51 U/ml after the optimization of culture conditions. Higher coefficient of determination (R2= 97.09 %) for RSM and lower values of MSE (0.039) and MAPE (3.46 %) for ANN proved the adequacy of both the models. The optimized culture conditions predicted by RSMvs. ANN were pH (7.5), inoculum age (48 h), inoculum size (11.25 % v/v), fermentation period (72 h), incubation temperature (30°C) and shaking speed (175 rpm).Conclusion:The modeling, optimization and prediction abilities of both RSM and ANN methodologies were compared. The values of Pearson correlation coefficient (r) (ANN0.98> RSM0.95), regression coefficient (R2) between experimental activity, RSM and ANN predicted ChOx activity, respectively (ANN0.96> RSM0.90) and Absolute Average Deviation (AAD) for (ANN3.46%< RSM9.87%) substantiated better prediction ability of ANN than RSM. These statistical values indicated the superiority of ANN in capturing the non-linear behavior of the system.

Cholesterol Degradation and Production of Extracellular Cholesterol Oxidase from Bacillus pumilus W1 and Serratia marcescens W8.

Cholesterol is a waxy substance present in all types of the body cells. The presence of higher concentration of low density lipoprotein (LDL) is characterized by abnormal cholesterol level and is associated with cardiovascular diseases which lead to the development of atheroma in arteries known as atherosclerosis. The transformation of cholesterol by bacterial cholesterol oxidase can provide a key solution for the treatment of diseases related to cholesterol and its oxidized derivatives. Previously isolated bacteria from oil-contaminated soil were screened for cholesterol degradation. Among fourteen, five isolates were able to utilize cholesterol. Two strains Serratia marcescens W1 and Bacillus pumilus W8 using cholesterol as only carbon and energy source were selected for degradation studies. Several parameters (incubation time, substrate concentration, pH, temperature, and different metal ions) for cholesterol decomposition by the selected bacterial strains were evaluated. Maximum cholesterol reduction was achieved on the 5th day of incubation, 1g/L of substrate concentration, pH 7, in the presence of Mg2+ and Ca2+ ions, and at 35°C. Cholesterol degradation was analyzed by enzymatic colorimetric method, thin layer chromatography (TLC), and high-performance liquid chromatography (HPLC). Under optimized conditions 50% and 84% cholesterol reduction were recorded with Serratia marcescens W1 and Bacillus pumilus W8, respectively. Cholesterol oxidase activity was assayed qualitatively and quantitatively. The results revealed that Serratia marcescens W1 and Bacillus pumilus W8 have great potential for cholesterol degradation and would be regarded as a source for cholesterol oxidase (CHO).

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.

Expression optimization of recombinant cholesterol oxidase in Escherichia coli and its purification and characterization

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.

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.

Scale up and optimization of cholesterol oxidase production from Streptomyces rimosus MTCC 10792 in a 3-L bioreactor

A scale-up approach was investigated for maximum production of cholesterol oxidase (CO) enzyme, due to its high demand in agricultural and pharmaceutical industries. The CO production from Streptomyces rimosus in submerged fermentation conditions at shake flask level was observed in yeast malt glucose (YMG) medium as 1.82 U/mL. Maximum specific growth rate (μmax) and generation time (g) in log phase of microbial growth were found as 0.107 h−1 and 6.46 h respectively. During the CO production in 3-L bioreactor, the optimum parameters were observed as agitation—200 rpm, aeration—1.5 vvm, pH—7.0 and temperature 30 °C, with maximum production (8.66 U/mL, yield 0.916 U/mg cells), which showed 4.75 fold increase as compared to shake-flask production (1.82 U/mL, yield 0.243 U/mg). The volumetric oxygen mass transfer rate (KLa) was found to be in the range of 16 h−1 by dynamic gassing out method under optimized aeration and agitation parameters. The production of CO from S. rimosus in bioreactor is not reported earlier.

Extracellular cholesterol oxidase production by Streptomyces aegyptia, in vitro anticancer activities against rhabdomyosarcoma, breast cancer cell-lines and in vivo apoptosis

In recent years, microbial cholesterol oxidases have gained great attention due to its widespread use in medical applications for serum cholesterol determination. Streptomyces aegyptia strain NEAE-102 exhibited high level of extracellular cholesterol oxidase production using a minimum medium containing cholesterol as the sole source of carbon. Fifteen variables were screened using Plackett–Burman design for the enhanced cholesterol oxidase production. The most significant variables affecting enzyme production were further optimized by using the face-centered central composite design. The statistical optimization resulted in an overall 4.97-fold increase (15.631 UmL−1) in cholesterol oxidase production in the optimized medium as compared with the unoptimized medium before applying Plackett Burman design (3.1 UmL−1). The purified cholesterol oxidase was evaluated for its in vitro anticancer activities against five human cancer cell lines. The selectivity index values on rhabdomyosarcoma and breast cancer cell lines were 3.26 and 2.56; respectively. The in vivo anticancer activity of cholesterol oxidase was evaluated against Ehrlich solid tumor model. Compared with control mice, tumors growth was significantly inhibited in the mice injected with cholesterol oxidase alone, doxorubicin alone and cholesterol oxidase/doxorubicin combination by 60.97%, 72.99% and 97.04%; respectively. These results demonstrated that cholesterol oxidase can be used as a promising natural anticancer drug.

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

BackgroundThere 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.ResultsThe 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.ConclusionsTaking 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.

Erratum to: Identification and statistical optimization of fermentation conditions for a newly isolated extracellular cholesterol oxidase producing Streptomyces cavourensis strain NEAE-42.

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.

Identification and statistical optimization of fermentation conditions for a newly isolated extracellular cholesterol oxidase-producing Streptomyces cavourensis strain NEAE-42.

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.

Optimization of cholesterol oxidase production and 16S rRNA partial sequence of Bacillus cereus strain KAVK4 isolated from butter

Endophytes isolated from passion fruit plants: molecular identification, chemical characterization and antibacterial activity of secondary metabolitesMariana Sanches Santos, Ravely Casarotti Orlandelli, Julio Cesar Polonio, Marcos Alessandro dos Santos Ribeiro, Maria Helena Sarragiotto, João Lúcio Azevedo, João Alencar Pamphile

Purification and Characterization of an Extracellular Cholesterol Oxidase of Bacillus subtilis Isolated from Tiger Excreta.

A mesophilic Bacillus sp. initially isolated from tiger excreta and later identified as a Bacillus subtilis strain was used to produce an extracellular cholesterol oxidase (COX) in cholesterol-enriched broth. This bacterial isolate was studied for the production of COX by manipulation of various physicochemical parameters. The extracellular COX was successfully purified from the cell-free culture broth of B. subtilis by successive salting out with ammonium sulfate, dialysis, and riboflavin-affinity chromatography. The purified COX was characterized for its molecular mass/structure and stability. The enzyme possessed some interesting properties such as high native Mr (105kDa), multimeric (pentamer of ∼21kDa protein) nature, organic solvent compatibility, and a half-life of ∼2h at 37°C. The bacterial COX exhibited ∼22% higher activity in potassium phosphate buffer (pH 7.5) in the presence of a nonionic detergent Triton X-100 at 0.05% (v/v). The K m and V max value of COX of B. subtilis COX were found to be 3.25mM and 2.17μmolminml(-1), respectively. The purified COX showed very little cytotoxicity associated with it.