Biochemical and biotechnological studies on a novel purified <i>bacillus</i> cholesterol oxidase tolerant to solvent and thermal stress

ElBaz, Fathy N.; Gamal, Rawia F.; El-Baz, Ashraf F.; Nasser, Ibrahim; ElMekawy, Ahmed

doi:10.1080/10242422.2017.1306742

Cited by 13 publications

(16 citation statements)

References 26 publications

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“…COX was quite stable at pH range 6.0–9.0 with maximum stability at pH values 7.0 and 8.0. Such findings were reported with other bacterial species ChOx, which were stable within the studied pH range . The thermostability profile of ChOx indicated that the enzyme was optimally stable at 37°C, exhibiting a half‐life of 8 h. While Doukyu et al reported an optimum temperature of 65°C for the ChOx from Chromobacterium sp.…”

Section: Discussionsupporting

confidence: 79%

“…Whereas, in other reports, the optimum temperature for ChOx from Bacillus pumilus, Rhodococcus sp. PKPD-CL, Streptomyces aegyptia NEAE 102, and E. hirae was found in the range of 37-40°C [3,8,36,41]. In some other studies, the optimum temperature for bacterial ChOx has been recorded in the range of 25-65°C [11,43].…”

Section: Discussionmentioning

confidence: 86%

“…Streptomyces aegyptia NEAE 102 produced extracellular ChOx of 46 kDa, which was purified by using techniques of ammonium sulfate precipitation and DEAE Sepharose CL-6B ion-exchange chromatography with an increase of 3.14-fold in its activity [36]. Also, ChOx of 55 kDa from Bacillus pumilus was purified using Sephadex G-100 gel filtration chromatography with 19.2% recovery [3].…”

Section: Discussionmentioning

confidence: 99%

“…Cholesterol oxidase (EC 1.1.3.6), a monomeric flavoenzyme (flavin adenine dinucleotide [FAD]-dependent enzyme) of the oxidoreductase family, catalyzes two reactions: oxidation and isomerization of cholesterol (cholest-5-en-3β-ol) to cholestenone (cholest-4-en-3-one; Figure 1) [1]. ChOx has been isolated and characterized from a vast variety of microbial sources, including Arthrobacter [2], Bacillus [3], Brevibacterium [4], Bordetella [5], Mycobacterium [6], Nocardia [7], Rhodococcus [8], Streptomyces [9], Burkholderia [10], Chromobacterium [11], and Pseudomonas spp. [12,13].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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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.

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Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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“…They reported that, yeast extract, pH, Tween 20 and temperature were the most significant factors and the maximum cholesterol oxidase production by Streptomyces badius was 2.05 U/mL in the optimized medium using the optimum levels of the four variables that were determined to be: yeast extract, 0.45%; pH, 6.5; Tween 20, 0.05% and 30°C. Moreover, ElBaz et al [44] used a two-step statistical approach to optimize the production of cholesterol oxidase from Bacillus pumilus. The maximum cholesterol oxidase production (90 U/mL) was obtained after 6 days of fermentation at pH 8 with medium/flask ratio of 0.35 and the concentrations of cholesterol, NH 4 NO 3 , yeast extract and Tween 80 were 0.2, 0.3, 1 and 0.2%; respectively.…”

Section: Optimization Of the Selected Significant Variables By Box-bementioning

confidence: 99%

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

El‐Naggar

El-Shweihy

2020

BMC Microbiol

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Background: Cholesterol 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. Results: The 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). Conclusions: Maximum cholesterol oxidase activity was obtained at the following fermentation conditions: g/L (cholesterol 4, yeast extract 5, NaCl 0.5, K 2 HPO 4 1, FeSO 4 .7H 2 O 0.01, MgSO 4 .7H 2 O 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.

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Bioprocess optimization of glutathione production by Saccharomyces boulardii: biochemical characterization of glutathione peroxidase

et al. 2021

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The well-known probiotic GRAS Saccharomyces boulardii (CNCM I-745) was used for the rst time to produce glutathione (GSH). The culture conditions affecting GSH biosynthesis were screened using a Plackett-Burman design (PBD). Analyzing the regression coe cients for 12 tested variables; 6 of them, including yeast extract, glucose, peptone and cysteine; temperature and agitation rate had a positive signi cant effect on GSH production with a maximum production of 192 mg/L. The impact of addition time of cysteine was investigated in 19 experiments during the growth time course (0-36 h), the best addition time was 8h post-inoculation producing 235 mg/L of GSH. The most signi cant variables were further explored at 5-levels using Central Composite Rotatable Design (CCRD), giving a maximum production of GSH (552 mg/L). Using ba ed asks, the GSH was increased to 730 mg/L, i.e 1.32-folds increment than obtained using CCRD. The two rate-limiting genes of GSH biosynthesis "γ-glutamyl cysteine synthetase (gsh1) and GSH-synthetase (gsh2) were ampli ed and sequenced to validate the GSH biosynthetic potency of S. boulardii. The sequences of genes showed 99% similarity with gsh1 and gsh2 genes of S. cerevisiae. Glutathione peroxidase was puri ed and characterized from S. boulardii with molecular mass and subunit structure of 80 kDa and 35 kDa as revealed from native and SDS-PAGE, ensuring its homodimeric identity. The activity of GPx was reduced by 2.5-folds upon demetallization con rming its metalloproteinic identity. The enzyme was strongly inhibited by hydroxylamine and DTNB, ensuring the implication of surface lysine and cysteine residues on the enzyme active site domains.

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Biochemical and biotechnological studies on a novel purified bacillus cholesterol oxidase tolerant to solvent and thermal stress

Cited by 13 publications

References 26 publications

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

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

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

Bioprocess optimization of glutathione production by Saccharomyces boulardii: biochemical characterization of glutathione peroxidase

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