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.     

Use of glucose and carbon isotope fractionation by microbial cells immobilized on solid-phase surface

By the example of glucose uptake by the soil bacteria Pseudomonas aureofaciens BS1393(pBS216) and Rhodococcus sp. 3–30 immobilized on a solid-phase surface (quartz sand), their growth parameters were determined: growth rate (doubling time), total CO₂ production, CO₂ production per cell, lag period with respect to substrate uptake, respiratory quotient. The growth of P. aureofaciens and Rhodococcus sp. on glucose revealed (1) differences of the lag period with respect to substrate (lag time of ～4 h for P. aureofaciens and ～26 h for Rhodococcus sp.); (2) differences between the maximal rates of CO₂ production (～50 μg C-CO₂ g^?1 sand h^?1 for P. aureofaciens and ～8.5 μg C-CO₂ g^?1 sand h^?1 for Rhodococcus sp.); (3) differences in CO₂ production per cell (～1.94 × 10^?9 μM CO₂/CFU for P. aureofaciens and more than ～3.4 × 10^?9 μM CO₂/CFU for Rhodococcus sp.). The kinetics of the metabolic CO₂ isotopic composition was shown to be determined by the difference in the carbon isotopic characteristics of products in the cell. Upon introduction of glucose into the medium (the preparatory stage of the metabolism), the uptake of intracellular ¹³C-depleted products (lipids) is noted; at the stage of the maximal cell growth rate, introduced glucose is mainly metabolized; and at the final stage, upon exhaustion of substrate, the “stored” products—the lipid fraction—get involved in the metabolism. At the maximal rate of glucose uptake, the CO₂ carbon isotopic fractionation coefficient relative to organic products of microbial biosynthesis was determined to be α = 1.009 ± 0.002.     

Production of cholesterol oxidase by a newly isolated Rhodococcus sp.

Fifteen strains of microorganisms with ability to degrade cholesterol were isolated. Among them a Gram-positive, non-motile, non-sporing bacterium with meso-DAP in the cell wall and with a rod-coccus cycle showed the highest ability for cholesterol degradation. It was identified as Rhodococcus sp. strain 2C and was deposited by code 1633 in Persian type culture collection (PTCC). This strain was able to produce high levels of both extracellular and cell-bound cholesterol oxidases in media containing cholesterol as a sole carbon source. The effects of medium composition and physical parameters on cholesterol oxidase production were studied. The optimized medium was found to contain cholesterol 0.15% (w/v), yeast extract 0.3% (w/v), diammonium hydrogen phosphate 0.1% (w/v), Tween 80 (0.05%). The optimum pH and temperature for cholesterol oxidase production in optimized medium were found to be 8–30 °C respectively. Triton X-100 showed the greatest effect in releasing the cell-bound enzyme. The first and most probably the main metabolite of cholesterol degradation was purified and identified as 4-cholestene-3-one.     

Ethyl tert-butyl ether (ETBE) biodegradation by a syntrophic association of Rhodococcus sp. IFP 2042 and Bradyrhizobium sp. IFP 2049 isolated from a polluted aquifer

Ethyl tert-butyl ether (ETBE) enrichment was obtained by adding contaminated groundwater to a mineral medium containing ETBE as the sole carbon and energy source. ETBE was completely degraded to biomass and CO₂ with a transient production of tert-butanol (TBA) and a final biomass yield of 0.37?±?0.08 mg biomass (dry weight).mg^?1 ETBE. Two bacterial strains, IFP 2042 and IFP 2049, were isolated from the enrichment, and their 16S rRNA genes (rrs) were similar to Rhodococcus sp. (99 % similarity to Rhodococcus erythropolis) and Bradyrhizobium sp. (99 % similarity to Bradyrhizobium japonicum), respectively. Rhodococcus sp. IFP 2042 degraded ETBE to TBA, and Bradyrhizobium sp. IFP 2049 degraded TBA to biomass and CO₂. A mixed culture of IFP 2042 and IFP 2049 degraded ETBE to CO₂ with a biomass yield similar to the original ETBE enrichment (0.31?±?0.02 mg?biomass.mg^?1 ETBE). Among the genes previously described to be involved in ETBE, MTBE, and TBA degradation, only alkB was detected in Rhodococcus sp. IFP 2042 by PCR, and none were detected in Bradyrhizobium sp. IFP 2049.     

Combinatorial approach of statistical optimization and mutagenesis for improved production of acidic phytase by Aspergillus niger NCIM 563 under submerged fermentation condition

Combination of statistical optimization and mutagenesis to isolate hypersecretory strains is studied to maximize phytase production from Aspergillus niger NCIM 563 under submerged fermentation. The overall results obtained show a remarkable 5.98-fold improvement in phytase production rates when compared to that using basal medium. Optimization of culture conditions from parent strain is studied first by the Plackett–Burman technique to evaluate the effects of 11 variables for phytase production. The results showed that glucose, MgSO₄, KCl, incubation period, and MnSO₄ are the most significant variables affecting enzyme production. Further optimization in these variables, using a central composite design technique, resulted in 3.74-fold increase in the yield of phytase production to 254,500 U/l when compared with the activity observed with basal media (68,000 U/l) in shake flask. Our experiments show that the phytase from A. niger NCIM 563 exhibits desirable activity in simulated gastric fluid conditions with low pH and also improved thermostability when compared to commercial phytase. The improved yield demonstrates the potential applicability of phytase enzyme as a source of phytase supplement for phosphorus nutrition and environmental protection in animal feed industry. Physical and chemical mutagenesis experiments were carried out in parallel to isolate hypersecretory mutants that could possibly further enhance the enzyme production. Using optimized media conditions of the parent strain, our results show that mutant strain A. niger NCIM 1359 increased the phytase activity by another 1.6-fold to 407,200 U/l.     

Isomerization of glucose to fructose: production and some properties of glucose isomerase from Streptomyces sp. strain C7

Nine strains of actinomycetes isolated from Iraqi soils were investigated for glucose isomerase production. Only one strain, Streptomyces sp., C₇, was active. The maximum conversion ratio of the enzyme for the cells grown in d-xylose medium after 24 h incubation at 70°C and pH 6.9, was 64 and 48% for crude extract and cell-bound enzyme, respectively. The optimum pH value and temperatures for both enzymes were 8.0 and 70°C.     

Identification of cholesterol oxidase from fast-growing mycobacterial strains and Rhodococcus sp.

Cholesterol oxidase synthesis ability by various fast-growing mycobacteria (M. fortuitum, M. vaccae, M. phlei, M. smegmatis) and Rhodococcus sp. IM 58 was investigated by thin-layer chromatography and Western blot analysis. In contrast with the case of cholesterol oxidases from Arthrobacter sp. IM 79 and Streptomyces sp., those from the strains tested were localized intracellularly. Western blot experiments revealed high antigenic similarity of those enzymatic proteins to cholesterol oxidase from Schizophyllum commune, and lack of homology to cholesterol oxidase from Streptomyces sp. Differences in the analyzed enzymes were due not only to a single antigenic determinant alteration but also to the significant distinctions on the genetic level as shown by Southern blot hybridization.     

Artificial Intelligence versus Statistical Modeling and Optimization of Cholesterol Oxidase Production by using Streptomyces Sp.

Cholesterol oxidase (COD) is a bi-functional FAD-containing oxidoreductase which catalyzes the oxidation of cholesterol into 4-cholesten-3-one. The wider biological functions and clinical applications of COD have urged the screening, isolation and characterization of newer microbes from diverse habitats as a source of COD and optimization and over-production of COD for various uses. The practicability of statistical/ artificial intelligence techniques, such as response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) have been tested to optimize the medium composition for the production of COD from novel strain Streptomyces sp. NCIM 5500. All experiments were performed according to the five factor central composite design (CCD) and the generated data was analysed using RSM and ANN. GA was employed to optimize the models generated by RSM and ANN. Based upon the predicted COD concentration, the model developed with ANN was found to be superior to the model developed with RSM. The RSM-GA approach predicted maximum of 6.283 U/mL COD production, whereas the ANN-GA approach predicted a maximum of 9.93 U/mL COD concentration. The optimum concentrations of the medium variables predicted through ANN-GA approach were: 1.431 g/50 mL soybean, 1.389 g/50 mL maltose, 0.029 g/50 mL MgSO₄, 0.45 g/50 mL NaCl and 2.235 ml/50 mL glycerol. The experimental COD concentration was concurrent with the GA predicted yield and led to 9.75 U/mL COD production, which was nearly two times higher than the yield (4.2 U/mL) obtained with the un-optimized medium. This is the very first time we are reporting the statistical versus artificial intelligence based modeling and optimization of COD production by Streptomyces sp. NCIM 5500.     

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.     

Cultural factors affecting biosurfactant production by Gordonia sp. BS29

Gordonia sp. BS29 is a hydrocarbon-degrading bacterium isolated from a site chronically contaminated by diesel. The strain produces extracellular bioemulsifiers, able to produce stable emulsions, and cell-bound glycolipid biosurfactants, able to reduce surface tension. The aims of this work were to investigate the cultural factors affecting the production of the cell-bound biosurfactants by Gordonia sp. BS29 and to find the optimal composition of growth medium for the production. The cultural factors which have a significant influence on surfactant biosynthesis, identified by a two level 2^(8-2) Fractional Factorial Design, were the type and concentration of the carbon source, the concentrations of phosphates and sodium chloride, and the interactions among these factors. On these factors, a flask-scale optimisation of cultural conditions was carried out. Then, a steepest ascent procedure and a Central Composite Design were applied to obtain a second order polynomial function fitting the experimental data near the optimum. In the optimised cultural condition we obtained a 5-fold increase in the biosurfactant concentration compared to the un-optimised medium (26.00), reaching a Critical Micelle Dilution value (129.43) among the highest in literature. The optimisation procedure did not change the number and type of the glycolipid biosurfactants produced by Gordonia sp. BS29.     

Isolation, molecular identification and statistical optimization of culture condition for a new extracellular cholesterol oxidase-producing strain using response surface methodology

The current work details the screening of about 100 isolates from various soil samples, from which 1 isolate was finally selected based on the productivity of cholesterol oxidase. Further biochemical identification tests and 16S rRNA gene sequencing identified this isolate as Streptomyces badius. A preliminary culture media optimization was carried out using the initial screening method of Plackett-Burman. Then, a Box-Behnken design was employed to investigate the optimum concentrations of medium components and interactive effects of main variables on cholesterol oxidase production. The regression analysis showed a significant coefficient of determination (R ²) value (91 %), which was in close agreement ensuring a satisfactory adjustment of the proposed model. Maximal enzyme production (2.38 U/mL, i.e., approximately more than 100 % activity in the basal medium) was obtained at: temperature 35 °C; Tween 20 0.1 %; pH 6.5 and yeast extract 0.15 %. This two-stage statistical approach provided rapid identification and integration of key medium parameters for Streptomyces sp., resulting in high cholesterol oxidase production.     

Thermotolerant oil-degrading bacteria isolated from soil and water of geographically distant regions

Oil-degrading bacteria were isolated from soil and water samples taken in Russia, Kazakhstan, and the Antarctic; 13 of 86 strains proved to be thermotolerant. These bacteria utilized crude oil at 45–50°C; their growth optimum (35–37°C) and range (20–53°C) differ from those of mesophilic bacteria. Thermotolerant strains were identified as representatives of the genera Rhodococcus and Gordonia. It was shown that their ability to degrade petroleum products does not differ at 24 and 45°C. The strains Rhodococcus sp. Par7 and Gordonia sp. 1D utilized 14 and 20% of the oil, respectively, in 14 days at 45°C. All of the isolated thermotolerant bacteria grew in a medium containing 3% NaCl; the medium for the strains Gordonia amicalis 1B and Gordonia sp. 1D contained up to 10% NaCl. The bacteria G. amicalis and Rhodococcus erythropolis were able to utilize crude oil and individual hydrocarbons at higher (up to 50°C) temperatures.     

Rhodococcus UKMP-5M, an endogenous lipase producing actinomycete from Peninsular Malaysia

Escalation in food industries unctuous wastes has led to serious anthropogenic problems to the environment. Parallel to “green strategy”, growing awareness in biological treatment emphasizes efficacy of enzymatic technology for bioremediation. Pertinently, researchers are in search for new lipase-lipid interaction for improved outcome. Rhodococcus species have documented inadequate evidences on lipase enzyme production. Consequent assessments on Rhodococcus isolates from Peninsular Malaysia have identified twelve promising strains as lipase producer. Interestingly, apart from usual lipolytic behaviour, Rhodococcus sp. exhibited significant level of lipase endogenously, while cryogenic grinding method effectively ruptured the cell. An isolate from petroleum-contaminated site, namely Rhodococcus UKMP-5M, projected the highest level of lipase specificity and has further been optimized. It was found out that the best specificity was apparent in acidic condition (pH 5) with 6% inoculum at 30°C for 72 hours of incubation. Due to high level of mycolic cell-surfactant developed in triacylglycerol supplements, cell lysis was employed with Triton X-100 detergent solubilisation. As a result, oil blend composed of various carbon-chain length fatty acids (composite 2) induces enzyme production extensively. Remarkably, R. UKMP-5M found to cater enzyme production without aid of inducer by nature, but additional carbon source like glucose represses lipase production. Further ability for biological treatment was revealed when the optimized R. UKMP-5M whole cell degraded waste cooking oil significantly by solubilizing fatty acids and commencing conversion into biomass. These qualities resemble practical new lipid-lipase biological lipid rich on-site treatment.     

Optimization of parameters for improvement of phenol degradation by Rhodococcus UKMP-5M using response surface methodology

Phenol is a toxic compound and is one of the major pollutants contained in the waste water from petroleum and its downstream industries. Response surface methodology (RSM) was used to optimize medium composition and culture condition for enhancement of growth of Rhodococcus UKMP-5M and phenol degradation rate in shake flask cultures. Phenol and (NH₄)₂SO₄ concentrations as well as temperature were the most significant factors that influenced growth and phenol degradation. Central composite design (CCD) was used for optimization of these parameters with growth, and degradation rates were used as the responses. Cultivation with 0.5 g/L phenol and 0.3 g/L (NH₄)₂SO₄ and incubation at 36 °C greatly enhanced growth of Rhodococcus UKMP-5M, where the final cell concentration increased from 0.117 g/L to 0.376 g/L. On the other hand, the degradation rate was greatly increased in cultivation with 0.7 g/L phenol and 0.4 g/L (NH₄)₂SO₄ and incubation at 37 °C. In this cultivation, the time taken to degrade 1 g/L phenol in the culture was reduced from 48 h to 27 h. The model for both responses was found significant and the predicted values were found to be in a good agreement with experimental values and subsequently validated. Increases in phenol degradation rate during Rhodococcus UKMP-5M cultivation corresponded well with increasing phenol hydroxylase activity.     

Improving culture conditions for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Bacillus sp. ND153, a bacterium isolated from a mangrove forest in Vietnam

The production of polyhydroxyalkanoate (PHA) by Bacillus sp. ND153, a bacterium strain isolated from a mangrove forest in Vietnam, was studied. Bacillus sp. ND153 was grown on HM-1 medium with different carbon sources (e.g. glucose, sucrose, maltose, dextrin, and starch). Glucose was found to be the most suitable carbon source for PHA accumulation, whereas starch and dextrin favored cell growth over PHA accumulation. Optimization of the culture medium for PHA production was investigated by applying factorial design, and a maximum PHA content of 79 % (w/w) was obtained with low concentrations of NH₄Cl and MgSO₄ and a high concentration of KH₂PO₄ in the medium. Propionate was used as the precursor for the production of copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and the amount of 3-hydroxyvalerate (3HV) in the polymer showed an increasing linear trend with the increase in propionate concentration from 0.2 g l^?1 to 1.0 g l^?1. Thus, the production of PHBV by Bacillus sp. ND153, with 3HV fraction ranging from 1 mol% to 30 mol%, was noted to be high, and the characteristics of fast cell growth and accumulation of PHA exhibited by Bacillus sp. ND153 make it a promising choice for biopolyester production.     

Actinobacteria may influence white truffle (Tuber magnatum Pico) nutrition,ascocarp degradation and interactions with other soil fungi

To test the hypothesis that truffle-associated bacteria may improve truffle nutrition, we isolated bacteria from white truffle ascocarps and tested Actinobacteria for their ability to solubilise phosphate and iron, nutrients that have limited availability in white truffle grounds. Two isolates with sequence similarities to Curtobacterium flaccumfaciens and Rhodococcus sp. were characterized in detail. Both solubilised Ca₃(PO₄)₂ in a way that was dependent on the nitrogen and carbon sources present. Neither strain broke down phytate, but both produced chelating compounds, performed ammonification, and broke down β-glucan. Additionally, C. flaccumfaciens decomposed chitin, pectin, lipids and proteins, while Rhodococcus sp. exhibited urease activity. Three potentially fungicolous fungi were isolated from diseased white truffle ascocarps and bioassayed against the isolated Actinobacteria. The Rhodococcus isolate inhibited Verticillium leptobactrum, neither bacterium affected Clonostachys rosea, while both isolates promoted growth of Trichoderma sp. The results suggest that Actinobacteria might be involved in improving truffle nutrition, ascocarp degradation and establishing relationships with other soil fungi.     

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. V_max (21.93 μM/min mg) and substrate affinity K_m (101.3 μM) suggested high affinity of the COD for cholesterol. In presence of Ba²⁺ and Hg²⁺ the enzyme activity was inhibited while Cu²⁺ 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.     

Modified ferric hydroxamate spectrophotometry for assaying glycolic acid from the hydrolysis of glycolonitrile by Rhodococcus sp. CCZU10-1

We successfully modified a ferric hydroxamate spectrophotometry method for assaying glycolic acid. Comparable to the high-performance liquid chromatography (HPLC)-based method, ferric hydroxamate spectrophotometry can be used to accurately monitor the time course of glycolonitrile bioconversion. Glycolic acid was assayed simply and rapidly at room temperature (25 ～ 35°C). Optimum culture conditions were obtained using this method to assay the glycolonitrile-hydrolyzing activity of Rhodococcus sp. CCZU10-1. The preferred carbon and nitrogen sources and ideal inducer were glucose (10 g/L), a composite of peptone (10 g/L) plus yeast extract (5 g/L), and ?-caprolactam (2 mmol/L), respectively. The optimal growth temperature and initial medium pH for Rhodococcus sp. CCZU10-1 glycolonitrile-hydrolyzing activity were 30°C and pH 7.0. Modified ferric hydroxamate spectrophotometry could potentially be employed to assay other carboxylic acids.     

Biodegradation of Crude Oil by Constructed Bacterial Consortia and the Constituent Single Bacteria Isolated From Malaysia

Three bacterial isolates identified as Pseudomonas aeruginosa (UKMP-8T), Rhodococcus sp. M15-2 (UKMP-5T), and Rhodococcus sp. ZH8 (UKMP-7T) based on biochemical, physiological, and morphological characteristics and on 16S rDNA sequences were isolated from groundwater of a crude oil refinery plant. From these three isolates, four bacterial consortia were designed by mixing the single bacterial cultures in the following ratios: (P. aeruginosa:Rhodococcus sp. M15-2, 1:1), (P. aeruginosa:Rhodococcus sp. ZH8, 1:1), (Rhodococcus sp. M15-2: Rhodococcus sp. ZH8, 1:1), and (P. aeruginosa: Rhodococcus sp. ZH8:Rhodococcus sp. M15-2, 1:1:1), respectively. Bacterial isolates and consortia showed differing preferences for nitrogen source (0.01% ammonium chloride, 0.10% yeast extract, or 0.50% peptone) to reach optimum growth. When fortified with the preferred nitrogen sources and grown in minimal salt medium, within 7 days all three single isolates and the four bacterial consortia biodegraded 97.6-99.9% of Tapis Massa oil without any significant differences.