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.     

Production of xylanases from a newly isolated alkalophilic thermophilic Bacillus sp.

A new thermophilic strain of Bacillus SPS-0 which produces thermostable xylanases was isolated from a hot spring in Portugal. Xylanase production was 50 nkat/ml in the presence of wheat bran arabinoxylan. The temperature and pH for optimum activity were 75°C and 6–9, respectively. The hydrolysis patterns demonstrated that crude xylanases yield mainly xylose and xylobiose from xylan, whereas xylose and arabinose were produced from destarched wheat bran. An increase in xylose release was observed when SPS-0 xylanase was supplemented by a ferulic acid esterase. © Rapid Science Ltd. 1998     

Production of moderately halophilic amylase by newly isolated Micrococcus sp. 4 from a salt-pan

A new moderately halophilic Micrococcus sp. 4, isolated from salt-pan water from India, produced extracellular amylase when cultivated aerobically in medium containing wheat bran, peptone, beef extract and sodium chloride. Other salts, such as sodium nitrate, potassium nitrate and sodium sulphate, were also found to be suitable for growth and enzyme production. Maximum amylase activity (1.2 IU ml^-1) was secreted in the presence of 1 mol 1^-1 sodium chloride. The enzyme requires the presence of either sodium chloride, potassium chloride, sodium nitrate, sodium citrate or sodium acetate for its activity. Maximum activity was found in the presence of 1 mol 1^-1 sodium chloride. The pH and temperature optima for enzyme activity were 7.5 and 50°C, respectively.     

Production of amylase by newly isolated moderate halophile,Halobacillus sp. strain MA-2

Production of extracellular amylase was demonstrated under stress conditions of high temperature and high salinity in aerobically cultivated culture of a newly isolated moderately halophilic bacterium of spore-forming Halobacillus sp. strain MA-2 in medium containing starch, peptone, beef extract, and NaCl. The maximum amylase production was secreted in the presence of 15% (w/v) Na(2)SO(4) (3.2 U ml(-1)). The isolate was capable of producing amylase in the presence of NaCl, NaCH(3)COOH, or KCl, with the results NaCl>NaCH(3)COOH>KCl. Maximum amylase activity was exhibited in the medium containing 5% (w/v) NaCl (2.4 U ml(-1)). Various carbon sources induced enzyme production. The potential of different carbohydrates in the amylase production was in the order: dextrin>starch>maltose>lactose>glucose>sucrose. In the presence of sodium arsenate (100 mM), maximum production of the enzyme was observed at 3.0 U ml(-1). Copper sulfate (0.1 mM) decreased the amylase production considerately, while lead nitrate had no significant enhancement on amylase production (p<0.05). The pH, temperature, and aeration optima for enzyme production were 7.8, 30 degrees C, and 200 rpm, respectively, while the optimum pH and temperature for enzyme activity was 7.5-8.5 and 50 degrees C, respectively.     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Biodegradation of nicotine by newly isolated Pseudomonas sp. CS3 and its metabolites

Aims: Isolation and characterization of nicotine‐degrading bacteria with advantages suitable for the treatment of nicotine‐contaminated water and soil and detection of their metabolites. Methods and Results: A novel nicotine‐degrading bacterial strain was isolated from tobacco field soil. Based on morphological and physiochemical properties and sequence of 16S rDNA, the isolate was identified as Pseudomonas sp., designated as CS3. The optimal culture conditions of strain CS3 for nicotine degradation were 30°C and pH 7·0. However, the strain showed broad pH adaptability with high nicotine‐degrading activity between pH 6·0 and 10·0. Strain CS3 could decompose nicotine nearly completely within 24 h in liquid culture (1000 mg L^?1 nicotine) or within 72 h in soil (1000–2500 mg kg^?1 nicotine) and could endure up to 4000 mg L^?1 nicotine in liquid media and 5000 mg kg^?1 nicotine in soil. Degradation tests in flask revealed that the strain had excellent stability and high degradation activity during the repetitive degradation processes. Additionally, three intermediates, 3‐(3,4‐dihydro‐2H‐pyrrol‐5‐yl) pyridine, 1‐methyl‐5‐(3‐pyridyl) pyrrolidine‐2‐ol and cotinine, were identified by GC/MS and NMR analyses. Conclusions: The isolate CS3 showed outstanding nicotine‐degrading characteristics such as high degradation efficiency, strong substrate endurance, broad pH adaptability, and stability and persistence in repetitive degradation processes and may serve as an excellent candidate for applications in the bioaugmentation process to treat nicotine‐contaminated water and soil. Also, detection of nicotine metabolites suggests that strain CS3 might decompose nicotine via a unique nicotine‐degradation pathway. Significance and Impact of the Study: The advantage of applying the isolated strain lies in broad pH adaptability and stability and persistence in repetitive use, the properties previously less focused in other nicotine‐degrading micro‐organisms. The strain might decompose nicotine via a nicotine‐degradation pathway different from those of other nicotine‐utilizing Pseudomonas bacteria reported earlier, another highlight in this study.     

Production of alkaline xylanase by a newly isolated alkaliphilic Bacillus sp. strain 41M-1

Alkaliphilic Bacillus sp. strain 41M-1, isolated from soil, produced xylan-degrading enzymes extracellularly. Optimum pH for the crude xylanase preparation was about pH 9, confirming the production of novel alkaline xylanase(s) by the isolate. Xylanases were induced by xylan, but were not produced in the presence of xylose, arabinose or glucose. Xylanase productivity was influenced by culture pH, and production at pH 10.5 was higher than that at pH 8.0. Zymogram analysis of the culture supernatant showed the alkaline xylanase with a molecular mass of 36 kDa.     

Biodegradation of disperse textile dye Brown 3REL by newly isolated Bacillus sp. VUS

Aims:  To isolate the potential micro-organism for the degradation of textile disperse dye Brown 3 REL and to find out the reaction mechanism.
Methods and Results:  16S rDNA analysis revealed an isolate from textile effluent contaminated soil as Bacillus sp. VUS and was able to degrade (100%) dye Brown 3REL within 8 h at static anoxic condition. A significant increase in the activities of lignin peroxidase, laccase and NADH-DCIP reductase was observed up to complete decolourization of Brown 3REL. The optimum temperature required for degradation was 40°C and pH 6·5–12·0. Phyto-toxicity and chemical oxygen demand revealed nontoxic products of dye degradation. The biodegradation was monitored by UV–VIS, FTIR spectroscopy and HPLC. The final products 6,8-dichloro-quinazoline-4-ol and cyclopentanone were characterized by gas chromatography-mass spectrometry. This Bacillus sp. VUS also decolourized (80%) textile dye effluent within 12 h.
Conclusions:  This study suggests that Bacillus sp. VUS could be a useful tool for textile effluent treatment.
Significance and Impact of the Study:  The newly isolated Bacillus sp. VUS decolourized 16 textile dyes and textile dye effluent also. It achieved complete biodegradation of Brown 3REL. Phytotoxicity study demonstrated no toxicity of the biodegraded products for plants with respect to Triticum aestivum and Sorghum bicolor .     

Ethanol and amylase production by a newly isolated Clostridium sp.

A newly isolated, anaerobic, mesophillic bacterium, Clostridium sp. strain YK-3, ferments pentoses, hexoses, oligosaccharides and polysaccharides, such as soluble starch and glycogen, to ethanol and acetate. The potential of this strain for ethanol and amylase production has been examined. Ethanol was the major product and acetate a minor one. The organism could grow with soluble starch in the presence of 40 g ethanol/l. Extracellular -amylase activity was detected when the strain was cultivated with soluble starch, glycogen or dextrin. The optimum pH of this amylase was 5.5 to 7.5 with an optimum temperature of 50°C.The authors are with the Laboratory of Applied Microbiology, Faculty of Agriculture, Yamagata University, Tsuruoka 997, Japan.     

Production of extracellular alkaline alpha-amylase by solid state fermentation with a newly isolated Bacillus sp

Production of alkaline alpha-amylase employing our laboratory isolate, Bacillus sp., under solid state fermentation, was optimized. The effect of wheat bran and lentil husk was examined. Lentil husk exhibited the highest enzyme production. The appropriate incubation time, inoculum size, moisture level, and buffer solution level were determined. Maximum yields of 216,000 and 172,800 U/g were achieved by employing lentil husk and wheat bran as substrates in 0.1 M carbonate/bicarbonate buffer at pH 10.0 with 30% initial moisture level at 24 h. Inoculum size and buffer solution level were found to be 20% and 1:0.5 for two solid substrates.     

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.     

Biodegrading of pyrene by a newly isolated Pseudomonas putida PL2

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic compounds derived from natural sources and anthropogenic processes, which have been recommended as priority pollutants. Degradation of PAHs in the environment is becoming more necessary and urgent. In the current study, strain PL2, which is capable of growing aerobically on pyrene (PYR) as the sole carbon source, was isolated from hydrocarbons-contaminated soil and then identified as Pseudomonas putida by morphological and physiological characteristics as well as 16S rDNA sequence. The strain PL2 was able to degrade 50.0% of the pyrene at 28°C within 6 days in the presence of 50 mg/L pyrene, while the strain PL2 degraded 50.0% of the pyrene within 2 days when a solution of 50 mg/L pyrene and 50 mg/L phenanthrene was used. In addition, phenanthrene was shown to increase the biodegradation efficiency of pyrene by the strain PL2. The order of degradation by the strain PL2 was pH 6.0 > pH 7.0 > pH 5.0 > pH 8.0. The degradation rate of PYR in the soil by the strain PL2 reached 70.0% at the 10^th day. The dynamics of PYR degradation in soil by PL2 was fit to the first order model and the strain PL2 was shown to efficiently degrade PYR in soil. The current study showed that P. putida PL2 was a novel bacterium that could degrade pyrene and holds great promise for use in PAHs bioremediation in soil.     

Production of fruity aroma by newly isolated yeast

Summary One strain of yeast (Geotrichum sp) which was isolated from papaya fruit produced 22 volatile compounds and 8 of these were chemically identified. Ethyl isovalerate and ethyl hexanoate showed as fruity flavor, whereas ethyl propionate and ethyl butyrate showed weak fruitiness. Production of two fruity compounds was further studied by using various culture media.     

Chitinase production by a newly isolated thermotolerant Paenibacillus sp. BISR-047

Chitinase is one of the important mycolytic enzymes with industrial significance, and is produced by a number of organisms, including bacteria. In this study, we describe isolation, characterization and media optimization for chitinase production from a newly isolated thermotolerant bacterial strain, BISR-047, isolated from desert soil and later identified as Paenibacillus sp. The production of extracellularly secreted chitinase by this strain was optimized by varying pH, temperature, incubation period, substrate concentrations, carbon and nitrogen source,etc. The maximum chitinase production was achieved at 45 °C with media containing (in g/l) chitin 2.0, yeast extract 1.5, glycerol 1.0, and ammonium sulphate 0.2 % (media pH 7.0). A three-fold increase in the chitinase production (712 IU/ml) was found at the optimized media conditions at 6 days of incubation. The enzyme showed activity at broad pH (3–10) and temperature (35–100 °C) ranges, with optimal activity displayed at pH 5.0 and 55 °C, respectively. The produced enzyme was found to be highly thermostable at higher temperatures, with a half-life of 4 h at 100 °C.     

Substrate induction of isomaltulose synthase in a newly isolated Klebsiella sp. LX3

AIMS: Production of isomaltulose by newly isolated Klebsiella sp. LX3. METHODS AND RESULTS: The bacterial isolate LX3, which transforms sucrose to isomaltulose and trehalulose, has been isolated from a soil sample in Singapore. Morphological and biochemical analysis, as well as 16s rRNA sequence demonstrated that the isolate could represent a new member of genus Klebsiella. The strain has several interesting features. The immobilized cells of Klebsiella sp. LX3 convert more than 99% of sucrose to products that consist of more than 87% of isomaltulose, 11.6% of trehalulose, and <1% of glucose. CONCLUSIONS: The production of isomaltulose synthase in isolate LX3 is inducible by its substrate sucrose and the sugars containing a fructofuranosyl group. SIGNIFICANCE AND IMPACT OF STUDY: It would be useful for future biotechnological applications to understand the structural features or motifs of the isomaltulose synthases that determine the sucrose conversion efficiency and the ratio of the conversion products.     

Production and partial characterization of lipases from a newly isolated Penicillium sp. using experimental design

Aims:  The objective of this work was to investigate the lipase production by a newly isolated Penicillium sp . , using experimental design technique, in submerged fermentation using a medium based on peptone, yeast extract, NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained.
Methods and Results:  Lipase activity values of 9·5 U ml⁻¹ in 96 h of fermentation was obtained at the maximized operational conditions of peptone, yeast extract, NaCl and olive oil concentrations (g l⁻¹) of 20·0, 5·0, 5·0 and of 10·0 respectively. The partial characterization of crude enzymatic extract obtained by submerged fermentation showed optimum activity at pH range from 4·9 to 5·5 and temperature from 37°C to 42°C. The crude extract maintained its initial activity at freezing temperatures up to 100 days.
Conclusions:  A newly isolated strain of Penicillium sp . used in this work yielded good lipase activities compared to the literature.
Significance and Impact of the Study:  The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. New lipase producers are relevant to finding enzymes with different catalytic properties of commercial interest could be obtained, without using genetically modified organisms (GMO).     

Biodegradation of nicotine by a newly isolated Agrobacterium sp. strain S33

Aims: To isolate and characterize bacteria capable of degrading nicotine from the rhizospheric soil of a tobacco plant and to use them to degrade the nicotine in tobacco solid waste. Methods and Results: A bacterium, strain S33, was newly isolated from the rhizospheric soil of a tobacco plant, and identified as Agrobacterium sp. based on morphology, physiological tests, Biolog MicroLog3 4·20 system and 16S rRNA gene sequence. Using nicotine as the sole source of carbon and nitrogen in the medium, it grew optimally with 1·0 g l^?1 of nicotine at 30°C and pH 7·0, and nicotine was completely degraded within 6 h. The resting cells prepared from the glucose‐ammonium medium or LB medium could not degrade nicotine within 10 h, while those prepared from the nicotine medium could completely degrade 3 g l^?1 of nicotine in 1·5 h at a maximal rate of 1·23 g nicotine h^?1 g^?1 dry cell. Using the medium containing nicotine, glucose and ammonium simultaneously to cultivate strain S33, the resting cells could degrade 98·87% of nicotine in tobacco solid waste with the concentration as 30 mg nicotine g^?1 dry weight tobacco solid waste within 7 h at a maximal rate of 0·46 g nicotine h^?1 g^?1 dry cell. Conclusions: This is the first report that Agrobacterium sp. has the ability to degrade nicotine. Agrobacterium sp. S33 could use nicotine as the sole source of carbon and nitrogen. The use of resting cells of the strain S33 prepared from the nicotine–glucose–ammonium medium was an effective method to degrade nicotine and detoxify tobacco solid waste. Significance and Impact of the Study: Nicotine in tobacco wastes is both toxic and harmful to human health and the environment. This study showed that Agrobacterium sp. S33 may be suitable for the disposal of tobacco wastes and reducing the nicotine content in tobacco leaves.     

Production of 2,3-butanediol by newly isolated Enterobacter cloacae

Enterobacter cloacae NRRL B-23289 was isolated from local decaying wood/corn soil samples while screening for microorganisms for conversion of l-arabinose to fuel ethanol. The major product of fermentation by the bacterium was meso-2,3-butanediol (2,3-BD). In a typical fermentation, a BD yield of 0.4 g/g arabinose was obtained with a corresponding productivity of 0.63 g/l per hour at an initial arabinose concentration of 50 g/l. The effects of initial arabinose concentration, temperature, pH, agitation, various monosaccharides, and multiple sugar mixtures on 2,3-BD production were investigated. BD productivity, yield, and byproduct formation were influenced significantly within these parameters. The bacterium utilized sugars from acid plus enzyme saccharified corn fiber and produced BD (0.35 g/g available sugars). It also produced BD from dilute acid pretreated corn fiber by simultaneous saccharification and fermentation (0.34 g/g theoretical sugars). Received: 17 December 1998 / Revision received: 9 March 1999 / Accepted: 20 March 1999     

Production of d-arabitol by a newly isolated Kodamaea ohmeri

A new yeast, isolated from natural osmophilic sources, produces d-arabitol as the main metabolic product from glucose. According to 18S rRNA analysis, the NH-9 strain belongs to the genus Kodamaea. The optimal culture conditions for inducing production of d-arabitol were 37 °C, neutral pH, 220 rpm shaking, and 5% inoculum. The yeast produced 81.2 ± 0.67 g L⁻¹ d-arabitol from 200 g L⁻¹ d-glucose in 72 h with a yield of 0.406 g g⁻¹ glucose and volumetric productivity Q_textP Q_{text{P}} of 1.128 g L⁻¹ h⁻¹. Semi-continuous repeated-batch fermentation was performed in shaker-flasks to enhance the process of d-arabitol production by Kodamaea ohmeri NH-9 from d-glucose. Under repeated-batch culture conditions, the highest volumetric productivity was 1.380 g L⁻¹ h⁻¹.     

Lactic acid production from sugar-cane juice by a newly isolated Lactobacillus sp.

A newly isolated sucrose-tolerant, lactic acid bacterium, Lactobacillus sp. strain FCP2, was grown on sugar-cane juice (125 g sucrose l⁻¹, 8 g glucose l⁻¹ and 6 g fructose l⁻¹) for 5 days and produced 104 g lactic acid l⁻¹ with 90% yield. A higher yield (96%) and productivity (2.8 g l⁻¹ h⁻¹) were obtained when strain FCP2 was cultured on 3% w/v (25 g sucrose l⁻¹, 2 g glucose l⁻¹ and 1 g fructose l⁻¹) sugar-cane juice for 10 h. Various cheap nitrogen sources such as silk worm larvae, beer yeast autolysate and shrimp wastes were also used as a substitute to yeast extract.