Lipase production by Aeromonas sobria LP004 in a medium containing whey and soybean meal

World Journal of Microbiology and Biotechnology -     

Thermo-acido-tolerant phytase production from a soil bacterium in a medium containing rice bran and soybean meal extract

A bacterial strain capable of producing a thermo-acido-tolerant phytase was isolated from soil around haystacks and designated as strain PH01. The phytase produced was purified to homogeneity as determined by native PAGE. From SDS-PAGE, it was 30 kDa in size. The purified phytase was a thermo-acido-tolerant enzyme. A complex medium for the PH01 phytase production was developed. The medium, "PheB", was composed of 2% glucose, 0.2% CaCl(2), 0.5% NH(4)NO(3), 0.05% KCl, 0.05% MgSO(4).7H(2)O, 0.001% FeSO(4).7H(2)O, 0.001% MnSO(4).H(2)O in rice bran plus soybean meal extract containing 3% (v/v) phosphate solution (7.3% NaHPO(4)+3.2%KH(2)PO(4), pH 7.2). Cultivation was done at 37 degrees C with aeration for 48 h which produced phytase at 10 U/ml. Exposure of the phytase to 1% bile salt; i.e., taurocholate or deoxycholate, caused less than 15% reduction of activity. Potential application of PH01 phytase as a feed supplement was suggested.     

Evaluation of the roles that alkyl hydroperoxide reductase and Ohr play in organic peroxide-induced gene expression and protection against organic peroxides in Xanthomonas campestris

Alkyl hydroperoxide reductase (ahpC) and organic hydroperoxide resistance (ohr) are distinct genes, structurally and regulatory, but have similar physiological functions. In Xanthomonas campestris pv. phaseoli inactivation of either gene results in increased sensitivity to killing with organic peroxides. An ahpC1-ohr double mutant was highly sensitive to both growth inhibition and killing treatment with organic peroxides. High level expression of ahpC or ohr only partially complemented the phenotype of the double mutant, suggesting that these genes function synergistically, but through different pathways, to protect Xanthomonas from organic peroxide toxicity. Functional analyses of Ohr and AhpC abilities to degrade organic hydroperoxides revealed that both Ohr and AhpC could degrade tert-butyl hydroperoxide (tBOOH) while the former was more efficient at degrading cumene hydroperoxide (CuOOH). Expression analysis of these genes in the mutants showed no compensatory alterations in the levels of AhpC or Ohr. However, CuOOH induced expression of these genes in the mutants was affected. CuOOH induced ahpC expression was higher in the ohr mutant than in the parental strain; in contrast, the ahpC mutation has no effect on the level of induced ohr expression. These analyses reveal complex physiological roles and expression patterns of seemingly functionally similar genes.     

Production of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus strain 1593 on media using a byproduct from a monosodium glutamate factory

Two newly developed media, H4 and H7, were found to be highly suitable for culturing Bacillus thuringiensis subsp. israelensis and B. sphaericus, respectively. These media contained 0.05% K₂HPO₄ and 4% HDL (H4 medium) or 0.05% K₂HPO₄ and 7% HDL (H7 medium); HDL is the by-product from a monosodium glutamate factory. Tests to compare endospore formation and toxicity values of B. thuringiensis subsp. israelensis in H4 medium and nutrient broth supplemented with salts and glucose (NBSG) medium were carried out in a 3-liter fermentor. The viable cell count and LC₅₀ value of B. thuringiensis subsp. israelensis in H4 medium at 48 hr were 2.5 × 10⁸ cells/ml and 10^?7.2 (dilution), respectively, while those in NBSG medium were 1.6 × 10⁸ cells/ml and 10^?6.5, respectively. In the case of B. sphaericus grown in H7 medium, the number of cells and LC₅₀ value were found to be 1.4 × 10⁹ cells/ml and 10^?7.8, respectively. B. sphaericus grown in nutrient broth supplemented with salt and yeast extract (NBSY) were found to produce 6.4 × 10⁸ cells/ml and an LC₅₀ value of 10^?6.8. The toxicity of B. thuringiensis subsp. israelensis was tested against Aedes aegypti larvae, while that of B. sphaericus was tested against Culex quinquefasciatus. The cost of 10 liters of medium for production of B. thuringiensis subsp. israelensis and in B. sphaericus and H4 and H7 was $0.02 and $0.03, respectively. The cost of these newly developed media was much less than that of NBSG medium ($7.05 per 10 liters) for cultivation of B. thuringiensis subsp. israelensis and NBSY medium ($11.67 per 10 liters) for cultivation of B. sphaericus.     

Purification and characterization of lipase from psychrophilic Acinetobacter calcoaceticus LP009

A lipase-producing bacterium, Acinetobacter calcoacetius LP009, was isolated from raw milk. The optimum conditions for growth and lipase production by A. calcoaceticus LP009 were 15 degrees C with shaking at 200 rpm in LB supplemented with 1.0% (v/v) Tween 80. The crude lipase was purified to homogeneous state by ultrafiltration and gel filtration chromatography on Sephadex G-100. Its molecular weight determined by SDS-PAGE was 23 kDa and it exhibited maximum activity at pH 7.0 and 50 degrees C. It was stable over the pH range of 4.0 to 8.0 and at temperatures lower than 45 degrees C. It was a metalloenzyme that is positionally non-specific and had the ability to improve fat hydrolysis in soybean meal and in premixed animals feed.     

Lipase from Pseudomonas aeruginosa LP602: biochemical properties and application for wastewater treatment

Lipase from Pseudomonas aeruginosa LP602, a bacterial strain isolated from a domestic wastewater sample, was preliminarily characterized. The enzyme exhibited maximum lipolytic activity at pH 8.0 where it was also stably maintained. At 55°C, the lipase had the highest activity but not stability. The enzyme was insensitive to EDTA and to many ions tested except Zn²⁺. It was sensitive to SDS but not to Tween-20, Tween-80 or Triton X-100. The enzyme was active towards a number of commercial food grade fats and oils. A suitable medium formula for lipase production was MMP containing 6.25% whey as a carbon source, 1% soybean oil as inducer and 0.5% yeast extract supplement. The culture was fed with glucose to a final concentration of 0.1% at the 15th hour of incubation. Lipase production under this condition was 3.5 U ml⁻¹. Both P. aeruginosa LP602 cells and the lipase were shown to be usable for lipid-rich wastewater treatment. Received 21 April 1998/ Accepted in revised form 6 August 1998     

Optimization of cyclodextrin production from sago starch

Cyclodextrin (CD) is synthesized by bacterial cyclodextrin glycosyltransferase (CGTase) and is widely used in food, pharmaceutical, cosmetic, and agricultural industries. In this study, Bacillus circulans CGTase was partially purified by ammonium sulfate precipitation at 50-70% saturation. The optimum pH and temperature for CD production from sago starch were found to be in the ranges of 4.5-5.0 and 55-60 degrees C, respectively. beta-CD was the predominant product, constituting 65% of all CD products. The beta-CD produced using partially purified and crude CGTase were compared and found to have no significant difference in yield and productivity. The appropriate proportion of CGTase to sago starch for beta-CD production was determined by response surface methodology. The most appropriate enzyme:substrate ratio was 50 U g sago starch(-1) CGTase and 60 g l(-1) sago starch.     

Lipase activity and gene cloning of Acinetobacter calcoaceticus LP009

The production of lipase from Acinetobacter calcoaceticus LP009, a bacterium isolated from raw milk, was found to be best induced by Tween-80 at 1.0% concentration. It was efficiently secreted, and only a minute amount of activity was detected at the cell surface and intracellularly. A. calcoaceticus LP009 lipase exhibited maximum activity at pH 7.0 and 50 degrees C, and was relatively stable upon storage at pH 5.0 to 7.0 and at 4, 30, or 37 degrees C. The enzyme was found to be inactivated by EDTA suggesting that it was a metalloenzyme. Its activity was reduced by less than 20% with the addition of various ions to reaction mixtures, but long storage with them caused approximately 50% reduction in subsequent reactions under standard conditions. By contrast, the addition of Fe(3+) enhanced activity. The enzyme was highly stable upon storage with 0.1% of Triton X-100, Tween-80 or Tween-20, but highly unstable with various organic solvents tested. PMSF, a serine enzyme inhibitor, and 2-mercaptoethanol, a reducing agent, did not affect enzyme activity. After extraction and transfer, the lipase gene was efficiently expressed in recombinant Aeromonas sobria. This recombinant strain was shown to have increased hydrolyzing efficiency and have high potential for lipid-rich wastewater treatment.