Purification of CBS 819.72 α-amylase by aqueous two-phase systems: Modelling using Response Surface Methodology

The partition and purification of α-amylase from a culture supernatant of Aspergillus oryzae CBS 819.72 was made in aqueous two-phase system (ATPS). According to bibliography and preliminary studies, the factors polyethylene glycol (PEG) molecular weight (M_PEG) and concentration (C_PEG), buffer type (BU) and concentration (C_BU), temperature (T), salt nature (SALT) and concentration (C_SALT), bioligand (BL) and concentration (C_BL) and pH were investigated using a Plackett–Burman design to identify the factors affecting separation. Taking into consideration a simultaneous increase in enzyme recovery (R_Y) and purification factor (P_F), the best performance of the system was obtained at 4 °C and pH 6 using PEG 8000 g/mol, citrate buffer, KCl and sucrose. Experimental Box–Behnken design together with the Response Surface Methodology (RSM) have been used to find optimum C_PEG, C_Citrate and C_SALT. Quadratic models were predicted for P_F and R_Y in the top phase and a better compromise between these two parameters can be found by superimposing the contour plots of P_F and R_Y for 8% citrate. A region in the experimental space can be defined where the purification factor is always higher than 3 with yields exceeding 65%.     

Optimization of medium composition and culture conditions for agarase production by Agarivorans albus YKW-34

Effect of medium composition and culture conditions on agarase production by Agarivorans albus YKW-34 was investigated in shake flasks. The most suitable carbon source, nitrogen source, and culture temperature were agar, yeast extract, and 25 °C, respectively, for agarase production by one-factor-at-a-time design. The nutritional components of the medium and culture conditions were analyzed by Plackett–Burman design. Among the nine factors studied, agar, yeast extract, and initial pH had significant effects on agarase production (p < 0.05). The optimum levels of these key variables were further determined using a central composite design. The highest agarase production was obtained in the medium consisting of 0.23% agar and 0.27% yeast extract at initial pH 7.81. The whole optimization strategy enhanced the agarase production from 0.23 U/ml to 0.87 U/ml. The economic medium composition and culture condition as well as the dominant occupation of agarase with high activity in culture fluid enlighten the potential application of A. albus YKW-34 for the production of agarase.     

Aerial mycelia of Aspergillus oryzae accelerate α-amylase production in a model solid-state fermentation system

Aspergillus oryzae is commonly used in solid-state fermentation (SSF) and forms abundant aerial mycelia. Previously, we have shown that aerial mycelia are extremely important for the respiration of this fungus during growth on a wheat-flour model substrate. In this paper, we show that aerial mycelia of this fungus give a strong increase in fungal biomass and α-amylase production. Cultures of A. oryzae on wheat-flour model substrate produced twice the amounts of fungal biomass and α-amylase, when aerial mycelia were formed. Utilization of these findings in commercial solid-state fermenters requires further research; results from packed beds of grain indicate that aerial mycelia are of limited importance there. Probably, substrate pre-treatment and an increase in bed voidage are required.     

Application of statistically-based experimental designs in medium optimization for spore production of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> from distillery effluent

Spore production of Bacillus subtilis from distillery effluent was optimized using statistically-based experimental designs. The two-level Plackett–Burman design was applied to choose the nutrient supplements significantly influencing spore production. Among the seven variables we tested, the most significant variables influencing spore production were statistically elucidated for optimization, and included (NH₄)₂SO₄, corn flour and MgSO₄. The optimum concentration of each significant variable was then predicted using Box–Behnken design. A second-order polynomial was determined by the multiple regression analysis of this experimental data. The optimum values for the critical nutrient supplements for the maximum were obtained as followed: (NH₄)₂SO₄, 4.54%; corn flour, 1.2%; MgSO₄, 0.56% with the corresponding value of maximum spore production of 7.24 × 10⁸ spores/ml. A verification experiment performed under the optimum conditions resulted in 6.95 × 10⁸ spores/ml. The determination coefficient (R ²) was 0.98, which ensure an adequate credibility of the model.     

Solid state fermentation for production of α-amylase by a thermotolerant Bacillus subtilis from hot-spring water

The production of extracellular α-amylase by thermotolerant Bacillus subtilis was studied in solid state fermentation (SSF). The effect of wheat bran (WB) and rice husk (RH) was examined. The appropriate incubation period, moisture level, particle size and inoculum concentration was determined. Maximum yields of 159,520 and 21,760 U g⁻¹ were achieved by employing WB and RH as substrates in 0.1 M phosphate buffer at pH 7 with 30% initial moisture content at 24 and 48 h. Particle size and inoculum concentration were found to be 1000 μm, 20% and 500 μm, 15% for WB and RH, respectively. Enzyme yield was 7.3-fold higher with WB medium compared with RH.     

Medium optimization for the production of a novel bioflocculant from Halomonas sp. V3a′ using response surface methodology

The novel exopolysaccharide bioflocculant HBF-3 is produced by Halomonas sp. V3a′, which is a mutant strain of the deep-sea bacterium Halomonas sp. V3a. Response surface methodology (RSM) was employed to optimize the production medium for increasing HBF-3 production. Using a Plackett–Burman experimental design to aid in the first step of optimization, edible glucose, MgSO₄·7H₂O, and NH₄Cl were found to be significant factors affecting HBF-3 production. To determine the optimal concentration of each significant variable, a central composite design was employed. Based on response surface and canonical analysis, the optimum concentrations of the critical components were obtained as follows: edible glucose, 16.14 g/l; MgSO₄·7H₂O, 2.73 g/l; and NH₄Cl, 1.97 g/l. HBF-3 production obtained by using the optimized medium was 4.52 g/l, which was in close agreement with the predicted value of 4.55 g/l. By scaling up fermentation from flask to fermenter, HBF-3 production was further increased to 5.58 g/l.     

Purification, biochemical characterisation and partial primary structure of a new α-amylase inhibitor from Secale cereale (rye)

Plant α-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the α-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13 756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional α-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus α-amylases was observed. The inhibitor is more effective against insect α-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional α-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.     

Optimization of alkaline protease production by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis> ES1 in <Emphasis Type="Italic">Mirabilis jalapa</Emphasis> tuber powder using statistical experimental design

Medium composition and culture conditions for the bleaching stable alkaline protease production by Aspergillus clavatus ES1 were optimized. Two statistical methods were used. Plackett-Burman design was applied to find the key ingredients and conditions for the best yield. Response surface methodology (RSM) including full factorial design was used to determine the optimal concentrations and conditions. Results indicated that Mirabilis jalapa tubers powder (MJTP), culture temperature, and initial medium pH had significant effects on the production. Under the proposed optimized conditions, the protease experimental yield (770.66 U/ml) closely matched the yield predicted by the statistical model (749.94 U/ml) with R (2)=0.98. The optimum operating conditions obtained from the RSM were MJTP concentration of 10 g/l, pH 8.0, and temperature of 30 degrees C, Sardinella heads and viscera flour (SHVF) and other salts were used at low level. The medium optimization contributed an about 14.0-fold higher yield than that of the unoptimized medium (starch 5 g/l, yeast extract 2 g/l, temperature 30 degrees C, and pH 6.0; 56 U/ml). More interestingly, the optimization was carried out with the by-product sources, which may result in cost-effective production of alkaline protease by the strain.     

Fusarium roseum and Aspergillus oryzae-mediated enantioselective reduction of benzils to benzoins

Aspergillus oryzae OUT5048 and Fusarium roseum OUT4019 were found to be effective biocatalysts in the reduction of benzils to optically active benzoins. Easily available symmetrical benzil derivatives were reduced to the corresponding benzoins [(S)-2-hydroxy-1,2-diphenylethanones] by A. oryzae OUT5048 with up to 94% ee and by F. roseum OUT4019 with up to 98% ee, respectively. In this work, first general method for whole-cell-mediated selective reduction of benzils to benzoins is reported. It is also shown that this method is applicable for benzils with both electron-withdrawing and electron-donating groups.     

Enhanced production of amidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> MTCC 1526 by medium optimisation using a statistical experimental design

In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R. erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket–Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g dry cells, respectively. Thus, use of RSM increased production of amidase from R. erythropolis MTCC 1526 by 6.88-fold.     

The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL⁻¹ affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL⁻¹ TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K_M values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL⁻¹, and V_max values were 0.038 and 0.047 mg min⁻¹, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg²⁺ and Cu²⁺ suggesting that N-glycosylation is involved in the folding of Amy1.     

Xylanase production by Aspergillus awamori. Development of a medium and optimization of the fermentation parameters for the production of extracellular xylanase and beta-xylosidase while maintaining low protease production

A growth medium was developed for maximal production in batch culture of extracellular xylanase and beta-xylosidase by Aspergillus awamori CMI 142717 and a mutant (AANTG 43) derived from the wild-type strain. The optimum pH for the production of xylanase and beta-xylosidase was 4.0. The best temperature of xylanase production was 30 degrees C; 35 degrees C was optimal for beta-xylosidase. Protease production was never completely suppressed under any of the conditions tested. However, protease titre was 3.5-fold less than the control in medium in which proteose peptone and yeast extract were omitted: the level of xylanase was not affected (8.6 U mL(-1)) but beta-xylosidase titre was increased 4.7-fold to 1.5 U mL(-1). When corn steep liquor was used as the sole nitrogen source, xylanse and beta-xylosidase titres were further increased by 1.5- and 1.9-fold, respectively. Of the carbon sources investigated, ball-milled oat straw or oat spelt xylan produced the highest titres of xylanse and beta-xylosidase. None of the soluble carbon sources investigated produced the high titres of xylanase or beta-xylosidase induced by either oat straw for xylanse and beta-xylosidase was 2% and the optimum spore inoculum was between 10(6) and 10(7) spores/mL(-1) final concentration. The level of xylanse activity obtained in the culture filtrates of the mutant was a remarkable 820 U mL(-1) when the reducing sugar released was measured by the dinitrosalicylic acid method. This enzyme titre would appear to be the highest reported so far. The xylanases system contained the correct balance of enzymes to effect extensive hydrolysis of oat spelt xylan. The protease titre was very low.     

Improvement of isoflavone aglycones production using β-glucosidase secretory produced in recombinant Aspergillus oryzae

β-Glucosidase (BGL1) from Aspergillus oryzae was efficiently produced in recombinant A. oryzae using sodM promoter-mediated expression system. The yield of BGL1 was 960 mg/l in liquid culture, which is 20-fold higher than the yield of BGL1 produced using the yeast Saccharomyces cerevisiae. Recombinant BGL1 converted isoflavone glycosides into isoflavone aglycones more efficiently than β-glucosidase from almond. In addition, BGL1 produced isoflavone aglycones even in the presence of the insoluble form of isoflavone glycosides.     

Heterogeneity of lotus rhizome starch granules as revealed by α-amylase degradation

Lotus (Nelumbo nucifera Gaertn.) rhizome starch granules have an elongated oval shape with the hilum located at one end. The morphologic characteristics were used as a direction anchor to study the heterogeneity of molecular organization of starch granules using microscopy before and after partial digestion by bacterial α-amylase (Bacillus sp.) The partially digested granule showed a single, big eroded hole at the end distant from the hilum. The enzyme-attacked end was revealed to be the loosely packed end and to be the weak point for enzyme hydrolysis. The α-amylase hydrolyzed the loosely packed central part of the granule faster than the densely packed periphery, and left an empty shell with a fish-bone-like tunnel inside. The periphery was more resistant to amylase hydrolysis and had strong birefringence. For the whole starch granule, the selectivity of α-amylase hydrolysis was low for the crystalline and amorphous regions and for amylose and amylopectin molecules. This study elucidated that the molecular organization of lotus rhizome starch granules was heterogeneous.     

The sequence and phylogenesis of the α-globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon) and Cyprus mouflon (Ovis aries ophion)

In order to investigate the polymorphism of α-globin chain of hemoglobin amongst caprines, the linked ^Iα and ^IIα globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5′ and 3′ untranslated regions. European and Cyprus mouflons, which do not show polymorphic α globin chains, had almost identical α globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different α genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the ^Iα-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.     

Seasonal reversibility of acclimation to irradiance in leaves of common box (Buxus sempervirens L.) in a deciduous forest

Understorey shade plants are seasonally exposed to dramatic changes in light conditions in deciduous forests related with the dynamics of the overstorey leaf phenology. These transitions are commonly followed by changes in herb plant communities, but shade-tolerant evergreen species must be able to adapt to changing light conditions. In this work we checked the photoprotective responses of evergreen species to acclimate to the shady summer environment and reversibly de-acclimate to a more illuminated environment after leaf fall on deciduous overstoreys. For that purpose we have followed the process of light acclimation in leaves of common box (Buxus sempervirens) during the winter to spring transition, which decrease irradiance in the understorey, and conversely during the transition from summer to autumn. Four parameters indicative of the structure and degree of acclimation of the photosynthetic apparatus have been studied: chlorophyll a/b ratio which is supposed to be inversely proportional to the antenna size, α/β-carotene which increases in shade acclimated leaves and the pools of α-tocopherol and xanthophyll cycle pigments (VAZ) which are two of the main photoprotection mechanisms in plants. Among these parameters, chlorophyll a/b ratio and VAZ pool responded finely to changes in irradiance indicating that modifications in the light harvesting size and photoprotective capacity contribute to the continuous acclimation and de-acclimation of long-lived evergreen leaves.     

Upregulation of tumor necrosis factor-alpha expression by trans10-cis12 conjugated linoleic acid enhances phagocytosis of RAW macrophages via a peroxisome proliferator-activated receptor gamma-dependent pathway

The aim of this study was to examine whether tumor necrosis factor (TNF)-alpha expression in the phagocytic activity of RAW macrophages by trans10-cis12 (10t-12c) conjugated linoleic acid (CLA) is associated with peroxisome proliferator-activated receptor gamma (PPARgamma) activation. 10t-12c CLA induced the TNF-alpha expression in RAW macrophages. Phagocytic activity of naive RAW macrophages was increased either by recombinant mouse (rm) TNF-alpha or by culture supernatant from 10t-12c CLA-treated RAW macrophages. This phagocytic activity was inhibited by addition of anti-rmTNF-alpha polyclonal antibody (pAb). 10t-12c CLA also increased the level of PPARgamma protein and mRNA in RAW macrophages. When naive RAW macrophages were incubated with the culture supernatant from RAW macrophages treated with 10t-12c CLA plus GW 9662, a PPARgamma antagonist, their phagocytic activity was significantly inhibited. In addition, GW 9662 antagonized the effect of 10t-12c CLA in stimulating TNF-alpha expression. These results suggest that 10t-12c CLA modulates the phagocytic activity of RAW macrophages by upregulating TNF-alpha expression via a PPARgamma-dependent pathway.     

Metabolism of prebiotic products containing beta(2-1) fructan mixtures by two Lactobacillus strains

The capacity of two probiotic strains, isolated from human breast milk, to use several beta(2-1) fructan mixtures as carbon and energy source in in vitro cultures has been tested. Results showed that both strains, Lactobacillus gasseri CECT5714 and Lactobacillus fermentum CECT5716, reached higher growth levels on culture media containing fructooligosaccharide mixtures produced by enzymatic synthesis, compared to those obtained by inulin hydrolysis. Furthermore, the shortest beta(2-1) fructan, kestose, was the only prebiotic compound in the mixtures significantly metabolized in all growth media tested. Analysis of short-chain fatty acid production showed no correlation between the fatty acid profile produced and the carbon source used in each experiment. These data could serve to select appropriate beta(2-1) fructans to be used as prebiotics for L. gasseri CECT5714 and L. fermentum CECT5716 and to design suitable symbiotic food products containing the mentioned lactobacilli.     

Design, synthesis, and evaluation of a novel series of alpha-substituted phenylpropanoic acid derivatives as human peroxisome proliferator-activated receptor (PPAR) alpha/delta dual agonists for the treatment of metabolic syndrome

A series of alpha-alkyl-substituted phenylpropanoic acids was prepared as dual agonists of peroxisome proliferator-activated receptors alpha and delta (PPARalpha/delta). Structure-activity relationship studies indicated that the shape of the linking group and the shape of the substituent at the distal benzene ring play key roles in determining the potency and the selectivity of PPAR subtype transactivation. Structure-activity relationships among the amide series (10) and the reversed amide series (13) are similar, but not identical, especially in the case of the compounds bearing a bulky hydrophobic substituent at the distal benzene ring, indicating that the hydrophobic tail part of the molecules in these two series binds at somewhat different positions in the large binding pocket of PPAR. alpha-Alkyl-substituted phenylpropanoic acids of (S)-configuration were identified as potent human PPARalpha/delta dual agonists. Representative compounds exhibited marked nuclear receptor selectivity for PPARalpha and PPARdelta. Subtype-selective PPAR activation was also examined by analysis of the mRNA expression of PPAR-regulated genes.     

Optimization of culture conditions for the production of an extracellular ribonuclease by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a benchtop bioreactor

SummarySelf-directing optimization was successfully employed to determine the optimal combination of engineering parameters, viz., pH, aeration rate and agitation rate, for extracellular ribonuclease production by Aspergillus niger SA-13-20 in a batch bioreactor. Maximal RNase production of 5.38 IU ml^–1 was obtained at controlled pH of 2.33, aeration rate of 1.67 v/v/m and agitation rate of 850 rev/min. The effect of oxygen on the fermentation was also investigated. With increase in volumetric oxygen transfer coefficients (K_La), cell growth and RNase production first increased and then decreased. RNase production was further increased to 7.10 IU ml^–1 and the fermentation time was shortened from 96 to 72 h by controlling dissolved oxygen concentration at 10% saturation by aerating oxygen after about 28 h of fermentation under the above optimal condition. The kinetic model showed that RNase production by A. niger SA-13-20 was growth-associated.