Mouna Sahnoun

Aspergillus oryzae S2 alpha-amylase production under solid state fermentation: Optimization of culture conditions

Aspergillus oryzae S2 was assayed for alpha-amylase production under solid state fermentation (SSF). In addition to AmyA and AmyB already produced in monitored submerged culture, the strain was noted to produce new AmyB oligomeric forms, in particular a dominant tetrameric form named AmyC. The latter was purified to homogeneity through fractional acetone precipitation and size exclusion chromatography. SDS-PAGE and native PAGE analyses revealed that, purified AmyC was an approximately 172 kDa tetramer of four 42 kDa subunits. AmyC was also noted to display the same NH2-terminal amino acid sequence residues and approximately the same physico-chemical properties of AmyA and AmyB, to exhibit maximum activity at pH 5.6 and 60 °C, and to produce maltose and maltotriose as major starch hydrolysis end-products. Soyabean meal was the best substitute to yeast extract compared to fish powder waste and wheat gluten waste. AmyC production was optimized under SSF using statistical design methodology. Moisture content of 76.25%, C/N substrate ratio of 0.62, and inoculum size of 10(6.87) spores allowed maximum activity of 22118.34 U/g of dried substrate, which was 33 times higher than the one obtained before the application of the central composite design (CCD).

Purification and biochemical characterization of a novel thermostable lichenase from Aspergillus niger US368.

New β-1,3;1,4-glucanase was purified from Aspergillus niger US368. The pure glucanase has a molecular mass of about 32 kDa. The N-terminal sequence of the purified enzyme (A-G-T-N-P-P-I-G-V) was determined. The optimum pH and temperature recorded for enzyme activity were 5 and 60 °C, respectively. It also displayed marked thermostability with a half-life of 30 min at 70 °C. At 37 °C, the enzyme showed 100% stability from pH 3 to 10. The Km and Vmax values exhibited by the enzyme on barley β-glucan were 0.62 mg ml(-1) and 34.46 U ml(-1), respectively. The enzyme is a retaining-one and was only active toward glucan containing β-1,3;1,4-linkages. The production of β-glucanase with barley flour as the sole carbon source was optimized. This is the first report on the purification and characterization of a β-1,3;1,4-glucanase from A. niger. This lichenase could be considered as a candidate for future application particularly in the animal feed industry.

Biocontrol of tomato plant diseases caused by Fusarium solani using a new isolated Aspergillus tubingensis CTM 507 glucose oxidase.

The present study focuses on the potential of glucose oxidase (GOD) as a promising biocontrol agent for fungal plant pathogens. In fact, a new GOD producing fungus was isolated and identified as an Aspergillus tubingensis. GOD (125 AU) has been found to inhibit Fusarium solani growth and spore production. Indeed, GOD caused the reduction of spores, the formation of chlamydospores, the induction of mycelial cords and the vacuolization of mycelium. In vivo assays, GOD acted as a curative treatment capable of protecting the tomato plants against F.xa0solani diseases. In fact, the incidence was null in the curative treatment with GOD and it is around 45% for the preventive treatment. The optimization of media composition and culture conditions led to a 2.6-fold enhancement in enzyme activity, reaching 81.48U/mL. This study has demonstrated that GOD is a potent antifungal agent that could be used as a new biofungicide to protect plants from diseases.

Improvement of cyclodextrin glycosyltransferase (CGTase) production by recombinant Escherichia coli pAD26 immobilized on the cotton

The cyclodextrin glycosyltransferase (CGTase) of the recombinants Escherichia coli pAD26 cells immobilized on cotton was optimally produced by statistical methodology. Primarily, carbon and nitrogen sources were selected by one-factor-at-a-time method. Wheat starch, Casamino acid, Edamin and Hy-soy were identified as the best nutrients. These sources were secondly confirmed by Plackett-Burman design (fifteen variables were studied with sixteen experiments), as the most significant components with respect to CGTase production. In the third step, concentration of most significant factors and their interaction were optimized with a Box-Behnken experimental design. Under the optimized conditions (agitation 200 rpm, yeast extract concentration 20 g/L, wheat starch concentration 10 g/L and Hy-soy concentration 2.5 g/L), CGTase yield 145.11 U/mL was 3.6 and 23 folds higher than those obtained by the use of the initial conditions (39.77 U/mL) and free cells (6.37 U/mL), respectively.

Effect of Aspergillus oryzae CBS 819.72 α-amylase on rheological dough properties and bread quality

An optimum Aspergillus oryzae CBS 819.72 α-amylase production in a laboratory-scale fermentor using a wheat grinding by-product as a sole carbon source (340 U/mL) was obtained after 48 h of batch fermentation under an agitation rate of 900 rpm and a pH maintained constant for 24 h. The application of this α-amylase preparation at an adequate concentration showed positive effects on dough properties and bread quality. Extensographic analysis revealed that while this addition induced a significant increase in maximal dough resistance to extension and area below the curve (energy), it brought a substantial decrease in extensibility. Farinographic results revealed small decreases in terms of time development, water absorption, and dough stability. Bread volume was also observed to undergo a significant increase.

Optimization of submerged Aspergillus oryzae S2 α-amylase production

Use of 4 agro-industrial by products and organic materials as nitrogen sources for production of Aspergillus oryzae S2 α-amylase in liquid culture was investigated. The 2 agro-industrial byproducts maltose and saccharose, and also lactose and starch were individually evaluated for use as carbon sources. A Box-Behnken experimental design was used to determine optimal conditions for production of α-amylase. A maximum amylase activity of 750 U/mL was obtained at a temperature of 24°C, a urea concentration of 1 g/L, and a C/N ratio of 2. Laboratory scale application of optimal conditions in a 7 L fermentor produced a final α-amylase activity of 770 U/mL after 3 days of batch cultivation. Addition of 10% starch to the culture medium each 12 h immediately after the stationary phase of cell growth led to a production yield of 1,220 U/mL at the end of fed-batch cultivation.

Purification, biochemical characterization and antifungal activity of a novel Aspergillus tubingensis glucose oxidase steady on broad range of pH and temperatures.

This study was carried out to evaluate the in vitro and in vivo antifungal efficiency of Aspergillus tubingensis CTM 507 glucose oxidase (GOD) against plant pathogenic fungi. GOD displayed a wide inhibitory spectrum toward different fungi at a concentration of 20xa0AU. The GOD had a strong inhibitor effect on mycelia growth and spore germination of Pythium ultimum. Interestingly, the GOD exhibited a potent in vivo antifungal effect against P. ultimum responsible for potato plants disease. The antifungal GOD was purified 13-fold with 27xa0% yield and a specific activity of 3435xa0U/mg. The relative molecular mass of the GOD was 180xa0kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GOD activity was optimum at pH 4.5 and 60xa0°C. It was found to be stable over a large pH range (3–9). It also displayed a marked thermostability with a 50-min half-life at 65xa0°C. The 10 residues of the N-terminal sequence of the purified GOD (S–K–G–S–A–V–T–T–P–D) showed no homology to the other reported GOD, identifying a novel GOD. FTIR spectroscopic analysis revealed the presence of C–O and C=O groups corresponding to a d-glucono-lactone. The findings indicated that GOD is the first A. tubingensis-produced fungicide ever reported to exhibit such promising biological properties. It could become a natural alternative to synthetic fungicides to control certain important plant microbial diseases.

Aspergillus Oryzae S2 α-Amylase Domain C Involvement in Activity and Specificity: In Vivo Proteolysis, Molecular and Docking Studies

We previously reported that Aspergillus oryzae strain S2 had produced two α-amylase isoforms named AmyA and AmyB. The apparent molecular masses revealed by SDS-PAGE were 50 and 42 kDa, respectively. Yet AmyB has a higher catalytic efficiency. Based on a monitoring study of the α-amylase production in both the presence and absence of different protease inhibitors, a chymotrypsin proteolysis process was detected in vivo generating AmyB. A. oryzae S2 α-amylase gene was amplified, cloned and sequenced. The sequence analysis revealed nine exons, eight introns and an encoding open reading frame of 1500 bp corresponding to AmyA isoform. The amino-acid sequence analysis revealed aY371 potential chymotrypsin cleaving site, likely to be the AmyB C-Terminal end and two other potential sites at Y359, and F379. A zymogram with a high acrylamide concentration was used. It highlighted two other closed apparent molecular mass α-amylases termed AmyB1 and AmyB2 reaching40 kDa and 43 kDa. These isoforms could be possibly generated fromY359, and F379secondary cut, respectively. The molecular modeling study showed that AmyB preserved the (β/α)8 barrel domain and the domain B but lacked the C-terminal domain C. The contact map analysis and the docking studies strongly suggested a higher activity and substrate binding affinity for AmyB than AmyA which was previously experimentally exhibited. This could be explained by the easy catalytic cleft accessibility.

Enzymatically hydrolysed, acetylated and dually modified corn starch: physico-chemical, rheological and nutritional properties and effects on cake quality

Corn starch was treated by enzymatic hydrolysis with Aspergillus oryzae S2 α-amylase, acetylation with vinyl acetate, and dual modification. The dual modified starch displayed a higher substitution degree than the acetylated starch and lower reducing sugar content than the hydrolysed starch. The results revealed that the cooling viscosity and amylose content of those products decrease (Pxa0<xa00.05). An increase in moisture, water, and oil absorption capacity was observed for the acetylated starch and, which was less pronounced for the enzymatically hydrolysed starch but more pronounced for the enzymatically hydrolysed acetylated product. The latter product underwent an increase in resistant starch content, which is induced by a rise in hydrolysis time to attain about 67xa0% after 1xa0h of reaction. The modified starch samples were added to cake formulations at 5 and 10xa0% concentrations on a wheat flour basis and compared to native starch. The results revealed that when applied at 5xa0% concentrations, the modified starches reduced the hardness, cohesion, adhesion and chewiness of baked cakes and enhanced their elasticity, volume, height, crust color, and appearance as compared to native starch. These effects were more pronounced for the cake incorporating the dually modified starch.

Citrus flavonoids collectively dominate the α-amylase and α-glucosidase inhibitions

Abstract A series of citrus flavonoids were evaluated for α-amylase and α-glucosidase inhibitory activities in vitro. The inhibitory capacities of flavanone glycosides were greater than those of polymethoxy flavones for α-amylase. Naringin exhibited the most potent α-glucosidase inhibitory activity with IC50 0.55 μM and was about 196.83 times more active than acarbose. Poncirin led to a 43-fold improvement in α-amylase inhibition over acarbose. The double-reciprocal (Lineweaver-Burk) plot confirms a competitive inhibition mode towards α-amylase and α-glucosidase activities. The inhibition activity was significantly lowered when citrus flavonoids were pre-incubated with starch. The binding site for naringin, poncirin, hesperidin, tangeretin in α-amylase showed a polar contact numbers, respectively, of 47, 54, 51, and 44 more than that involving acarbose. The hydrogen bonds formed between nobilitin and the key residue of Lys506 of α-glucosidase, namely Asn475:N-A:Lys506:O, might provide its extra affinity when compared to tangeritin. These findings provided a strong and rational reason to establish the selected citrus flavonoids capability as a therapeutic target for postprandial hyperglycaemia modulation.