Mining and identification of a biosynthetic gene cluster producing xanthocillin analogues from Penicillium chrysogenum MT-40, an endophytic fungus of Huperzia serrata / 生物工程学报

Xanthocillin is a unique natural product with an isonitrile group and shows remarkable antibacterial activity. In this study, the genome of an endophytic fungus Penicillium chrysogenum MT-40 isolated from Huperzia serrata was sequenced, and the gene clusters with the potential to synthesize xanthocillin analogues were mined by local BLAST and various bioinformatics analysis tools. As a result, a biosynthetic gene cluster (named for) responsible for the biosynthesis of xanthocillin analogues was identified by further heterologous expression of the key genes in Aspergillus oryzae NSAR1. Specifically, the ForB catalyzes the synthesis of 2-formamido-3-(4-hydroxyphenyl) acrylic acid, and the ForG catalyzes the dimerization of 2-formamido-3-(4-hydroxyphenyl) acrylic acid to produce the xanthocillin analogue N, N'-(1, 4-bis (4-hydroxyphenyl) buta-1, 3-diene-2, 3-diyl) diformamide. The results reported here provide a reference for further discovery of xanthocillin analogues from fungi.

Which are the best murine models to study Eosinophilic Chronic Rhinosinusitis? A contemporary review

Abstract Objective: Mechanisms that lead to Eosinophilic Chronic Rhinosinusitis (ECRS) are not fully established in the literature. It is desirable to assess ECRS in a model that embraces most of the related events. This article reviewed the murine models for ECRS and compared them regarding eosinophilic polypoid formation. Methods: The authors reviewed the articles that included the terms "chronic rhinosinusitis" OR "chronic sinusitis" AND "animal model". We analyzed articles in English that evaluated both the number of polyps and the number of eosinophils in the sinus mucosa of mouse models. Results: We identified a total of 15 articles describing different models of ECRS that used BALB/c or C57BL/6 mice, and different triggers/stimulants such as Staphylococcus aureus Enterotoxin B (SEB) + Ovalbumin (OVA); House Dust Mite (HDM) ± Ovalbumin (OVA); and Aspergillus oryzae Protease (AP) + Ovalbumin (OVA). OVA associated with SEB was the commonest protocol to induce ECRS in both BALB/c and C57BL/6 mice, and it produced a robust response of eosinophilic nasal polyps in both. AP + OVA protocol also led to a good ECRS response. The other models were not considered adequate to produce eosinophilic polyps in mice. Conclusion: In conclusion, OVA associated with SEB seems to produce the most robust eosinophilic sinonasal inflammation.

Improved protease activity of Pixian broad bean paste with cocultivation of Aspergillus oryzae QM-6 and Aspergillus niger QH-3

BACKGROUND: The preparation of broad bean koji is a key process in the production of Pixian broad bean paste (PBP). Protease is essential for the degradation of proteins during PBP fermentation. To obtain broad bean koji with high protease activity using the cocultivated strains of Aspergillus oryzae QM-6 (A. oryzae QM-6) and Aspergillus niger QH-3 (A. niger QH-3), the optimization of acid and neutral protease activities was carried out using Box­Behnken design with response surface methodology (RSM). RESULTS: The optimum conditions were found to be as follows: inoculation proportion (X1), 3:1 (A. oryzae QM-6: A. niger QH-3, w/w); culture temperature (X2), 33°C; inoculum size (X3), 0.5% (w/w); incubation time (X4), 5 d. The acid and neutral protease activities were 605.2 ± 12.4 U/g and 1582.9 ± 23.7 U/g, respectively, which were in good agreement with the predicted values. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles revealed that the broad bean koji extracellular proteins in the case of cocultivation were richer compared to those in the case of A. oryzae QM-6 or A. niger QH-3 strain only. In addition, the free amino acids (FAAs) in the fermentation product were 55% higher in the cocultivation process than in that involving only A. oryzae QM-6, further confirming the diversity of proteases in the fermentation products. CONCLUSIONS: The optimal conditions of koji-making in PBP were obtained using RSM. The cocultivation of A. oryzae and A. niger increases the overall enzyme activities in the culture medium and the FAAs content, which would thus have potential application in the PBP industry.

A new polyketide from endophytic fungus Aspergillus oryzae derived from Paris polyphylla var. yunnanensis / 中草药

Objective: To study the secondary metabolites of the endophytic fungus Aspergillus oryzae from Paris polyphylla var. yunnanensis in order to find new compounds. Methods: The endophytic fungus A. oryzae was fermented by liquid fermentation. After extraction, silica gel and macroporous adsorption resin were used to separate and purify the extract. The structures of the compounds were identified according to their physical and chemical properties and spectroscopic data. Results: Three compounds were isolated and their structures were identified as 3-amino-4,5-dihydroxy-4,6-dimethyl-2-(2-methylbutanoyl)cyclohex-2-enone (1), 12-N-methyl- cyclo-(L-tryptophyl-L-phenylalanyl) (2) and ditryptophenaline (3). Conclusion: Compound 1 is a new polyketide named asperpolyketide A.

Two new α-pyronoids from endophytic fungus Aspergillus oryzae derived from Paris polyphylla var. yunnanensis / 中草药

Objective: To study the chemical composition and structure of the secondary metabolites of the endophytic fungus Aspergillus oryzae from Paris polyphylla var. yunnanensis. Methods: A. oryzae was fermented by liquid fermentation. After extraction, it was separated and purified by various chromatography methods. The structure of the compounds was identified according to the physical and chemical properties and spectral data. Results: Four compounds were isolated and their structures were identified as 4-hydroxy-6-[(2S,3S)-3-hydroxybutan-2-yl]-3-methyl-2H-pyran-2-one (1), (R)-4-hydroxy-6-(1-hydroxy-2- methylpropyl)-3- methyl-2H-pyran-2-one (2), flufuran (3) and flufuran methyl ester (4). Conclusion: Compounds 1 and 2 are new α-pyronoids named asper-α-pyranone A and asper-α-pyranone B.

Desarrollo y capacidad de remoción de los pesticidas atrazina, clorpirifós y endosulfán por parte de cepas de Aspergillus sección Flavi no toxigénicas / Development and removal ability of non-toxigenic Aspergillus section Flavi in presence of atrazine, chlorpyrifos and endosulfan

This study evaluated the in vitro effect of three concentrations of atrazine, chlorpyrifos and endosulfan on the growth parameters of four non-toxigenic Aspergillus section Flavi strains. The ability of the strains to remove these pesticides in a synthetic medium was also determined. Growth parameters were measured on soil extract solid medium supplied with 5,10 and 20mg/l of each pesticide, and conditioned to -0.70, -2.78, -7.06 and -10.0 water potential (MPa). Removal assays were performed in Czapek Doc medium (CZD) supplied with 20mg/l of each pesticide under optimal environmental conditions (-2.78 of MPa and 25 °C). The residual levels of each pesticide were detected by the reversed-phase HPLC/fluorescence detection system. The lag phases of the strains significantly decreased in the presence of the pesticides with respect to the control media. This result indicates a fast adaptation to the conditions assayed. Similarly, the mycelial growth rates in the different treatments increased depending on pesticide concentrations. Aspergillus oryzae AM 1 and AM 2 strains showed high percentages of atrazine degradation (above 90%), followed by endosulfan (56 and 76%) and chlorpyrifos (50 and 73%) after 30 days of incubation. A significant (p <0.001) correlation (r = 0.974) between removal percentages and growth rate was found. This study shows that non-toxigenic Aspergillus section Flavi strains from agricultural soils are able to effectively grow in the presence of high concentrations of atrazine, chlorpyrifos and endosulfan under a wide range of MPa conditions. Moreover, these strains have the ability to remove high levels of these pesticides in vitro in a short time.

En este estudio se evaluó los efectos in vitro de 3 concentraciones de atrazina, clorpirifós y endosulfán sobre los parámetros de crecimiento de 4 cepas no toxigénicas de Aspergillus sección Flavi. También se evaluó la capacidad de las cepas de remover los pesticidas. Los parámetros de crecimiento se ensayaron en medio agar extracto de suelo suplementado con 5, 10 y 20mg/l de cada pesticida y acondicionado a -0.70, -2.78, -7.06 y -10.0 de potencial de agua (MPa). Los ensayos de remoción se realizaron en medio Czapek Dox con 20mg/l de cada pesticida bajo condiciones óptimas de crecimiento (-2.78 de MPa y 25 °C). Los niveles residuales de atrazina, clorpirifós y endosulfán se detectaron en un sistema HPLC con detección por fluorescencia. La fase de latencia de las cepas disminuyó significantemente en presencia de los pesticidas, indicando una rápida adaptación a dichas condiciones. La velocidad de crecimiento se incrementó considerablemente dependiendo de la concentración de pesticida. Las cepas Aspergillus oryzae AM1 y AM2 mostraron porcentajes elevados de degradación de atrazina (aproximadamente el 90%), seguidos por endosulfán (56 y 76%) y clorpirifós (50 y 73%). Se observó una correlación (r = 0.974) significante (p <0.001) entre el porcentaje de pesticida removido y la velocidad de crecimiento. Este estudio muestra que cepas no-toxigénicas de Aspergillus sección Flavi aisladas de suelos agrícolas desarrollan eficientemente en presencia de altas concentraciones de atrazina, clorpirifós y endosulfán en un amplio rango de MPa. Además, presentan capacidad de remover in vitro altos niveles de pesticidas en corto tiempo.

Brewer’s rice - A potential substrate for cosmeceutical bio-ingredient production by solid state fermentation using Aspergillus oryzae

Aims@#Brewer’s rice is one of the by-products from rice processing industry that is rich in bioactive compounds but currently underutilized. Exploitation of agro-industrial by-products as substrates in solid-state fermentation processes provides value-addition to these underutilized by-products. The purpose of this research is to evaluate the potentiality of brewer’s rice as a source of cosmeceutical or cosmetic bio-ingredient by utilizing solid-state fermentation process. @*Methodology and results@#Brewer’s rice was submitted to solid-state fermentation with Aspergillus oryzae from MARDI’s Collection of Functional Food Culture (CFFC). Extracts of unfermented and fermented brewer’s rice were later subjected to determination of biological content and biological activities, as well as measurement of their phenolic and organic acids content. The extract of fermented brewer’s rice exhibited an increase in total phenolic and total flavonoid content and showed enhanced 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging and ferric-reducing activities. Additionally, it was also found that the tyrosinase and elastase inhibition activities of fermented brewer’s rice extract is significantly higher with nearly 7- and 57-fold, respectively, than the unfermented extract. Ferulic and kojic acid – two of the most important compounds in cosmeceutical formulations, were also detected in fermented brewer’s rice extract. @*Conclusion, significance and impact of study@#Antioxidant, anti-pigmentation and anti-wrinkle properties of brewer’s rice were successfully enhanced by fermentation with A. oryzae. Fermented brewer’s rice extract has high potential to be developed as functional bio-ingredient for cosmeceutical as well as nutraceutical products.

Local strains Aspergillus oryzae KKB4 and Rhizopus oryzae KP1R1 as a reducing and detoxifying agents for deoxynivalenol

Aims@#Deoxynivalenol is a type B trichothecene produced by Fusarium graminearum that can cause serious health problems in human and livestock. The present study aimed to reduce and detoxify deoxynivalenol using a local strain Aspergillus oryzae KKB4 and Rhizopus oryzae KP1R1. @*Methodology and results@#Corn as solid substrate artificially inoculated with F. graminearum bio 163252 to produce deoxynivalenol. Deoxynivalenol contaminated corn then inoculated with A. oryzae KKB4 and R. oryzae KP1R1. During fermentation, a decrease in deoxynivalenol levels is analyzed including loss of dry matter and glucosamine content. Deoxynivalenol was extracted from the substrate by solid phase extraction and quantified using high-performance liquid chromatography. The reduction of deoxynivalenol by A. oryzae KKB4 and R. oryzae KP1R1 were 65.91% and 56.82%, respectively after ten days of fermentation. Toxicity analysis revealed that residues of deoxynivalenol were not toxic to growth of Saccharomyces cerevisiae cells. @*Conclusion, significance and impact of study@#Local strains A. oryzae KKB4 and R. oryzae KP1R1 were able to reduce and detoxify deoxynivalenol in solid substrates. This study provides supporting data to control mycotoxin that is critical for food and feed safety.

The role of Aspergillus oryzae KKB4 in reducing and detoxifying aflatoxin B1 applied in moist-heated corn

Aims: The growth and metabolic activity of Aspergillus oryzae KKB4 in AFB1-contaminated corn and later coincided with AFB1 reduction and detoxification were investigated. Methodology and results: The decreasing of AFB1 amount by A. oryzae KKB4 could be clearly observed if the initial AFB1 concentration in corn was increased. Thus, moist-heated corn was artificially inoculated with Aspergillus flavus FNCC 62C7 to increase AFB1 content. AFB1-contaminated corn was applied as solid substrate and then inoculated with A. oryzae KKB4. During fermentation periods, the growth, metabolic activity, and AFB1 decline were investigated by glucosamine content, water content loss, and AFB1 concentration, respectively. The maximum growth was occurred in 4 thday at 1.499 ± 0.028 g glucosamine/ 100 g dry matter. The metabolic activity was going on up to the end of fermentation days, as shown as water content loss at 0.175 ± 0.007 g/g dry matter. In accordance with the growth and metabolic activity, the amount of AFB1 reduction was 37.04 ng AFB1/g dry matter during 5 days fermentation. According to toxicity analysis, it was found that the residues of AFB1 were not toxic to Bacillus megaterium cells. Conclusion, significance and impact of study: A. oryzae KKB4 is able to be applied in solid substrates as AFB1 reduction and detoxification agent. These lucrative effects are also important in relation with food and feed safety.

Characteristics of Korean Alcoholic Beverages Produced by Using Rice Nuruks Containing Aspergillus oryzae N159-1

Herein, nuruks derived from non-glutinous and glutinous rice inoculated with Aspergillus oryzae N159-1 (having high alpha-amylase and beta-glucosidase activities) were used to produce Korean alcoholic beverages. The resultant beverages had enhanced fruity (ethyl caproate and isoamyl alcohol) and rose (2-phenethyl acetate and phenethyl alcohol) flavors and high taste scores.

Purification, characterization and antiproliferative activity of L-asparaginase from Aspergillus oryzae CCT 3940 with no glutaminase activity

Objective To explore the anti-proliferative activity of purified L-asparaginase from Aspergillus oryzae CCT 3940 (A. oryzae). Methods L-asparaginase was produced by submerged fermentation and purified to electrophoresis homogeneity by ionic exchanged chromatography in a fast protein liquid chromatographic system. The purified enzyme was characterized and used for the antiproliferative assay against nine tumor cell lines and one non-tumor cell line. Results The free glutaminase L-asparaginase was purified 28.6 fold. L-asparaginase showed high stability under physiological condition, remaining stable in the pH range 7.0–8.0 after 1 h incubation at temperature range 30–45 °C. The Km and Vmax values of purified L-asparaginase were estimated as 0.66 mmol/L and 313 IU/mL, respectively. The purified enzyme could inhibit the growth of a broad range of human tumor cell lines at the concentrations studied. Also, the enzyme from A. oryzae CCT 3940 could inhibit tumor growth of leukemia cell line (K562) with a total growth inhibition value of (3.2 ± 2.5) IU/mL and did not inhibit the non-carcinogenic human cell line growth at the concentrations studied. Conclusions The sensitivity of the cells lines to purified L-asparaginase from A. oryzae CCT 3940 appeared to be concentration dependent affording a more significant decrease in cell growth than that observed for the commercial L-asparaginase from Escherichia coli. The L-asparaginase from A. oryzae CCT 3940 has a high potential for pharmaceutical exploitation in the treatment of leukemia.

Purification, characterization and antiproliferative activity of L-asparaginase from Aspergillus oryzae CCT 3940 with no glutaminase activity

Objective: To explore the anti-proliferative activity of purified L-asparaginase from Aspergillus oryzae CCT 3940 (A. oryzae). Methods: L-asparaginase was produced by submerged fermentation and purified to electrophoresis homogeneity by ionic exchanged chromatography in a fast protein liquid chromatographic system. The purified enzyme was characterized and used for the anti-proliferative assay against nine tumor cell lines and one non-tumor cell line. Results: The free glutaminase L-asparaginase was purified 28.6 fold. L-asparaginase showed high stability under physiological condition, remaining stable in the pH range 7.0–8.0 after 1 h incubation at temperature range 30–45 °C. The Km and Vmax values of purified L-asparaginase were estimated as 0.66 mmol/L and 313 IU/mL, respectively. The purified enzyme could inhibit the growth of a broad range of human tumor cell lines at the concentrations studied. Also, the enzyme from A. oryzae CCT 3940 could inhibit tumor growth of leukemia cell line (K562) with a total growth inhibition value of (3.2 ± 2.5) IU/mL and did not inhibit the non-carcinogenic human cell line growth at the concentrations studied. Conclusions: The sensitivity of the cells lines to purified L-asparaginase from A. oryzae CCT 3940 appeared to be concentration dependent affording a more significant decrease in cell growth than that observed for the commercial L-asparaginase from Escherichia coli. The L-asparaginase from A. oryzae CCT 3940 has a high potential for pharmaceu-tical exploitation in the treatment of leukemia.

Extracted substrate colour as an indicator of fungal growth in solid state fermentation

Aims: In solid state fermentation (SSF), estimation of biomass is difficult as fungal mycelium penetrates deep and remains attached to the solid substrate particles. This study examines and evaluates a new technique based on colour changes of fermented substrates during SSF as an indicator for fungal growth. Methodology and Results: SSF refers to microbial fermentation, which takes place in the absence or near absence of free water, thus being close to the natural environment in which the selected microorganisms, especially fungi, are naturally adapted. Although many promising methods are available, the evaluation of microbial growth in SSF may sometimes become difficult, impractical, and inaccurate. Essentially, this remains another critical issue for monitoring growth. In this study, measurements of colour changes of fermented substrates during SSF are used as indicators for growth and this technique has a potential to be used to quantify growth of microbes. For the growth of Aspergillus awamori and A. oryzae on wheat bran, soybean hulls, and rapeseed meal, it was confirmed that colour changes were directly proportional to the fungal growth. This new approach is an important complementation to the existing techniques, especially for basic studies. The advantages of this method are its ease of use, fast, non-destructive, cheap, and requires no special and expensive reagents. The key finding is that the colorimetric technique demonstrated in this study provides good means to estimate growth than that obtained by visual observation or spores counting.

Solid-state fermentation of Acanthogobius hasta processing by-products for the production of antioxidant protein hydrolysates with Aspergillus oryzae

Functional properties and antioxidative activity of a protein hydrolysate prepared from Acanthogobius hasta processing by-product protein during solid-state fermentation with Aspergillus oryzae were investigated. Overall, protease activity increased with the degree of hydrolysis (DH) decreased during solid-state fermentation. All the protein hydrolysate had excellent solubility, possessed interfacial properties, and varying degrees of antioxidant activity which were governed by their concentrations and DH, molecular weight distribution and amino acid composition. After 5 days fermentation, the DH of the protein hydrolysate was 31.23%. The protein hydrolysate had the highest total hydrophobic amino acid content, the highest DPPH scavenging activity, reducing power, and the chelating activity. The radical-scavenging activity of the hydrolysates at 6 mg/mL was 78.6%. The reducing power of protein hydrolysate at the range of 0-6 mg/mL was lower than that of BHA at the range of 0-60 µg/mL, while the chelating activity of APs was similar to that of BHA at the range of 0-60 µg/mL. Moreover, the protein hydrolysate showed good emulsifying and foaming properties over a wide pH range from 2 to 12. Therefore, solid state fermentation provided a suitable and low-cost method for converting Acanthogobius hasta processing by-product protein into antioxidant protein hydrolysates.

Soluble extract of soybean fermented with Aspergillus oryzae GB107 inhibits fat accumulation in cultured 3T3-L1 adipocytes

BACKGROUND/OBJECTIVES: This study was conducted to investigate the effects of fermented soybean (FS) extract on adipocyte differentiation and fat accumulation using cultured 3T3-L1 adipocytes. MATERIALS/METHODS: 3T3-L1 adipocytes were treated with FS and nonfermented soybean (NFS) extract during differentiation for 10 days in vitro. Oil red O staining was performed and glycerol-3-phosphate dehydrogenase (GPDH) activity was measured for analysis of fat accumulation. Expressions of adipogenic genes were measured. RESULTS: Soluble extract of soybean fermented with Aspergillus oryzae GB107 contained higher levels of low-molecular-weight protein than conventional soybean protein did. FS extract (50 microg/ml) inhibited adipocyte differentiation and fat accumulation during differentiation of 3T3-L1 preadipocytes for 10 days in vitro. Significantly lower GPDH activity was observed in differentiated adipocytes treated with the FS extract than those treated with NFS extract. Treatment with FS extract resulted in decreased expression levels of leptin, adiponectin, and adipogenin genes, which are associated with adipogenesis. CONCLUSIONS: This report is the first to demonstrate that the water-soluble extract from FS inhibits fat accumulation and lipid storage in 3T3-L1 adipocytes. Thus, the soybean extract fermented with A. oryzae GB107 could be used to control lipid accumulation in adipocytes.

Aspergillus Associated with Meju, a Fermented Soybean Starting Material for Traditional Soy Sauce and Soybean Paste in Korea

Aspergillus is an important fungal genus used for the fermentation of Asian foods; this genus is referred to as koji mold in Japan and China. A. oryzae, A. sojae, and A. tamari are used in the production of miso and shoyu in Japan, but a comprehensive taxonomic study of Aspergillus isolated from Meju, a fermented soybean starting material for traditional soy sauce and soybean paste in Korea, has not been conducted. In this study, various Aspergillus species were isolated during a study of the mycobiota of Meju, and the aspergilli were identified based on phenotypic characteristics and sequencing of the beta-tubulin gene. Most strains of Aspergillus were found to belong to the following sections: Aspergillus (n = 220), Flavi (n = 213), and Nigri (n = 54). The most commonly identified species were A. oryzae (n = 183), A. pseudoglaucus (Eurotium repens) (n = 81), A. chevalieri (E. chevalieri) (n = 62), A. montevidensis (E. amstelodami) (n = 34), A. niger (n = 21), A. tamari (n = 15), A. ruber (E. rubrum) (n = 15), A. proliferans (n = 14), and A. luchuensis (n = 14); 25 species were identified from 533 Aspergillus strains. Aspergillus strains were mainly found during the high temperature fermentation period in the later steps of Meju fermentation.

Optimization of Culture Conditions for the Production of Extracellular Cellulases via Solid State Fermentation.

Aim: The aims of the present study were to screen different filamentous fungi for extracellular cellulases production and to optimize solid-state fermentation medium and culture conditions to enhance cellulases production. Study Design: Using agro-industrial waste as raw material for the production of cellulases by a hyper cellulase producing fungus and evaluating the influence of various parameters to design a suitable SSF process for cellulase production. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between January 2013 and October 2013. Methodology: Different filamentous fungi were grown and maintained on potato dextrose agar slants at 28ºC for 7 days. The spores were washed down by distilled water. Then, 2.0 ml aliquots were used to inoculate 250 ml Erlenmeyer flasks, containing rice straw as the only carbon source. The inoculated flasks were incubated for 5 days at 28ºC. The enzymes were extracted by mixing homogenously the fermented substrate with 50 ml citrate phosphate buffer (0.1 M, pH 5.0) and agitated (150 rpm) for 1 hr. Pooled extracts were centrifuged at 5000 rpm for 15min and the clear supernatant was used as a source of extracellular enzyme. Results: Aspergillus oryzae NRRL 3484 exhibited relatively higher cellulases production. The optimum incubation period, temperature, and initial moisture level were reported on the 7th day, at 28°C, and 70%, respectively. Peptone proved to be the suitable nitrogen source followed by yeast extract, while pH 5.0 was ideal for cellulases production. Conclusion: Using ligninolytic fungi, including their enzymes, may be one potential alternative to provide a more practical and environmental-friendly approach for enhancing the nutritive value of rice straw. Moreover, the application of ligninolytic fungi or their enzymes combined with chemical pre-treatments to rice straw may be an alternative way to shorten the period of the incubation times and (or) decrease the amount of chemicals, effecting some synergy.

Utilization of Agro-Industrial Waste Residues for the Production of Amylase from Aspergillus oryzae IIB-6.

Aims: The aim of this study was to evaluate different solid substrates and moistening agents for the production of an extracellular 1, 4-α-D-glucan glucohydrolase from a newly isolated Aspergillus oryzae IIB-6 by solid state fermentation and optimization of culture conditions for the maximal production of enzyme. Study Design: Six different agro-industrial waste residues (rice straw, rice bran, corn flakes, wheat bran, wheat flakes, and grinded wheat kernel) were procured from the local market. These substrates (10 g) were moistened (1:1) with different moistening agents (distilled water, tap water, mineral salts solution (FeSO4.7H2O 0.02, MgSO4.7H2O 1.0, (NH4)2SO4 4.0, KH2PO4 0.6, K2HPO4 1.4 mg/gds at pH 5), 0.1 N HCl, sodium acetate buffer (pH 5.5), sodium phosphate buffer pH 7.5) and screened for the production of 1,4-α- D-glucan glucohydrolase for 96 hours in static cultures. The substrate and moistening agent that gave maximum enzyme production were selected and their fermentation conditions were further optimized. Place and Duration of Study: Institute of Industrial Biotechnology (IIB), GC University Lahore, Kachery Road Lahore, Pakistan, between February 2011 and March 2012. Methodology: The levels of selected solid substrate, moistening agent and fermentation conditions such as pH, temperature, time of incubation, inoculum size etc. were optimized by one variable at a time method. Results: Wheat bran at the level of 5 g and mineral salts solution containing FeSO4.7H2O 0.016, MgSO4.7H2O 0.8, (NH4)2SO4 3.5, KH2PO4 0.48, K2HPO4 1.12 mg/gds gave relatively best enzyme production. The maximum enzyme activity 7800 U/gds (407 Umg-1 protein) were achieved when wheat bran with 80% moisture content was incubated at 30°C, pH 5, after inoculating with 10% spore suspension (1.2×106 CFU/gds) for 72 h. Conclusion: Aspergillus oryzae IIB-6 was a good producer of 1,4-α-D-glucan glucohydrolase in wheat bran medium containing mineral salts as an additional trace elements so that it can be used for biotechnological purposes.

Purification and Partial Characterization of a Non-specific Acid Phosphatase Degrading NAD from Aspergillus oryzae NRRL447.

A non specific acid phosphatase from Aspergillus oryzae NRRL447 catalyzes the phosphate hydrolysis from nicotinamide adenine dinucleotide forming nicotinamide riboside, adenosine and Pi as the final products of the reaction. The enzyme was purified to homogeneity by a sequential treatment of acetone fractionation, DEAE-cellulose chromatography and gel filtration chromatography. The enzyme was purified 400-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 52 kDa. The enzyme displayed maximum activity at pH 5.0 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg2+, Ca2+ whereas inhibited strongly by F-, Mo04 -, Cu2+ and Fe2+. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The substrate concentration-activity relationship is the hyperbolic type and the apparent Km for NAD+ was 6.25 x 10-4 M.

Action of a pancreatin and an Aspergillus oryzae protease on whey proteins: correlation among the methods of analysis of the enzymatic hydrolysates

The objectives of this study were to hydrolyze whey proteins using a pancreatin and an Aspergillus oryzae protease; to evaluate the degree of hydrolysis (DH) and the peptide profile; and to establish the correlations among the analytical methods. Ten hydrolysates were prepared at different reaction times and the highest DH was obtained by the protein content method. Good correlations (r > 0.87) between the methods of formaldehyde and orthophthalaldehyde (OPA), formaldehyde and osmometry as well as osmometry and OPA were observed using pancreatin. Similar results were obtained between OPA and soluble protein content for the A. oryzae protease. The action of pancreatin produced the highest contents of di- and tripeptides (9.07, 7.12 and 6.46%) and the lowest of large peptides (42.43, 41.33 and 41.13%), after 3, 4 and 5 h of hydrolysis, respectively. Using pancreatin, the DH measured by formol titration and OPA was positively correlated with medium peptide content and negatively correlated with large peptide content. For the A. oryzae protease, a strong negative correlation was observed between the large peptide content and the DH measured by the OPA method.