Visualization of polypeptides including fragmented α-amylase in rice koji grains using mass spectrometry imaging.

The quality of rice koji greatly affects the quality of sake. To accurately evaluate the quality of rice koji, various approaches for the evaluation of rice koji are required. In this study, we directly and simultaneously visualized the distribution of polypeptides in rice koji using mass spectrometry imaging. We demonstrated four koji-specific polypeptides at m/z 4660, 6140, 8170, and 11,840 and one rice-derived polypeptide at m/z 5330. To identify the koji-specific polypeptides, extracts from rice koji were separated using tricine SDS-PAGE, and the band appeared to coincide with the polypeptide at m/z 11,840 was identified to be the N-terminal fragment of α-amylase. The polypeptide seemed to have no hydrolytic activity based on the primary structure of α-amylase. The polypeptide at m/z 11,840 seemed to coincide with the fragmented α-amylase was detected at the later stage of koji making (after 42 h). At the same period during koji making, the increasing rate of α-amylase activity decreased compared to that of glucoamylase activity, suggesting that α-amylase fragmentation possibly leads to the deceleration of the increase in α-amylase activity at the later stage of koji making. This is the first study to directly and simultaneously demonstrate the distribution of polypeptides in rice koji using mass spectrometry imaging and imply the relationship between α-amylase fragmentation and activity in rice koji.

Anticancer properties of dried-pericarp water extracts of Camellia japonica L. fermented with Aspergillus oryzae through regulation of IGFBP-2/mTOR pathway.

This study aimed to investigate the anticancer activity of dried-pericarp water extract of fermented C. japonicus (CJ). The dried-pericarp water extracts of CJ were fermented using Aspergillus oryzae and Saccharomyces cerevisiae at 30 °C and 35 °C. The anticancer activities of both water extracts fermented at 30 °C and 35 °C using A. oryzae against FaDu cells were remarkably changed compared with unfermented dried-pericarp water extract of CJ, which has no anticancer activity. Cleaved-PARP, caspase 3, and apoptotic cells stained with annexin V/PI were significantly increased by treatment with A. oryzae extracts fermented at 30 °C. The insulin-like growth factor-binding protein 2 (IGFBP-2) protein level and mTOR phosphorylation by A. oryzae fermented extracts (AOFE) were dramatically reduced, and the expression levels of IGFBP-2 and phosphorylated mTOR were significantly increased depending on the glucose concentrations in FaDu cells. These results suggested that the cell viabilities in AOFE were restored as the glucose concentrations increased. Furthermore, it was confirmed LC/MS/MS that the content of gallic acid was increased by fermentation of Aspergillus oryzae (5.596 ± 0.1746 µg/mg) compared to the unfermented extract (1.620 ± 0.0432 µg/mg). Based on these results, the anticancer effect of AOFE was achieved through inhibition of the IGFBP-2/mTOR signaling pathway. These results suggest that AOFE may be a potential treatment for head and neck cancer.

Biosynthetic Studies of Phomopsins Unveil Posttranslational Installation of Dehydroamino Acids by UstYa Family Proteins.

UstYa family proteins (DUF3328) are widely and specifically distributed in fungi. They are known to be involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and nonribosomal peptides, and possibly catalyze various reactions, including oxidative cyclization and chlorination. In this study, we focused on phomopsin A, a fungal RiPP consisting of unique nonproteinogenic amino acids. Gene knockout experiments demonstrated that three UstYa homologues, phomYc, phomYd, and phomYe, are essential for the desaturation of amino acid moieties, showing unprecedented function among UstYa family proteins. Sequence similarity network analysis indicated that their amino acid sequences are highly diverged and that most remain uncharacterized, paving the way for genome mining of fungal metabolites with unique modifications.

Selenium chalcogen bonds are involved in protein-carbohydrate recognition: a combined PDB and theoretical study.

In this manuscript the ability of selenium carbohydrates to undergo chalcogen bonding (ChB) interactions with protein residues has been studied at the RI-MP2/def2-TZVP level of theory. An inspection of the Protein Data Bank (PDB) revealed SeA (A = O, C and S) intermolecular contacts involving Se-pyranose ligands and ASP, TYR, SER and MET residues. Theoretical models were built to analyse the strength and directionality of the interaction together with "Atoms in Molecules" (AIM), Natural Bonding Orbital (NBO) and Non Covalent Interactions plot (NCIplot) analyses, which further assisted in the characterization of the ChBs described herein. We expect that the results from this study will be useful to expand the current knowledge regarding biological ChBs as well as to increase the visibility of the interaction among the carbohydrate chemistry community.

Anti-aging derivatives of cycloastragenol produced by biotransformation.

In this study, the microbial transformation of cycloastragenol (CA) by the fungi Mucor subtilissimus AS 3.2456 and Aspergillus oryzae AS 3.407 yielded 19 metabolites. Their structures were established based on extensive NMR and HR-MS data analyses, and six of them are new compounds. The two fungal strains exhibited distinct biocatalytic features. M. subtilissimus could catalyse hydroxylation and carbonylation reactions meanwhile the fragile 9,19-cyclopropane ring remained intact. A. oryzae preferred to catalyse hydroxylation, acetylation and ring expansion reactions. These highly specific reactions are difficult to achieve by chemical synthesis, particularly under mild conditions. Furthermore, we found that most of the metabolites could significantly extend the lifespan of Caenorhabditis elegans at 50 µM. These biotransformed derivatives of CA could be potential anti-aging agents.

Effect of agitation speed and aeration rate on fructosyltransferase production of Aspergillus oryzae IPT-301 in stirred tank bioreactor.

OBJECTIVE: Fructooligosaccharides (FOS) are prebiotic substances that have been extensively incorporated in different products of food industry mostly for their bifidogenic properties and economic value. The main commercial FOS production comes from the biotransformation of sucrose and intracellular and extracellular microbial enzymes-fructosyltransferases (FTase). Aspergillus oryzae IPT-301 produces FTase. In order to increase its production, this study focuses on evaluating the effects of different agitation speed and aeration rates which affect yields in a stirred tank bioreactor. RESULTS: Agitation had more influence on cell growth than aeration. The maximum intracellular FTase activity and the volumetric productivity of total intracellular FTase were obtained at 800 rpm and 0.75 vvm, and reached values of 2100 U g-1 and 667 U dm-3 h-1, respectively. The agitation speed had a strong influence on the activity of extracellular FTase produced which reached the maximum amount of 53 U cm-3. The higher value of total activity obtained was 22,831 U dm-3 at 0.75 vvm and 800 rpm. CONCLUSION: Aeration rates and agitation speed showed strong influence upon the growth and production of fructosyltransferase from Aspergillus oryzae IPT-301 in media containing sucrose as carbon source. The control of aeration rate and agitation speed can be a valuable fermentation strategy to improve enzyme production.

Solar activation of fungus coated in photothermal cloth.

Bioorthogonal reactions based on manipulating the physicochemical and biological behavior of natural cells have gained tremendous attention for meeting the demands for multifunctional microorganisms without decreasing cell viability. Described herein is a novel bioorthogonal method for microorganism (Aspergillus oryzae) modification which coats the microorganism with a photothermal conversion cloth for staying bioactive in cold environments. Two steps, including ferric ions primarily binding to the microorganism cell surface, followed by in situ polymerization of pyrrole, are adopted to actualize highly efficient polypyrrole modification on the microorganism surfaces. The production of α-amylase by Aspergillus oryzae and α-amylase catalytic ability are two representative indexes of cold adaptation as confirmed by a starch decomposition test. This strategy for coating microorganisms with photothermal cloth is biocompatible and cost-effective, and can achieve non-contact modulation, which also offers great promise for generating living cell-polymer hybrid structures based on other microorganism systems for low-temperature environmental adaptation.

Enhancing Activities of Salt-Tolerant Proteases Secreted by Aspergillus oryzae Using Atmospheric and Room-Temperature Plasma Mutagenesis.

Aspergillus oryzae 3.042 was mutagenized using atmospheric and room-temperature plasma (ARTP) technology to enhance its salt-tolerant proteases activity. Compared to the starting strain, mutant H8 subjected to 180 s of ARTP treatment exhibited excellent genetic stability (15 generations), growth rate, and significantly increased activities of neutral proteases, alkaline proteases, and aspartyl aminopeptidase during fermentation. Mutant H8 significantly enhanced the contents of 1-5 kDa peptides, aspartic acid, serine, threonine, and cysteine in soy sauce by 16.61, 7.69, 17.30, 8.61, and 45.00%, respectively, but it had no effects on the contents of the other 14 free amino acids (FAAs) due to its slightly enhanced acidic proteases activity. Analyses of transcriptional expressions of salt-tolerant alkaline protease gene (AP, gi: 217809) and aspartyl aminopeptidase gene (AAP, gi: 6165646) indicated that their expression levels were increased by approximately 30 and 27%, respectively. But no mutation was found in the sequences of AP and AAP expression cassettes, suggesting that the increased activities of proteases in mutant H8 should be partially attributed to the increased expression of proteases. ARTP technology showed great potential in enhancing the activities of salt-tolerant proteases from A. oryzae.

Multiwalled carbon nanotubes bound beta-galactosidase: It's activity, stability and reusability.

Carbon nanotubes (CNTs) based biosensors are recognized to be a next generation building block for ultrasensitive and fast biosensing systems. This article starting with a brief history on CNTs provides an overview on the recent expansion of research in the field of CNT-based biosensors. This is followed by the discussion on structure and properties related to CNTs. Furthermore, the basic and some newly developed synthetic methods of CNTs are summarized. In this chapter, we used polyaniline cobalt multiwalled CNTs to immobilize ß-galactosidase, by adopting both noncovalent and covalent strategies. Herein, the methodologies of both techniques have been discussed in detail. The η (effectiveness factor) values for nanocomposite bound ß-galactosidase by physical adsorption and covalent method were calculated to be 0.93 and 0.97, respectively. The covalently bound ß-galactosidase retained 92% activity even after its 10th successive reuse as compared to the adsorbed enzyme which exhibited only 74% of its initial activity. CNT armored enzymes demonstrated remarkably high catalytic stability at both sides of temperature and pH-optima along with easy recovery from the reaction medium which can be utilized in various biotechnological applications. Lastly, the scientific and technological challenges in the field are discussed at the end of this chapter.

Secretory production of N-glycan-deleted glycoprotein in Aspergillus oryzae.

The pharmaceutical industry has a high demand for glycoprotein production. The glycoform of glycoproteins is crucial for pharmacological activity. However, in general, cells produce glycoproteins with a heterologous glycoform, which is unfavorable for making uniform, efficacious therapeutic proteins. Here, to produce more glycoproteins with N-glycan uniformity, we applied the GlycoDelete strategy, in which endo-ß-N-acetylglucosaminidase (ENGase) from the fungus Hypocrea jecorina (EndoT) is expressed at the Golgi membrane to cleave N-glycan from secretory glycoproteins, to Aspergillus oryzae cells. First, we selected candidate transmembrane domains to target EndoT to the Golgi membrane in A. oryzae cells, generated constructs for expressing the transmembrane-fused EndoT proteins and produced four potential AoGlycoDelete strains. We then confirmed that these strains produced α-amylase with a molecular weight lower than that of native α-amylase without an effect on growth. To test whether the A. oryzae α-amylase proteins had been cleaved by EndoT, we expressed and purified HA-tagged α-amylase AmyB and glucoamylase GlaA proteins from the AoGlycoDelete strain. MS and N-glycan analyses of the intact proteins confirmed neither AmyB-HA nor GlaA-HA produced from the AoGlycoDelete strain contained N-glycan. Lastly, we determined the enzymatic activities of the amylases produced by the AoGlycoDelete strain, which showed that the lack of N-glycan did not affect their activity under the conditions tested. Collectively, our findings demonstrate successful generation of an AoGlycoDelete strain that might be a good candidate for producing pharmaceutical glycoproteins with a uniform N-glycan structure.

Cloning, expression and characterization of a thermo-alkali-stable xylanase from Aspergillus oryzae LC1 in Escherichia coli BL21(DE3).

In the present investigation, cloning and overexpression of xylanase (XynF1), the main xylanase of A. oryzae LC1, was performed in prokaryotic system E. coli BL21(DE3) to produce recombinant xylanase with high titer of specific activity (1037.3 U/mg), which was 9.3-fold higher than the native strain. Further, the recombinant XynF1 of size 37 kDa was purified using Ni2+-NTA resins followed by cation exchange chromatography, which showed an 1.8-fold increase in purity with 71.4% yield. The r-XynF1 exhibited a wide range of activity at different pH (3.0-10.0) range and temperature (30-70 °C) with an optimum pH at 5.0 and temperature at 30 °C. The results from the current study, clearly demonstrate that this is an effective method to generate a recombinant enzyme with improved activity, making it useful for possible industrial applications.

Mapping haze-komi on rice koji grains using ß-glucuronidase expressing Aspergillus oryzae and mass spectrometry imaging.

In sake brewing, the quality of rice koji is evaluated by experienced sake brewers based on visual inspection of the haze, which is defined by the extent of fungal hyphae spread on/into the rice grains. There is an increasing interest in understanding the factors that affect the quality of rice koji, which is dependent on its making process. Several studies have focused on the degree of mycelial penetration (haze-komi) and enzyme production during rice koji production. However, there are limited analytical methods available to monitor hyphal growth on a solid surface. Here we used a ß-glucuronidase (GUS)-expressing strain of Aspergillus oryzae to visualize and map the fungal growth on rice koji grains. Observation of indigo color revealed that A. oryzae hyphae penetrated the steamed rice grain in the early stage (24 h) of rice koji-making before spreading on the surface during the later stages. Additionally, hyphae penetrated along the endosperm cells and penetrated the cells to form the sou-haze. Furthermore, mass spectrometry imaging (MSI) of glucose demonstrated that the area of mycelial penetration is directly correlated with the spread of glucose during fermentation. This is the first report on utilizing new tools such as GUS-body-mapping of A. oryzae and MSI to monitor fungal growth during rice koji making.

Polyphasic Assessment of Aflatoxin Production Potential in Selected Aspergilli.

This study investigated the aflatoxin production potentials of selected fungi using a polyphasic approach. Internally transcribed spacer region of the fungi was amplified using the polymerase chain reaction. Forty-five Aspergillus strains were further assessed for aflatoxin production using the conventional methods such as growth on yeast extract sucrose, ß-cyclodextrin neutral red desiccated coconut agar (ß-CNRDCA); expression of the aflatoxin regulatory genes and the use of both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A large proportion (82.22%) of the isolates harbored the Nor-1 gene while 55.56%, 68.89%, and 80% possessed the ver-1, omt-A, and aflR genes, respectively. All 100% the isolates harbored the aflJ gene. Twenty-three isolates were positive for aflatoxin production based on the yeast extract sucrose medium (YES) test; ammonium vapor test (51%), yellow pigment production (75.5%), and ß-CNRDCA tests; and blue/green fluorescence (57.7%). Based on TLC detection 42.2% produced aflatoxins while in the HPLC, total aflatoxin (AFTOT) production concentrations ranged from 6.77-71,453 µg/g. Detectable aflatoxin B1 (AFB1) concentrations obtained from the HPLC ranged between 3.76 and 70,288 µg/g; 6.77 and 242.50 µg/g for aflatoxin B2 (AFB2); 1.87 and 745.30 µg/g for aflatoxin G1 (AFG1); and 1.67 and 768.52 µg/g for aflatoxin G2 (AFG2). AFTOT contamination levels were higher than European Union tolerable limits (4 µg/kg). The regression coefficient was one (R2 = 1) while significant differences exist in the aflatoxin concentrations of Aspergillus (p ≤ 0.05). This study reports the potentials of Aspergillus oryzae previously known as a non-aflatoxin producer to produce AFG1, AFG2, AFB1, and AFB2 toxins. Aspergillus species in feedlots of animals reared for food are capable of producing aflatoxins which could pose hazards to health.

Difference in microbial community and taste compounds between Mucor-type and Aspergillus-type Douchi during koji-making.

Douchi has attracted people's attention because of its unique taste and rich health function. The microbes participated in the koji-making process contribute to taste compounds of Douchi. However, the majority of studies on Douchi focused on their functional components and the microbial community in single type of Douchi during koji-making so far. In the present study, the taste components of Mucor-type and Aspergillus-type Douchi were measured initially and the results showed that the amino acid and organic acid levels as well as the percentage of unsaturated fatty acids in Mucor-type Douchi were significantly higher than those in Aspergillus-type. The investigation of the microbial composition in two types of Douchi showed that Aspergillus, Candida, Meyerozyma and Lecanicillium were shared by >50% of samples during koji-making. Comparison of the microbial community between the two types of Douchi revealed that Meyerozyma and Lecanicillium were the main microbial community with significant difference during the initial stage of koji-making, while Candida was significantly different during the later stage of koji-making. When supplemented with Meyerozyma and Candida in Aspergillus-type Douchi, the level of all amino acid and organic acids as well as the percentage of unsaturated fatty acid was significant improved, which further validated the importance roles of the two microorganisms in enhancing the taste components of Douchi during koji-making. The results provide useful information on optimizing the microbial community structure of Douchi during the process of koji-making and improving the product quality.

A new method for determining the mycelial weight of the koji-mold Aspergillus oryzae by measuring its glycosylceramide content.

At present, the quantitation of the mycelial weight of the industrially important non-pathogenic fungus Aspergillus oryzae, which is used for manufacturing koji, is performed by quantitating N-acetylglucosamine. However, since N-acetylglucosamine is a cell wall component, the extraction procedure is costly and tedious, and its quantitative performance is poor. Here, we report a novel method for the quantitation of A. oryzae mycelial weight. The amount of glycosylceramide significantly correlated with both the mycelial weight of A. oryzae and the amount of N-acetylglucosamine, an established index of the mycelial weight of A. oryzae in koji. This new method is simple and efficient and can be used in the brewing and food industries to determine the mycelial weight of A. oryzae.

Effect of temperature on saccharification and oligosaccharide production efficiency in koji amazake.

Koji amazake, prepared from rice koji, is a traditional Japanese sweet beverage. The main source of sweetness is glucose derived from rice starch following digestion by enzymes of Aspergillus oryzae during saccharification. The temperature of this process was empirically determined as 45°C-60°C, but no studies have systematically investigated the effect of temperature on saccharification efficiency. We addressed this in the present study by evaluating saccharification efficiency at various temperatures. We found that glucose content was the highest at 50°C (100%) and was reduced at temperatures of 40°C (66.4%), 60°C (91.9%), and 70°C (76.6%). We previously reported that 12 types of oligosaccharides are present in koji amazake; the levels of eight of these, namely nigerose, kojibiose, trehalose, isomaltose, gentiobiose, raffinose, panose, and isomaltotriose, were the highest at 50°C-60°C, whereas sophorose production was maximal at 70°C. Based on these findings, we initially performed saccharification at 50°C and then switched the temperature to 70°C. The maximum amount of each saccharide including sophorose that was produced was close to the values obtained at these two temperatures. Thus, oligosaccharide composition of koji amazake is dependent on saccharification temperature. These findings provide useful information for improving the consumer appeal of koji amazake by enhancing oligosaccharide content.

Gene dosage and coexpression with endoplasmic reticulum secretion-associated factors improved the secretory expression of α-galactosidase.

The α-galactosidases, which can catalyze the removal of α-1,6-linked terminal galactose residues from galactooligosaccharide materials, have good potential for industrial applications. The high-level and efficient secretion of the α-galactosidases into the extracellular space has greatly simplified the downstream bioengineering process, facilitating their bioapplications. In this study, the effects of gene dosage and endoplasmic reticulum secretion-associated factors (ERSAs) on the secretory expression of an α-galactosidase gene derived from a Aspergillus oryzae strain were investigated by constructing multicopy expression cassettes and coexpressing the α-galactosidase gene with ERSAs. With the increase in the gene copy-number in the host genome, the expression of GalA was improved. However, the secretory expression level was not linearly related to the copy number. When the number was higher than four copies, the expression level of GalA gene declined. The ERSAs factors HAC1, PDI, and Ero1 improved the secretory expression of α-galactosidase, while Hsp40 inhibited its secretion. After methanol-induced expression in a bench-top bioreactor, Pichia recombinants carrying four copies of GalA genes reached 3520 U/mL in the supernatant of the culture. We further optimized the parameters for α-galactosidase to hydrolyze two types of galactooligosaccharides: raffinose and stachyose. This study has fulfilled the scale-up production of α-galactosidase, thus facilitating its industrial applications.

Changes in Polyamine Content in Rice Bran due to Fermentation with Aspergillus oryzae Analyzed by LC/ESI-MS/MS Combined with Derivatization.

Many studies have demonstrated that the dietary supplementation of polyamines, especially spermidine (SPD), prevents age-related diseases. Rice bran is rich in polyamines and their amounts could be increased by fermentation with Aspergillus oryzae (A. oryzae). In this study, we developed a method for the determination of putrescine (PUT), SPD and spermine (SPM) in rice bran samples by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) after derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F). The derivatization improved the LC retention and ESI-MS/MS detectability of the polyamines, and consequently enabled precise and accurate quantification. Using this method, we found that the SPD content increased to 158% due to fermentation with A. oryzae, while the content of PUT and SPM decreased. SPD is known as the polyamine playing a central role in cell proliferation and growth, and therefore has health benefits. The fermented rice bran might be a good material for functional foods aimed at SPD supplementation.

Separation, biochemical characterization and salt-tolerant mechanisms of alkaline protease from Aspergillus oryzae.

BACKGROUND: The salt tolerance of proteases secreted by Aspergillus oryzae 3.042 closely relates to the utilization of raw materials and the quality of soy sauce. However, little is known about the salt-tolerant proteases and their salt-tolerant mechanisms. RESULTS: In this study, we isolated and identified a salt-tolerant alkaline protease (AP, approximately 29 kDa) produced by A. oryzae 3.042. It was considered as a metal-ion-independent serine protease. The optimum and stable pH values were both pH 9.0 and the optimum temperature was 40 °C. Over 20% relative activity of AP remained in the presence of 3.0 mol L-1 NaCl after 7 days, but its Km and Vmax were only mildly influenced by the presence of 3.0 mol L-1 NaCl, indicating its outstanding salt tolerance. Furthermore, AP was more stable than non-salt-tolerant protease at high salinity. The salt-tolerant mechanisms of AP could be due to more salt bridges, higher proportion of ordered secondary structures and stronger hydrophobic amino acid residues in the interior. CONCLUSIONS: The above results are vital for maintaining, activating and/or modulating the activity of AP in high-salt environments. They would also provide theoretical guidance for the modification of AP and the engineering of A. oryzae 3.042 so as to secrete more AP. © 2018 Society of Chemical Industry.

Impact of Iron-Enriched Aspergillus oryzae on Iron Bioavailability, Safety, and Gut Microbiota in Rats.

Iron deficiency is a leading global nutritional problem. Ferrous sulfate (FeSO4) is the most common iron source used for supplementation. Because of many side effects associated with its consumption, it is important to identify new forms of iron. The objectives of this study were to assess the bioavailability of iron-enriched Aspergillus oryzae, Aspiron (ASP), evaluate the toxicity of high-dose iron supplementation with ASP, and determine the ASP impact on gut microbiota in rats. In this study, we investigated iron bioavailability using the hemoglobin repletion test. Aspartate aminotransferase, alanine aminotransferase, and blood urea nitrogen levels were determined to evaluate the effect on liver and kidney functions. Protein carbonyls were measured to assess oxidative damage to proteins. Fecal samples at the end of the 14 day repletion period were used for 16S rRNA sequencing for gut microbiota analysis. The slope ratio method using a common intercept linear regression model was used to compare the bioavailability of ASP to FeSO4. Iron repletion increased hemoglobin concentrations with both ASP and FeSO4 treatments compared to the control group, except in the lowest ASP group. The slope ratio indicated that relative iron bioavailability of ASP was 60% of that of FeSO4 when hemoglobin change was compared to iron in the diet. Similar results were obtained when absolute iron intake was compared on the basis of food consumption. In comparison to the control, protein carbonyl concentrations were significantly ( p < 0.05) higher in the FeSO4 group but not with the ASP group. Supplementation with both sources of iron reduced the Enterobacteriaceae population in the gut microbiota of the rats. A higher relative abundance of bacteria from the phylum Verrucomicrobia was also observed with the highest dose of ASP. Iron-enriched A. oryzae with 60% relative bioavailability of FeSO4 did not show any signs of adverse effects after 14 days of iron supplementation. Future human studies are needed to understand the ASP detailed effect on gut microbiota.