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More than 2,000 varieties of cheese currently exist in the world, and cheese manufacture continues to flourish. To develop the cheese ripening process, additional ingredients are used during cheese production. In this study, the effect of sake lees as an additional ingredient on the fermentation of cheese using Aspergillus oryzae (koji mold), known as koji cheese, was investigated. Aspergillus oryzae is used in the fermentation of Japanese traditional foods, such as sake and soy sauce, given its strong enzymatic activities, as well as in cheese production (i.e., koji cheese). Sake lees, a by-product of the fermentation of rice with A. oryzae and yeasts in the sake brewing process, contains various metabolites, such as amino acids. Here, supplementation with sake lees enhanced the activities of lactic acid bacteria and affected the color of the cheese. Metabolome analysis revealed that sake lees altered the balance of carbohydrates and fatty acids in the cheese. Remarkably, supplementation with sake lees enhanced the production of umami-enhancing γ-glutamyl (kokumi-active) peptides. This study suggests that a new type of cheese can be produced using A. oryzae and sake lees, and information on the synergistic effects of A. oryzae and sake lees will aid the development of cheese production.

The influence of light on the development of asexual spores is an example to elucidate the effect of environmental factors on fungi. Well-studied fungus Aspergillus nidulans produces more conidia under the light condition than under the dark condition, whereas RIB 40, a standard strain of A. oryzae , produces more conidia under the dark condition. In this study, we found that an A. oryzae strain (RIB 1187) responded positively to incubation under light and resulted in better conidia count as compared to that in the dark. We studied the gene expression of RIB 1187 and found that four homologous genes of A. nidulans (up-regulated genes by light) were also up-regulated in RIB 1187 under the light condition

Miso is a traditional Japanese seasoning paste produced by fermenting soybeans using the power of koji mold. A recent Japanese cohort study has shown that increased consumption of fermented soybean products is associated with a reduced risk of death in both men and women. In this review, we briefly explain what miso means in the Japanese culture and food industry, varieties of miso available today, and steps involved in miso making. Then, we review early and latest scientific researches in koji mold species, their safety, and beneficial enzymes they produce during fermentation and maturation processes, which play a major part in determining the quality and sensory profile of miso.

Xaa-Pro aminopeptidases are peptidases responsible for the cleavage of any amino acid N-terminally adjacent to a proline residue. We identified a gene encoding a putative Xaa-Pro aminopeptidase in the genome of the filamentous fungus Aspergillus oryzae (genome database number: AO090701000720) and named this gene xpmA . We produced its enzyme in a C-terminally His 6 -tag-fused form in an Escherichia coli expression system and purified it. The purified recombinant XpmA (rXpmA) showed hydrolysis activity toward Xaa-Pro-oligopeptides, especially the two dipeptides Ala-Pro and Phe-Pro. The molecular weight of rXpmA was estimated to be 69 kDa by SDS-PAGE and 126 kDa by gel filtration, suggesting that it is a homodimer. The enzyme was activated by various divalent metal ions such as Mn 2+ , Co 2+ , and Mg 2+ ; in particular, the enzyme activity was increased 27.6-times relative to the no-addition control by 1 mM Mn 2+ . Additionally, 10 mM EDTA suppressed its activity to 0.26-times of the control level. Therefore, rXpmA was a metalloprotease. Optimal hydrolytic activity of rXpmA was observed at 50°C and pH 8.5–9.0. The enzyme was stable up to 50°C and from pH 4.0 to 11.0. rXpmA showed substrate inhibition by Leu-Pro, Ser-Pro and Arg-Pro at concentrations over 4 mM, 10 mM, and 3 mM, respectively. NaCl increased the enzyme activity in the concentration range 0.5–3.0 M, suggesting that the enzyme is halophilic.

The A.oryzae strains isolated from coffee were screened to evaluate their potential for production of acid aspartic protease. The strains with highest activity were then subjected to UV-mutagenesis and the lethality was determined by spore spread plate method. The mutant strain CPO 025 produced 1.3 × 104 U/ml, which is 1.53 fold higher than the parent with survival rate of 76 ± 2%. The Central Composite Rotatable Design(CCRD) based Response Surface Methodology(RSM) was employed to optimize the parameters for Aspartic protease production. The mutant strain subjected to solid-state fermentation (SSF) using wheat bran as substrate yielded 1820 U/mg specific activity with functional conditions of moisture (35%), inoculum (6.5 CFU/ml), fermentation duration (108 h), and temperature (28 °C). The partially purified proteases by DEAE-Sepharose had attributes with 6082 U/mg specific activity, optimum pH 3.6, temperature 35 °C and MW of 35 kDa. Enhanced activity by β-mercaptoethanol and inhibition by iodoacetic acid confirmed cysteine protease. Application of cysteine protease with cocoa beans upsurged flavour profile compared to the untreated beans. The elevated level of dimethylpyzarine and ketone3,5-Bis(tert-butyl)-4-hydroxy-propiophenone indicated efficient protein hydrolysis with desirable cocoa flavour. Thus cocoa organoleptic enhancement with flavourzyme can camouflage poorly fermented cocoa and economy of the industry.

Potato pulp powder is a major byproduct generated in huge amounts globally from potato processing industry. The disposal of these residues causes environmental pollution. Considering the use of agro byproduct as economic and environmental benefits, biotranformation of Potato pulp powder by solid state fermentation for the production of acid protease a valuable food enzyme is explored. Screening of food grade and industrial Aspergillus oryzae strains on enriched media identified A. oryzae RIB 40 (ATCC 42149) yielding 20.5 U/ml of acid protease as a potential candidate. Further, the productivity of the parent strain was improved by UV mutagenesis with a lethality of 1.8 × 10 −4 . The mutant strain (F6) produced 5.6 fold higher than the parent. The solid state fermentation of A. oryzae (F6) with potato pulp powder as a sole source and along with supplementation of nitrogen yielded 2.85 ± 5.0 and 18.68 U/g of acid protease, respectively. Statistical optimization of biovariables, namely moisture (50%), temperature (30 °C) and fermentation time (120 h) accomplished using a central composite rotatable experimental design, led to an 11-fold increase in protease activity (31 U/g dry substrate). Model validations indicated 95% agreement between the experimental results and the predicted responses. The acid protease from A. oryzae liberated glycine (0.57 ± 0.25 U/g), which can enhance food palatability. Thus, the present study established fermenting potato pulp, with a starter culture of A. oryzae F6 strain and optimized variables can produce acid protease that has great potential for use in the food industry. This will also contribute to sustainable management in the potato industry and adds value-addition.

We truncated the short arm of chromosome 3 to delete the aflatoxin biosynthesis gene homolog cluster using telomeric repeats in Aspergillus oryzae. The predicted deletion was confirmed by Southern blot analyses. This telomere-mediated chromosomal truncation method enables the development of an artificial chromosome in A. oryzae.

A bacterial community analysis, using a culture-independent method (polymerase chain reaction-denaturing gradient gel electrophoresis), detected 17 species of bacteria including species of the genera Tetragenococcus, Lactobacillus, Pediococcus, Weissella Halanaerobium, Clostridium, and Sphingomonas in a traditional salty-fermented fish paste known as pla-ra or pa-daek in Thailand and Laos, which is used as a storage-stable multi-purpose seasoning. The representative genus of lactic acid bacteria seemed to vary in the 10 products collected from Thailand and Laos. Tetragenococci were common in products from central Thailand and Vientiane in Laos which had salinities of not less than 11% and pH values ranging from 5.6 to 6.1. However, lactobacilli were common in products from northern Thailand which had the lowest salinities (8.3–8.6%) and pH values (4.5–4.8) of all the samples examined. Two Lactobacillus and one Tetragenococcus species were detected in one product from northeastern Thailand containing 10% salt. These results suggest that salinity in pla-ra/pa-daek is an important determinant of the representative genus of lactic acid bacteria such as, Tetragenococcus or Lactobacillus. Additionally, differences in the acidity between these two groups seemed to be related to the production of d-/l-lactic acid in the lactic acid bacteria in each product. This is the first study to report a correlation between bacterial community structure and taste components in pla-ra/pa-daek products from various regions. This scientific work on a traditional fermented food will be useful in helping local producers meet differing consumer preferences in various regions.

Rationale The acylglycerols in miso have not been studied although it is known that they are important to the taste. In order to determine the fatty acid constituents in the acylglycerols and analyze them individually, multiple reaction monitoring (MRM) was performed utilizing a single platform, typically using both gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Methods Acylglycerols and fatty acids (FAs) in miso were extracted using the Bligh-Dyer method. Supercritical fluid chromatography (SFC) with a C30 column was conducted for separation, and mass spectrometric (MS) analysis was performed with electrospray ionization using a triple quadrupole mass spectrometer (QqQMS) in the MRM mode. Results The detection of FAs from the hydrolysis of acylglycerols and individual acylglycerols was achieved using only an SFC/MS platform. From the quality control (QC) sample of miso, we determined the main FA constituents, and then performed wide target analysis using MRM. In total, 23 triacylglycerols, 10 diacylglycerols, two monoacylglycerols, and five FAs were annotated effectively. Furthermore, the important compounds related to taste were determined through the analysis using both the relative quantitative data of acylglycerols and FAs and the quantitative descriptive analysis data of miso. Conclusions A method for the determination of the FA constituents in acylglycerols after hydrolysis and the comprehensive analysis of acylglycerols and FAs using MRM with SFC/QqQMS was developed. Using the data from the comprehensive analysis of acylglycerols and quantitative descriptive data, the key compounds related to taste were investigated. This type of research on lipids and the taste of food is expected to progress hereafter. Copyright © 2017 John Wiley & Sons, Ltd.

Pa-som is a traditional salty fermented food made from varieties of freshwater fish with garlic and rice in Laos. A similar product, plaa-som, is made in Thailand. To investigate the fermentation process of pa-som, lactic acid production and pH were monitored during laboratory-scale production of pa-som. Furthermore, the variety of bacteria involved and their succession during fermentation were examined using culture-independent polymerase chain reaction (PCR) denaturing gel gradient electrophoresis targeting the 16S ribosomal RNA (rRNA) gene. d- and l-lactic acid production and a decrease in pH were observed in the samples after 1 day of fermentation. The lactic acid content and pH continued to increase and decrease, respectively, until 4 days of fermentation. Overall, six lactic acid bacteria and eight other indigenous bacteria species were detected during pa-som fermentation. Among the lactic acid bacteria detected, two Lactococcus and one Weissella species were detected in samples after 1–4 days of fermentation, suggesting that these three species play major roles in pa-som fermentation from the initial phase. This is the first report to investigate the fermentation process in pa-som at the microbial level. This research approach should be applicable to a wide variety of pa-som and related fermented fishery products in Laos to help understand their microbial diversity and to identify beneficial bacterial species for improving quality.

Research on the relationship between mitochondrial membrane potential and fermentation profile is being intensely pursued because of the potential for developing advanced fermentation technologies. In the present study, we isolated naturally occurring strains of yeast from sake mash that produce high levels of malic acid and demonstrate that variations in mitochondrial membrane potential correlate with malic acid production. To define the underlying biochemical mechanism, we determined the activities of enzymes required for malic acid synthesis and found that pyruvate carboxylase and malate dehydrogenase activities in strains that produce high levels of malic acid were elevated compared with the standard sake strain K901. These results inspired us to hypothesize that decreased mitochondrial membrane potential was responsible for increased malic acid synthesis, and we present data supporting this hypothesis. Thus, the mitochondrial membrane potential of high malic acid producers was lower compared with standard strains. We conclude that mitochondrial membrane potential correlates with malic acid production.

We isolated 2,4-dinitrophenol (DNP)-resistant sake yeast strains by UV mutagenesis. Among the DNP-resistant mutants, we focused on strains exhibiting high malic acid and low acetic acid production. The improved organic acid composition is unlikely to be under the control of enzyme activities related to malic and acetic acid synthesis pathways. Instead, low mitochondrial activity was observed in DNP-resistant mutants, indicating that the excess pyruvic acid generated during glycolysis is not metabolized in the mitochondria but converted to malic acid in the cytosol. In addition, the NADH/NAD(+) ratio of the DNP-resistant strains was higher than that of the parental strain K901. These results suggest that the increased NADH/NAD(+) ratio together with the low mitochondrial activity alter the organic acid composition because malic acid synthesis requires NADH, while acetic acid uses NAD(+).

Aspergillus oryzae was isolated from coffee pulp, screened for the production of carboxypeptidase and confirmed by molecular studies. Coffee pulp posses high protease activity (1.4 × 103 nkat/mL) among the agro-substrates used for the production of proteases but wheat bran was used for further studies considering its easy availability in large volumes. Proximal composition, pre-treatment, auxiliary carbon and nitrogen with growth studies indicated carboxypeptidase activity (2.6 × 103 nkat/g). The RSM was employed to generate a predictive model of the combined effects of independent variables and indicated a significant impact on acid carboxypeptidase with 35% moisture, 30 °C temperature, 108 CFU mL−1 inoculum and 100 h duration with 4.8 × 103 nkat/g activity. The partially purified peptidase had a specific activity of 6.1 × 104 nkat/mg with a molecular weight of 50 kDa, optimal pH 3.6, temperature 35 °C and feature of a serine protease. Enzyme stability was maintained with 50% glycerol and 2 mg/mL BSA. The protease application to develop flavor profiles of coffee was noteworthy with an organoleptic score of 7.5 compared to 4.5 of the untreated sample. GC-MS highlighted enhanced flavor annotations of pyrazines contributing sweet fruit notes. Carboxypeptidase augmented the coffee bean with specialized flavor notes and has been reported for the first time.

Antibody display methods are increasingly being used to produce human monoclonal antibodies for disease therapy. Rapid screening and isolation of specific human antibody genes are valuable for producing human monoclonal antibodies showing high specificity and affinity. In this report, we describe a novel mammalian cell display method in which whole human IgG is displayed on the cell surface of CHO cells. Cells expressing antigen-specific human monoclonal IgGs with high affinity on the cell surface after normal folding and posttranscriptional modification were screened using a cell sorter. The membrane-type IgG-expressing CHO cells were then converted to IgG-secreting cells by transfection with a plasmid coding Cre recombinase. This mammalian cell display method was applied to in vitro affinity maturation of monoclonal C9 IgG specific to the human high-affinity IgE receptor (FcεRIα). The CDR3 of the C9 heavy chain variable region gene was randomly mutated and inserted into pcDNA5FRT/IgG. A C9 IgG (CDRH3r)-expressing CHO cell display library consisting of 1.1×10(6) independent clones was constructed. IgG-displaying cells showing high reactivity to FcεRIα antigen were screened by the cell sorter, resulting in the establishment of a CHO cell line producing with higher reactivity than the parent C9 IgG.

Three putative deuterolysin (EC 3.4.24.29) genes (deuA, deuB, and deuC) were found in the Aspergillus oryzae genome database (http://www.bio.nite.go.jp/dogan/project/view/AO). One of these genes, deuA, was corresponding to NpII gene, previously reported. DeuA and DeuB were overexpressed by recombinant A. oryzae and were purified. The degradation profiles against protein substrates of both enzymes were similar, but DeuB showed wider substrate specificity against peptidyl MCA-substrates compared with DeuA. Enzymatic profiles of DeuB except for thermostability also resembled those of DeuA. DeuB was inactivated by heat treatment above 80° C, different from thermostable DeuA. Transcription analysis in wild type A. oryzae showed only deuB was expressed in liquid culture, and the addition of the proteinous substrate upregulated the transcription. Furthermore, the NaNO3 addition seems to eliminate the effect of proteinous substrate for the transcription of deuB. DeuB was a non-thermostable deuterolysin from A. oryzae. The transcription of the gene was promoted by protein substrates, but the promotion was eliminated by adding inorganic nitrogen source such as NaNO3.

Japanese traditional fermented foods are centerpieces of taste of Washoku cuisine which was added to UNESCO’s Intangible Cultural Heritage list in 2013. Miso is fermented soybean paste. It is used for every day’s miso soup and as a seasoning in many kinds of cooking. Shoyu – soy sauce – is a liquid-type seasoning made of soybeans, wheat, koji, and salt water. Natto is soybeans fermented with Bacillus, with sticky but tasty γ-polyglutamic acid. Su is a traditional vinegar of cereal or fruit. Rice vinegar which is usually made directly from rice and rice koji is the most popular in Japan. Tsukemono is pickled vegetables, of which the traditional one is fermented with lactic acid bacteria. Tsukemono is usually served daily as a side dish with boiled rice. A variety of fermented seafoods, salted type (gyoshyo, shiokara, kusaya), pickled type (nare-zushi, nuka-zuke), and molded type (fushi) are also developed. The last one “fushi” is an indispensable ingredient in Japanese cuisine. Two kinds of alcoholic beverages, famous sake (rice wine) and shochu (spirits), are also important gifts of fermentation. The outline, including information of microbes, preparation methods and functionality and health benefits, of these traditional fermented foods and beverages popular in Japan are introduced.

Aspergillus oryzae has an ortholog of Saccharomyces cerevisiae KEX1 , termed kexA . A truncated form of KexA protein showed serine-type carboxypeptidase activity and somewhat broader substrate specificity than Kex1 protease. Furthermore, our results indicated that KexA is required for normal growth of A. oryzae and that it might be involved in hyphal branching.

Aspergillus oryzae is a filamentous fungus used as a starter microorganism in the process of manufacturing various kinds of traditional fermented food. It is also used as a producer of enzymes for both food processing and pharmaceuticals, such as proteases and taka-amylase. In order to clarify the function of this microorganism in fermentation industries and utilize the new function of A. oryzae, it was subject to genomic analysis. During the analysis, the telomere of A. oryzae exhibited a novel repeated sequence distinct from other fungi. Genomic analysis also revealed that the genome of A. oryzae consists of eight chromosomes containing 12,074 genes and is larger in size than Aspergillus nidulans and Aspergillus fumigatus. A. oryzae contains sequences specific for A. oryzae for genes involved in primary and secondary metabolism, when compared with A. nidulans and A. fumigatus. Among them, the function of one of the peptidase-like gene products was investigated with a post-genomic approach, and determined as an aminopeptidase specific for acidic amino acids (aspartate and glutamate) which was first identified in filamentous fungi. It also demonstrated that the addition of cobalt ion to the growth medium greatly improved the specific activity. Using the same approach, some of the novel aminopeptidases and carboxypeptidases of A. oryzae were identified and characterized.

Gene AO090103000153 is unique to Aspergillus oryzae RIB40 and A. flavus NRRL3357, and is speculated to encode a serine-type carboxypeptidase. In this study, we purified and characterized a heterologously expressed gene product of AO090103000153. 5'-Rapid amplification of cDNA ends indicated that the translation start site of the gene is located 1,586 bp downstream of the translation start site predicted by the genome sequencing project. The gene, starting from the revised translation start codon, termed ocpC, was transcribed constantly in A. oryzae RIB40. Purified recombinant OcpC exhibited the enzymatic properties of a serine-type carboxypeptidase. This protease was stable at temperatures below 45°C and a low pH, and had broad substrate specificity for N-acylpeptides, but it exhibited significantly lower specific activity and a lower k(cat) value for substrates than previously reported serine-type carboxypeptidases from A. oryzae.

Didodecyldimethylammonium bromide (DDAB) is widely used for an efficient delivery system into mammalian cells. However, the biological activities of DDAB nanoparticles in mammalian cells are insufficiently understood. The purpose of this study was to establish a critical role of DDAB in cellular response. Here, we demonstrate that DDAB is a potent inducer of cell death in a wide range of tumor cell lines, wherein leukemia cells (HL-60 and U937) and neuroblastoma cells (Neuro2a) were more sensitive to DDAB than carcinoma cells such as HepG2 and Caco-2 cells. Moreover, in HL-60 cells, treatment with DDAB led to increased numbers of apoptotic cells with fragmented DNA (99.6%) and high levels of caspase-3 activation in comparison to that with actinomycin D. Cotreatment with a caspase-8 inhibitor (Z-IETD-FMK) or a polyethylene glycol (Mr 2000) (PEG 2000) effectively prevented the activation of caspase-3 induced by DDAB. These results suggest that DDAB can trigger caspase-3-mediated apoptosis through the extrinsic caspase-8 pathway and cytotoxic pore formation in cell membrane. Therefore, the present findings provide new insight into the biological activity of DDAB to induce caspase-mediated apoptosis.

Aims: Prolyl aminopeptidase (PAP) degrades only amino-terminal proline from peptides. The food-grade fungus Aspergillus oryzae produces this enzyme only in small amounts. In this paper, we present efficient production of recombinant PAP with an overexpression system of A. oryzae and characterization of its biochemical properties. Methods and Results: The gene encoding PAP was overexpressed as a His-tag fusion protein under a taka-amylase gene (amyB) promoter with a limited expressing condition in A. oryzae. The PAP activity in the mycelia grown in rich medium containing glucose (repressing condition) was twice that in starch (inducing condition). The enzyme prepared as cell-free extract was partially purified through two-step column chromatography. The PAP was estimated to be a hexameric protein and exhibited salt tolerance against NaCl of up to 4 mol l−1. Conclusions: Aspergillus oryzae PAP was produced under the repressing condition of amyB promoter in a PAP-overexpressing strain and purified 1800-folds. Overproduction of PAP under promoter-inducing conditions led to an increase in inactive PAP, possibly because of irregular folding. Significance and Impact of the Study: PAP with a high specific activity and salt tolerance may be used effectively in the manufacturing processes of fermented foods.

Carboxypeptidase O from Aspergillus oryzae IAM2640 is a serine-type carboxypeptidase. In this study, we cloned and sequenced cDNA and genomic DNA carrying ocpO encoding carboxypeptidase O. The results showed that the length of ocpO was 1,816 bp, and the open reading frame encoded a putative preproenzyme composed of 472 amino acid residues of the mature carboxypeptidase O and an additional N-terminal sequence of 50 amino acid residues. A BLASTN search revealed that a gene, AO090020000351, in A. oryzae RIB40, which is strain used in genome-wide sequencing, is a homolog of ocpO. The difference between AO090020000351 and ocpO was only one nucleotide. The difference caused substitution of Ala for Pro at the 277th position of the enzyme; therefore the protein encoded by AO090020000351 was overproduced and purified. The purified protein showed enzymatic properties similar to carboxypeptidase O, indicating that carboxypeptidase O and protease encoded by AO090020000351 are same enzyme.

The aim of this study was to produce valuable products from potato pulp, which is a byproduct of potato starch production and contains a high concentration of starch. We used the pulp as a substrate for polygalacturonase (PG) production in solid-state fermentation by a recombinant Aspergillus oryzae strain, PGB3. To generate PGB3, we constructed the PG gene (pgaB) overexpression vector pGBmR, and used that vector to transform the A. oryzae strain, RIB40. PGB3 carries a PG gene under control of the Taka-amylase gene promoter. When cultured in potato pulp solid medium, PGB3 grew more rapidly and produced 4.5 times more PG than RIB40. We observed maximum production (173 U/g) of PG after 2 days of culture of PGB3 in potato pulp solid medium. This high-level PG production from potato pulp demonstrates its potential as a useful substrate in fermentation.

In the genome of Aspergillus oryzae, 12 genes have been predicted to encode serine-type carboxypeptidases. However, the carboxypeptidase activities of the proteins encoded by these genes have not yet been confirmed experimentally. In this study, we have constructed three of these 12 genes overexpressing strains using Aspergillus nidulans and characterized their overproduced recombinant proteins. Of these three genes, one was previously named cpI; the other two have not been reported yet, and hence, we named them ocpA and ocpB. The recombinant proteins released amino acid residues from the C terminus of peptides, and the activity of the enzymes was inhibited by phenylmethylsulfonyl fluoride, indicating the enzymes to be serine-type carboxypeptidases. Recombinant OcpA, OcpB, and CpI were stable at 45 degrees C, 55 degrees C, and 55 degrees C, respectively, at a low pH. The enzymatic properties of recombinant OcpB were different from those of any reported serine-type carboxypeptidase. On the other hand, recombinant OcpA had similar enzymatic properties to A. oryzae carboxypeptidases O1 and O2. The DNA and N-terminal amino acid sequences of carboxypeptidases O1 and O2 from A. oryzae IAM2640 were similar to those of OcpA. Result of transcriptional analysis of ocpA, ocpB, and cpI suggest differences in transcriptional regulation between these genes.

Understanding of materials for small interfering RNA (siRNA) delivery system is essential for the effective design and selection of appropriate materials as potential applications in basic and therapeutic research. However, little attempt has been made to investigate how the structural features of lipidic materials are essential for delivery function. The purpose of this study was to identify the structural features of glutamate-based lipidic materials as potential siRNA carriers for intracellular delivery. Totally designed lipid didodecylacetylglutamatetrimethylammonium (TMA-C2-Glu-C12) and its analogs were synthesized and estimated by silencing effects of fluorescent protein (EGFP) expression in CHO-EGFP cells. Here, we demonstrate that TMA-C2-Glu-C12 among a series of lipidic materials is able to perform efficient siRNA delivery with low cytotoxicity into the cells; the transfer ability resulted in 92.2% ± 1.16% of the transferred cells with Cy3-labeled siRNA. The chemical structure of TMA-C2-Glu-C12 having both hydrophobic chain (CH2CO) in head region, hydrophobic chain ((CH2)11CH3) in tail regions, and ethylene (CH2 CH2) in connector region played a crucial role to enhance the siRNA transfer efficiency. Moreover, compared with commercial reagents, TMA-C2-Glu-C12 was found to have an effective activity for siRNA delivery. These results may improve the novel design and development of lipidic materials on siRNA delivery system. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009

In order to find a promoter that could be influenced by temperature shift, we explored and isolated an Aspergillus oryzae gene expressed at high temperatures (37-42 degrees C) by the cDNA subtraction method. Of the 96 cDNA clones isolated from the subtraction library, one cDNA clone showed 73% identity with Aspergillus nidulans heat shock protein 30 (hsp30). Based on this, we designated the isolated gene hsp30 of A. oryzae. A. oryzae hsp30 was weakly expressed at 30 degrees C, but strongly at 40 degrees C. We showed that the promoter of this hsp30 induced heterologous gene expression at high temperatures using beta-glucuronidase (GUS) gene as a reporter. Regarding elucidation of the region essential for heat shock response, we showed that the minimum length of the promoter region that was essential for heat shock response was located between -388 and -272 (+1 indicated the first position of the translation initiation codon) of the hsp30 promoter. This promoter region harbors several putative transcription factor binding sites, including heat shock elements (HSEs), a CCAAT box, and a TATA box. Furthermore, site-directed mutagenesis of this promoter revealed that HSE1 (aTTCgtcGAAacgcccaGAAa) and HSE2 (cGAAagTTCtcGACg), located between -342 and -272 of the hsp30 promoter, were its cis-acting elements for heat shock response.

Aims: Aspartyl aminopeptidase (DAP) has a high degree of substrate specificity, degrading only amino-terminal acidic amino acids from peptides. Therefore, attention is focused here on the efficient production of this enzyme by a recombinant Aspergillus oryzae and characterization of its biochemical properties. Methods and Results: The gene encoding DAP was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a Co2+-containing medium. The enzyme was extracted from the mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high reactivity towards peptide substrate rather than synthetic substrates. Conclusions: Recombinant A. oryzae DAP was purified to homogeneity with an increased specific activity, when cultivated in a Co2+-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help in increasing the specific activity of other metalloproteases and their functional analysis. Significance and Impact of the Study: Recombinant DAP produced using a cobalt ion in culture media of A. oryzae and purification process allow high yield of the enzyme activity.

From a library of glyco-lipid mimics with muconic amide as the spacer, we found that 1, a glyco-lipid that has N-acetyl glucosamine and methyl cyclohexyl groups as its hydrophilic head and hydrophobic tails, respectively, formed a stable hydrogel (0.05 wt %) through hierarchical self-assembly of the lipid molecules into supramolecular nanofibers. The formation of the supramolecular hydrogel was verified by rheological measurements, and the supramolecular nanofiber was characterized as the structural element by transmission electron microscopy and atomic force microscopy observations. Absorption and circular dichroism spectroscopic measurements revealed that the muconic amide moieties of 1 are arranged in a helical, stacked fashion in the self-assembled nanofibers. Moreover, we unexpectedly found that the homogeneous distribution of the supramolecular nanofibers of 1 was greatly facilitated by the addition of polystyrene nanobeads (100-500 nm in diameter), as evaluated by confocal laser scanning microscopic observations. It is interesting that the obtained supramolecular hybrid matrix can efficiently encapsulate and distribute live Jurkat cells in three dimensions under physiological conditions. This supramolecular hybrid matrix is intriguing as a unique biomaterial.

Three extracellular dipeptidyl peptidase genes, dppB, dppE, and dppF, were unveiled by sequence analysis of the Aspergillus oryzae genome. We investigated their differential enzymatic profiles, in order to gain an understanding of the diversity of these genes. The three dipeptidyl peptidases were expressed using Aspergillus nidulans as the host. Each recombinant enzyme was purified and subsequently characterized. The enzymes displayed similar optimum pH values, but optimum temperatures, pH stabilities, and substrate specificities varied. DppB was identified as a Xaa-Prolyl dipeptidyl peptidase, while DppE scissile substrates were similar to the substrates for Aspergillus fumigatus DPPV (AfDPPV). DppF was found to be a novel enzyme that could digest both substrates for A. fumigatus DPPIV and AfDPPV. Semi-quantitative PCR revealed that the transcription of dppB in A. oryzae was induced by protein substrates and repressed by the addition of an inorganic nitrogen source, despite the presence of protein substrates. The transcription of dppE depended on its growth time, while the transcription of dppF was not affected by the type of the nitrogen source in the medium, and it started during the early stage of the fungal growth. Based on these results, we conclude that these enzymes may represent the nutrition acquisition enzymes. Additionally, DppF may be one of the sensor peptidases responsible for the detection of the protein substrates in A. oryzae environment. DppB may be involved in nitrogen assimilation control, since the transcription of dppB was repressed by NaNO3, despite the presence of protein substrates.

Gene transfer is an important tool to explore genomic, cell biologic, or gene therapeutic research. In this paper we report that several cationic amphiphiles have the potential to efficiently deliver DNA into CHO cells, which is one of the cell lines considered to be important for production of proteins including therapeutic proteins. We have found that O,O′-ditetradecanoyl-N-(trimethylammonio acetyl) diethanolamine chloride (14Dea2), among 29 types of cationic amphiphiles tested, shows a transfection efficiency of more than 40% in CHO cells. In addition, the results from a series of hydrocarbon chains of varying lengths bound to a connector have shown that an optimal chain length is important for the efficient delivery of DNA into cells. Moreover, flow cytometer analysis has shown that 14Dea2 transfection leads to high levels of expression of the reporter gene (green fluorescent protein) in individual cells. These findings have suggested that 14Dea2 is able to effectively deliver a number of plasmids into a cell nucleus. Thus, our system might be a powerful tool for high efficiency gene transfer and production of high levels of recombinant protein.

Parasporal inclusion proteins produced by Bacillus thuringiensis strain A1470 exhibit strong cytotoxicity against human leukemic T cells when activated by protease treatment. One of the cytotoxic proteins was separated by anion exchange chromatography and gel filtration chromatography and designated Cry45Aa. Its gene was then expressed in recombinant Escherichia coli, in which the Cry45Aa precursor was accumulated in an inclusion body. It was solubilized in sodium carbonate buffer and processed with proteinase K, and cytotoxic activities of the protein against various mammalian cell lines were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay. The protein exhibited high cytotoxic activity against CACO-2, Sawano, MOLT-4, TCS, and HL60 cells and moderate activity against U-937 DE-4, PC12, and HepG2 cells. On the other hand, the EC50 values against Jurkat, K562, HeLa, A549, Vero, COS-7, NIH3T3, CHO, and four normal tissue cells (human primary hepatocyte cells, UtSMC, MRC-5, and normal T cells) were >2 microg/mL.