Heterologous expression and characterization of salt-tolerant ß-glucosidase from xerophilic Aspergillus chevalieri for hydrolysis of marine biomass.

A salt-tolerant exo-ß-1,3-glucosidase (BGL_MK86) was cloned from the xerophilic mold Aspergillus chevalieri MK86 and heterologously expressed in A. oryzae. Phylogenetic analysis suggests that BGL_MK86 belongs to glycoside hydrolase family 5 (aryl-phospho-ß-D-glucosidase, BglC), and exhibits D-glucose tolerance. Recombinant BGL_MK86 (rBGL_MK86) exhibited 100-fold higher expression than native BGL_MK86. rBGL_MK86 was active over a wide range of NaCl concentrations [0%-18% (w/v)] and showed increased substrate affinity for p-nitrophenyl-ß-D-glucopyranoside (pNPBG) and turnover number (kcat) in the presence of NaCl. The enzyme was stable over a broad pH range (5.5-9.5). The optimum reaction pH and temperature for hydrolysis of pNPBG were 5.5 and 45 °C, respectively. rBGL_MK86 acted on the ß-1,3-linked glucose dimer laminaribiose, but not ß-1,4-linked or ß-1,6-linked glucose dimers (cellobiose or gentiobiose). It showed tenfold higher activity toward laminarin (a linear polymer of ß-1,3 glucan) from Laminaria digitata than laminarin (ß-1,3/ß-1,6 glucan) from Eisenia bicyclis, likely due to its inability to act on ß-1,6-linked glucose residues. The ß-glucosidase retained hydrolytic activity toward crude laminarin preparations from marine biomass in moderately high salt concentrations. These properties indicate wide potential applications of this enzyme in saccharification of salt-bearing marine biomass.

Improvement in salt-tolerance of Aspergillus oryzae γ-glutamyl transpeptidase via protein chimerization with Aspergillus sydowii homolog.

γ-Glutamyl transpeptidase is one of the key enzymes involved in glutamate production during high-salt fermentation of soy sauce and miso by koji mold, Aspergillus oryzae. However, the activity of γ-glutamyl transpeptidase from A. oryzae (AOggtA) is markedly reduced in the presence of NaCl, thus classifying it as a non-salt-tolerant enzyme. In contrast, the homologous protein from the xerophilic mold, A. sydowii (ASggtA) maintains its activity under high-salt conditions. Therefore, in this study, a chimeric enzyme, ASAOggtA, was designed and engineered to improve salt-tolerance in AOggtA by swapping the N-terminal region, based on sequence and structure comparisons between salt-tolerant ASggtA and non-salt-tolerant AOggtA. The parental AOggtA and ASggtA and their chimera, ASAOggtA, were heterologously expressed in A. oryzae and purified. The chimeric enzyme inherited the superior activity and stability from each of the two parent enzymes. ASAOggtA showed > 2-fold greater tolerance than AOggtA in the presence of 18% NaCl. In addition, the chimera showed a broader range of pH stability and greater thermostability than ASggtA. AOggtA and ASAOggtA were sy over the range pH 3.0 to pH 10.5. Thermal stability was found to be in the order AOggtA (57.5 °C, t1/2 = 32.5 min) > ASAOggtA (55 °C, t1/2 = 20.5 min) > ASggtA (50 °C, t1/2 = 12.5 min). The catalytic and structural characteristics indicated that non-salt-tolerant AOggtA would not undergo irreversible structural changes in the presence of NaCl, but rather a temporary conformational change, which might result in reducing the substrate binding and catalytic activity, on the basis of kinetic properties. In addition, the chimeric enzyme showed hydrolytic activity toward L-glutamine that was as high as that of AOggtA. The newly-designed chimeric ASAOggtA might have potential applications in high-salt fermentation, such as miso and shoyu, to increase the content of the umami-flavor amino acid, L-glutamate.

Isolation and characterization of a salt-tolerant γ-glutamyl transpeptidase from xerophilic Aspergillus sydowii.

Xerophilic Aspergillus molds isolated from halo-alkaliphilic and dry environments are attractive genetic resources for obtaining salt- and osmo-adaptive enzymes. A. sydowii MA0196 secreted the largest amount of γ-glutamyl transpeptidase (GGT) during solid-state fermentation at a low initial water activity (a w = 0.85). Gel filtration analysis revealed that the molecular mass of the purified native enzyme (MA0196 GGT) was 120 kDa. SDS-PAGE analysis showed that MA0196 GGT consists of two subunits with molecular masses of 56.4 and 33 kDa, indicating production from a proenzyme via autoproteolysis. Deglycosylation of the subunits by N-glycosidase F yielded 40.9 and 19.6 kDa species. MA0196 GGT retained transpeptidase and hydrolysis activities and their catalytic efficiency (k cat/K m) under high salt and low water activity. The enzyme displayed broad substrate specificity toward γ-glutamyl acceptors such as amino acids and the imidazole dipeptides, carnosine and anserine. Carnosine and L-glutamine were converted into γ-glutamyl-ß-alanyl-L-histidine by MA0196 GGT with a 32.9% yield in the presence of 2% (v/v) dimethyl sulfoxide. Phylogenetic analysis indicated that MA0196 GGT forms a distinct lineage from A. oryzae and A. sojae GGTs. These excellent properties indicate that MA0196 GGT can be used in salted fermentation and for producing bioactive peptides. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03259-3.

Identification and characterization of extracellular enzymes secreted by Aspergillus spp. involved in lipolysis and lipid-antioxidation during katsuobushi fermentation and ripening.

A mild-flavored soup stock made from katsuobushi is an important element of traditional Japanese cuisine and is the basic seasoning responsible for the taste. Fermented and ripened katsuobushi, known as karebushi, is manufactured by simmering skipjack tuna that is then smoke-dried, fermented, and ripened in a repeated molding process by five dominant Aspergillus species. Here, our aim was to characterize and identify the lipolytic enzymes secreted by the dominant Aspergillus species, especially A. chevalieri and A. pseudoglaucus, which are involved in hydrolyzing lipids during the molding process. The crude enzyme preparations from the five Aspergillus spp. cultivated on katsuobushi solid medium hydrolyzed triglycerides in fish oil, and more saturated and unsaturated fatty acids (C16:0, C16:1, C18:0, C18:1) were produced than major polyunsaturated fatty acids (C20:5, C22:6). On the basis of ion exchange chromatograms, the composition of the lipolytic enzymes was different in the five species. There was at least one active fraction with high hydrolytic activity toward fish oil in four of the Aspergillus spp., but not A. sydowii; the lipolytic enzyme secreted by A. sydowii had quite high activity toward the artificial substrate p-nitrophenyl butyrate, but low activity toward the natural oil. The lipolytic fractions from A. chevalieri and A. pseudoglaucus were further purified by hydrophobic interaction chromatography then gel-filtration chromatography; LC-MS-MS Mascot analysis identified a variety of lipolytic enzymes, including cutinase, esterase, phospholipase, and carboxyl esterase in the lipolytic fractions from these species. The identified enzymes had 30%-70% identity to previously reported or manually annotated lipases or esterases from taxa other than Aspergillus. The different lipolytic enzymes likely acted on triglycerides in the katsuobushi fish oil. Furthermore, catalase B and Cu/Zn superoxide dismutase, which limit oxidative damage of lipids, were also identified. These antioxidant enzymes may prevent lipid oxidation and rancidity as the lipolytic enzymes hydrolyze lipids during the long fermentation and ripening process. Umami and richness tastes tended to increase in extracts from culture of protease- and peptidase-producing A. sydowii. Our results will aid in the selection and application of desirable strains of Aspergillus species as starter cultures to improve the storage and quality of fermented and ripened karebushi.

Characterization of surface Aspergillus community involved in traditional fermentation and ripening of katsuobushi.

A soup stock made from katsuobushi is an important element of, and the basic seasoning responsible for the taste of, traditional Japanese cuisine. Fermented and ripened katsuobushi, called karebushi, is manufactured via a repeated molding process on the katsuobushi surface. Our aim was to characterize the surface Aspergillus community and their enzymes involved in the fermentation and ripening. Five dominant Aspergillus species isolated from the karebushi surface were identified-A. amstelodami, A. chevalieri, A. pseudoglaucus, A. ruber, and A. sydowii. Analyses were performed on final molding stage-samples from different manufacturers, and 1st to 4th molding stage-samples from the same manufacturer. The composition ratios of the five Aspergillus spp. varied according to the manufacturer of the karebushi. A. amstelodami and A. chevalieri tended to be detected as dominant species when the water content of the karebushi fillet was >15% and the fat content was >3.5%, respectively. In samples from a given manufacturer, the dominant species in the final molding stage tended to be A. chevalieri and A. pseudoglaucus. Mixed molds were cultured by solid-state fermentation using katsuobushi powder medium at two different water activity (aw) levels. Crude extracts from each culture showed lipase, aminopeptidase, carboxypeptidase, and protease activities. Notably, the crude extracts cultivated at 0.85 aw showed higher protease activity toward hemoglobin and lipase activity toward p-nitrophenyl palmitate than those at 0.95 aw. These hydrolytic enzymes are probably involved in decolorization of katsuobushi and lipid degradation during the long fermentative and ripening period. In addition, mixed cultures could transform 2,6-dimethoxyphenol into 1,2,3-trimethoxybenzene, previously reported as an attractive and mild flavor component. Our results may help promote the use of desirable Aspergillus spp. as starter cultures for manufacturers to stabilize and improve the quality of fermented and ripened karebushi.

Isolation and characterization of an aspartic protease able to hydrolyze and decolorize heme proteins from Aspergillus glaucus.

BACKGROUND: The xerophilic Aspergillus molds, Aspergillus glaucus and Aspergillus repens, have been used in the ripening and fermentation of dried tuna bonito (katsuobushi). These molds, and especially their extracellular hydrolytic enzymes, may also be of wider industrial value. RESULTS: Aspergillus glaucus strain MA0196 produces different types of hydrolytic enzymes, including amylase, serine protease, aspartic protease, lipase and cellulase, depending on the composition of the medium. We characterized several of these enzymes, focusing on a glycosylated aspartic protease. The results showed that the lower the d-glucose concentration in the medium, the higher the degree of protease glycosylation, with excess glycosylation tending to decrease protease activity. The molecular mass of the glycosylated protease as determined by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis was 243 and 253 kDa, respectively. The chemically deglycosylated protease had a molecular mass of only 46 kDa. The amount of myoglobin-decolorizing activity was similar to that of a previously reported aspartic protease from A. repens strain MK82. However, the strain MA0196 protease more broadly hydrolyzed myoglobin and hemoglobins than did the strain MK82 protease. CONCLUSION: The results of the present study demonstrate the potential utility of Aspergillus molds as a functionally new microbial resource for industrial applications such as the bleaching of heme proteins. © 2018 Society of Chemical Industry.

Heterologous expression and characterisation of the Aspergillus aspartic protease involved in the hydrolysis and decolorisation of red-pigmented proteins.

BACKGROUND: Aspergillus repens strain MK82 produces an aspartic protease (PepA_MK82) that efficiently decolorises red-pigmented proteins during dried bonito fermentation. However, further expansion of the industrial applications of PepA_MK82 requires the high-level production and efficient preparation of the recombinant enzyme. RESULTS: The genomic DNA and cDNA fragments encoding the protease were cloned from strain MK82 and sequenced. Phylogenetic analysis of PepA_MK82 and comparisons with previously reported fungal aspartic proteases showed that PepA_MK 82 clusters with different groups of these enzymes. Heterologous expression of PepA_MK82 in Pichia pastoris yielded preparations of higher purity than obtained with an Escherichia coli expression system. Total protease activity in a 100-mL culture of the P. pastoris transformant was 14 times higher than that from an equivalent culture of A. repense MK82. The recombinant PepA_MK82 was easily obtained via acetone precipitation; the final recovery was 83%. PepA_MK82 and its recombinant had similar characteristics in terms of their optimal pH, thermostability, and decolorisation activity. The recombinant was also able to decolorise flaked, dried bonito and to bleach a blood-stained cloth. CONCLUSION: Given its ability to hydrolyse and decolorise red-pigmented proteins, recombinant PepA_MK8 can be exploited in the food industry and as a stain-removal agent in laundry applications. © 2016 Society of Chemical Industry.

Jellyfish collagen stimulates production of TNF-α and IL-6 by J774.1 cells through activation of NF-κB and JNK via TLR4 signaling pathway.

We previously reported that jellyfish collagen stimulates both the acquired and innate immune responses. In the acquired immune response, jellyfish collagen enhanced immunoglobulin production by lymphocytes in vitro and in vivo. Meanwhile, in the innate immune response jellyfish collagen promoted cytokine production and phagocytotic activity of macrophages. The facts that jellyfish collagen plays several potential roles in stimulating cytokine production by macrophages have further attracted us to uncover its mechanisms. We herein describe that the cytokine production-stimulating activity of jellyfish collagen was canceled by a Toll-like receptor 4 (TLR4) inhibitor. Moreover, jellyfish collagen stimulated phosphorylation of inhibitor of κBα (IκBα), promoted the translocation of nucleus factor-κB (NF-κB), and activated c-Jun N-terminal kinase (JNK). A JNK inhibitor also abrogated the cytokine production-stimulating activity of jellyfish collagen. These results suggest that jellyfish collagen may facilitate cytokine production by macrophages through activation of NF-κB and JNK via the TLR4 signaling pathways.

Aspartic protease from Aspergillus (Eurotium) repens strain MK82 is involved in the hydrolysis and decolourisation of dried bonito (Katsuobushi).

BACKGROUND: Katsuobushi is a dried, smoked and fermented bonito used in Japanese cuisine. During the fermentation process with several Aspergillus species, the colour of Katsuobushi gradually changes from a dark reddish-brown derived from haem proteins to pale pink. The change in colour gives Katsuobushi a higher ranking and price. This study aimed to elucidate the mechanism of decolourisation of Katsuobushi. RESULTS: A decolourising factor from the culture supernatant of Aspergillus (Eurotium) repens strain MK82 was purified to homogeneity. The purification was monitored by measuring the decolourising activity using equine myoglobin and bovine haemoglobin as substrates. It was found that the decolourising factor had protease activity towards myoglobin and haemoglobin. Complete inhibition of the enzyme by the inhibitor pepstatin A and the internal amino acid sequence classified the protein as an aspartic protease. The enzyme limitedly hydrolysed myoglobin between 1-Met and 2-Gly, 43-Lys and 44-Phe, and 70-Leu and 71-Thr. The purified enzyme decolourised blood of Katsuwonus pelamis (bonito) and a slice of dried bonito. CONCLUSION: It is proposed that aspartic protease plays a role in the decolourisation of Katsuobushi by the hydrolysis of haem proteins that allows the released haem to aggregate in the dried bonito.

Immunostimulatory effects of collagen from jellyfish in vivo.

We focused on the biological activity of the collagen extracts obtained from the giant edible jellyfish, Nemopilema nomurai. Jellyfish collagen extracts stimulates the production of immunoglobulins (Igs) and cytokines by human hybridoma cells and human peripheral blood lymphocytes. Therefore, we examined the immunoregulatory function of jellyfish collagen extracts in mice. Intake of jellyfish collagen extracts facilitated the Ig production activity of lymphocytes from spleen and Peyer's patch. Furthermore, the levels of Igs in the serum clearly increased after the administration of jellyfish collagen extracts. Intake of bovine collagen from Achilles' tendon also activated lymphocytes activity in mice. The activity of total and antigen-specific Ig production in splenocytes from OVA-challenged mice was also enhanced by collagen intake. However, the total and OVA-specific IgE levels in the serum were not affected by the collagen intake. These results suggested that jellyfish collagen extracts stimulates an immune response in vivo, without inducing allergic complications.

Mode of action of the immunostimulatory effect of collagen from jellyfish.

We have previously demonstrated that collagen from jellyfish simulated immunoglobulin and cytokine production by human-human hybridoma line HB4C5 cells and by human peripheral blood lymphocytes (hPBL). The mode of action of the collagen as an immunostimulatory factor was investigated. The expression levels of immunoglobulin mRNAs in HB4C5 cells, and those of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta in hPBL were up-regulated by jellyfish collagen. In addition, this collagen activated IgM production by transcription-suppressed HB4C5 cells that had been treated with actinomycin D. This collagen also enhanced IgM production by translation-suppressed HB4C5 cells that had been treated with sodium fluoride, but was ineffective in accelerating IgM production by HB4C5 cells treated with cycloheximide. Moreover, the intracellular IgM level in HB4C5 cells treated with the post-translation inhibitor, monensin, was increased by this collagen. These results suggest that collagen from jellyfish stimulated not only the transcription activity, but also the translation activity for enhanced immunoglobulin and cytokine production.

Protease-resistant fraction of smoked, dried bonito alleviates atopic dermatitis-like skin lesions in NC/Nga mice.

The effect of smoke-dried bonito undigested fraction remaining after microbial protease treatment (SDBR) on a spontaneously occurring mouse model of atopic dermatitis was studied in male 5-wk-old, NC/Nga mice. Smoke-dried bonito, Katsuobushi, is a traditional Japanese food. SDBR contains 2 major components: bonito oil and protease-undigested proteins. Mice were fed a casein diet containing corn oil (C diet) or a diet containing SDBR (SDBR diet) for 18 wk. In comparison with the C diet, the SDBR diet alleviated the increase in skin severity score and plasma IgE concentration in a time-dependent manner, and lowered leucotriene B(4) (LTB(4))-releasing ability upon calcium ionophore A23187 stimulation. The SDBR diet did not affect scratching time. These results demonstrate that SDBR diet alleviates atopic dermatitis-like skin lesions in NC/Nga mice.

Hypocholesterolemic effect of katsuobushi, smoke-dried bonito, prevents ovarian hormone deficiency-induced hypercholesterolemia.

The purpose of this study was to examine whether katsuobushi, smoked-dried bonito (KB), which is a traditional Japanese food, prevents ovarian hormone deficiency-induced hypercholesterolemia. In experiment 1, ovariectomized rats (OVX-rats) were fed a purified diet containing casein or KB. Compared with the casein diet, the KB diet reduced the plasma cholesterol concentration and apparent protein digestibility, and increased the fecal dry weight and fecal bile acid excretion. In experiment 2, OVX-rats were fed one of the following four diets: casein diet containing corn oil or fish oil (CA/CO or CA/FO), or a diet containing the digested or undigested fraction of KB after treatment with microbial protease (KBE or KBR). KBR contains mainly two components: oil and protease-undigested protein of KB origin. In comparison with the CA/CO diet, the KBE diet did not affect the plasma and liver lipids concentrations, apparent protein digestibility nor fecal bile acid excretion. However, the KBR and CA/FO diets reduced the plasma cholesterol and triacylglycerol (TAG) concentrations and the liver total lipid and TAG concentrations, but increased the liver total and esterified cholesterol concentrations. The KBR diet increased fecal bile acid excretion and fecal dry weight, whereas the CA/FO diet did not. Thus, the preventive effect of KB on the ovarian hormone deficiency-associated increase in plasma cholesterol concentration appears to be mediated by an increase in bile acid excretion through a promoted secretion of bile acids by the binding of bile acids to resistant proteins.

Hypocholesterolemic effects of microbial protease-resistant fraction of Katsuobushi in ovariectomized rats depend on the both oil and undigested protein.

The purpose of this study was to examine which component in the microbial protease-resistant fraction of Katsuobushi (KBR), smoke-dried bonito, is hypocholesterolemic in ovariectomized rats (OVX-rats). KBR contains two major components: oil and protease-resistant protein. Oil extracted from KBR (EX) was rich in palmitic, oleic and docosahexaenoic acids. OVX-rats were fed one of the following diets for 28 d: diets containing casein as the protein source (C or C+EX diet), a diet containing KBR as the protein source (KBR diet) or diets containing degreased KBR as the protein source (DF/KBR or DF/KBR+EX diet). The C and DF/KBR diets contained soybean oil as the oil source. In the C+EX, KBR and DF/KBR+EX diets, soybean oil was replaced by oil extracted from KBR (EX). Plasma total- and low density lipoprotein-cholesterol concentrations in the C+EX, KBR and DF/KBR+EX groups, but not in the DF/KBR group, were significantly lower than that in the C group. Fecal bile acid excretion was significantly greater in the C+EX, KBR, DF/KBR and DF/KBR+EX groups in comparison to the C group, whereas excretion in the KBR and DF/KBR+EX groups was significantly greater than in the C+EX and DF/KBR groups. Cholesterol 7alpha-hydroxylase activity was higher in the C+EX, KBR, DF/KBR and DF/KBR+EX groups than the C group. In OVX-rats fed C, C+EX or KBR for 28 d, bile acid flux into the small intestine increased in KBR and C+EX groups in comparison to the C group. The hypocholesterolemic effect of KBR in OVX-rats reflected in increased fecal bile acid excretion may be mediated by increased bile acid flux caused by EX and the binding of bile acids by protease-resistant proteins.

Immunostimulation effect of jellyfish collagen.

Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 degrees C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles' tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.

Evaluation of iron bioavailability from bonito dark muscle using anemic rats.

The bioavailability of iron from ferrous sulfate (FeII-S), heme iron prepared from hemoglobin (HIP), and bonito dark muscle (BDM) was assessed in anemic rats using a hemoglobin regeneration efficiency (HRE) method. Freeze-dried BDM (FD), boiled and freeze-dried BDM (B/FD), and boiled and smoke-dried BDM (B/SD) were used as BDM source. Rats were made anemic by feeding on an iron-deficient diet for 28 days. To replete their iron levels, anemic rats were then fed on a diet containing iron at a level of 17 ppm for 14 days. Rats receiving FeII-S gained significantly more weight and had greater food intake and higher HRE compared to the other four groups. The bioavailability of iron from HIP was poor compared with that from FeII-S and BDM. When the HRE of rats fed FeII-S was 100, that of rats fed BDF was approximately 80. These results suggest that BMD is an effective dietary source of iron.