A salt-tolerant exo-β-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-β-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-β-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 β-1,3-linked glucose dimer laminaribiose, but not β-1,4-linked or β-1,6-linked glucose dimers (cellobiose or gentiobiose). It showed tenfold higher activity toward laminarin (a linear polymer of β-1,3 glucan) from Laminaria digitata than laminarin (β-1,3/β-1,6 glucan) from Eisenia bicyclis, likely due to its inability to act on β-1,6-linked glucose residues. The β-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.

Dried bonito broth, made from katsuobushi flakes, is a commonly used seasoning in Japanese cuisine. While extracting the broth, high-quality proteins are left behind in the katsuobushi grounds, which could be a valuable source of bioactive substances via a hydrolysis process. The xerophilic Aspergillus sydowii showed potential for protein hydrolysis and production of amino acids and soluble peptides through solid-state fermentation. Several peptidases involved in katsuobushi protein hydrolysis were purified from the crude enzyme preparations and identified as aminopeptidase, trypsin-like serine protease, and elastinolytic serine protease. The hydrolysates from the culture contained glutamic acid, leucine, and lysine and markedly increased soluble peptides with molecular weights ranging from 0.2 to 17 kDa. Electronic tongue analysis indicated that the hydrolysates had substantially stronger salty and umami tastes. Moreover, the hydrolysates exhibited radical-scavenging activities, ferric ion reducing antioxidant power, and angiotensin converting enzyme inhibitory activity, as well as promoting growth in lactic acid bacteria. In addition, de novo peptide sequencing was performed for peptides fractionated from the hydrolysates, and eight peptides were identified. The hydrolysates produced by A. sydowii fermentation could have potential applications in the food industry as natural flavor enhancers and functional ingredients with several bioactivities.

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.

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.

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.

Abstract Aspergillus glaucus MA0196 produces a highly glycosylated aspartic protease (PepA_MA0196) that shows hydrolytic and decolorization activities toward hemoglobin. Sequence analysis of PepA_MA0196 indicated two potential N-glycosylation consensus sites, at Asn131 and Asn275. To elucidate the role of N-glycosylation in the biochemical properties of PepA_MA0196 and the resulting effects, recombinant PepA_MA0196 and three mutant proteases deglycosylated via site-direct mutagenesis were heterologously expressed in Pichia pastoris. After hydrolysis of the enzymes by PNGase F or Endo H, the molecular masses on SDS-PAGE of the enzymatically deglycosylated wild-type PepA_MA0196 and recombinant and single-mutant proteases were identical to that of the double-mutant protease (42.2 kDa). PepA_MA0196 and its recombinant were stable over a broader pH range and more thermostable than the deglycosylated mutant proteases, especially the double-mutant protease. In addition, PepA_MA0196 retained a higher activity at low water activity than did the double-mutant protease. The commercial applications of the glycosylated protease from strain MA0196 include hydrolysis of the heme proteins in dried tuna meat under low water content. Hemoprotein lysis is also a property of interest to the meat processing and detergent industries.

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. 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. 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.