Kinetics of alpha-amylase secretion in Aspergillus oryzae.

Pulse and pulse-chase experiments have been performed to study L-[(35)S] methionine incorporation and protein secretion kinetics in Aspergillus oryzae. Pulse experiments confirmed the mechanism of methionine uptake reported previously for Penicillium chrysogenum (Benko et al., 1967). Pulse-chase experiments were carried out to investigate the alpha-amylase secretion kinetics in A. oryzae. No unglycosylated alpha-amylase was detected neither intracellularly nor extracellularly demonstrating that glycosylation was not the rate controlling step in the secretory pathway. The pulse chase experiments indicated that there are two pools of intracellular alpha-amylase: a fast secreted and a slow secreted. The secretion of those two pools were described with a kinetic model, which was fitted to the pulse chase experiments.     

Septum-directed secretion in the filamentous fungus Aspergillus oryzae

Although exocytosis in fungal cells takes place at hyphal tips, there also seems a line of circumstantial evidence suggesting the occurrence of exocytosis at other sites of cells, such as septa. To investigate whether exocytosis takes place at fungal septa, we monitored dynamics of EGFP‐fused α‐amylase (AmyB–EGFP), the representative secretory enzyme of the filamentous fungus Aspergillus oryzae. We found that AmyB–EGFP accumulates in Spitzenkörper at hyphal tips as well as septal periplasm between the plasma membrane and cell walls. The septal accumulation of AmyB–EGFP was a rapid process, and required microtubules but not F‐actin. Thus, this process is independent of exocytosis at hyphal tips that requires both microtubules and F‐actin. In addition, fluorescence recovery after photobleaching (FRAP) analysis of EGFP‐fused AoSnc1 revealed that secretory vesicles constitutively fuse with the septal plasma membrane. These results demonstrated that exocytosis takes place at septa in addition to hyphal tips. Analysis of two plasma membrane transporters, AoUapC and AoGap1, revealed that they preferentially accumulate at septa and the lateral plasma membrane with no clear accumulation at apical Spitzenkörper, suggesting that non‐tip directed exocytosis is important for delivery of these proteins.     

Characterization of a novel lipolytic enzyme from Aspergillus oryzae

In this study, we report the characterization of a protein from Aspergillus oryzae, exhibiting sequence identity with paraben esterase from the genus Aspergillus. The coding region of 1,586 bp, including a 77-bp intron, encoded a protein of 502 amino acids. The gene without the signal peptide of 19 amino acids was cloned into a vector, pPICZαC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0–8.0 and 30 °C, respectively, and was stable at the pH range of 7.0–10.0 and up to 40 °C. The optimal substrate for hydrolysis by the purified recombinant protein, among a panel of α-naphthyl esters (C2–C16), was α-naphthyl butyrate (C4), with activity of 0.16 units/mg protein. The considerable hydrolytic activity of the purified recombinant enzyme toward tributyrin was determined. However, no paraben esterase activity was detected toward the ethyl, propyl, and butyl esters of 4-hydroxybenzoic acid. In addition, no activity was detected toward the methyl esters of ferulic, p-coumaric, caffeic, and sinapic acids that would indicate feruloyl esterase activity.     

Culture conditions of Aspergillus oryzae for production of enzyme preparation

Submerged culture was better than solid culture in the production of proteinase and peptidases from Aspergillus oryzae 460. On the contrary, solid culture was better than submerged culture in the production of α-amylase, carboxymethyl cellulase, and pectinlyase from the same fungus.The soy souce mash (moromi) made with the enzyme preparation from submerged culture was highly viscous and the soy sauce produced was characteristic in low contents of alcohol and reducing sugar, low pH value, and less aroma. Soy sauce made with the enzyme preparation from solid culture was superior on these points to that from submerged culture.Wheat bran was best as the raw material for the enzyme preparation in easy koji making, large amount produced, and low cost.In enzyme production from a solid culture, addition of urea (0.8% to wheat bran) nearly doubled the leucine aminopeptidase for Leu-Gly-Gly. The incubation period was reduced to 30 to 40 h from 50 to 60 h using germinated spores and moisture-controlled culture with forced aeration.     

A novel enzyme producing isoprimeverose from oligoxyloglucans of Aspergillus oryzae

An enzyme producing isoprimeverose from xyloglucan fragment oligosaccharides has been purified to the electrophoretically pure state from a commercial enzyme preparation of Aspergillus oryzae (Sanzyme 1000). The purified enzyme showed approximately 1,280-fold increase of the specific activity over the original preparation. The purified enzyme was shown to be an oligomeric protein consisting of two subunits, each of which had a molecular weight of 115,000. The enzyme showed the highest activity at pH 5.0 and 60 degrees C, and was stable in the pH range from 5 to 7 and at up to 50 degrees C. The isoelectric point of this enzyme was pH 3.9. The purified enzyme was highly specific for xyloglucan fragment oligosaccharides and split off isoprimeverose units from the non-reducing end of the backbone of the substrate.     

Secretory enzyme production and conidiation of Aspergillus oryzae in submerged liquid culture

Summary The production of - and -galactosidases, proteinase and -amylase and also conidiation of Aspergillus oryzae were examined in liquid soybean meal culture. In a culture of soybean meal only, conidiation of the fungus was not induced and the production of the enzymes was not significant, although fungal growth was abundant. When phosphate was added to the medium at concentrations above 0.2 M, enzyme production was significantly increased and the cells formed conidiophores after enzyme production had attained maximum level. Increase in production of galactosidases was the most marked.K- or Na-salts other than phosphate were not effective stimulants for enzyme production, while conidiation was not induced under these growth conditions. Conidiation and production of enzymes were repressed by the addition of glucose or casamino acids to the soybean meal medium containing KH₂PO₄.Conidiation and enzyme production were also studied in modified Czapek media in which sucrose was replaced by other carbon sources. Lactose, lactulose, melibiose and polysaccharides composed of galactosyl linkage such as arabinogalactan induced both conidiation and production of the enzymes.     

Immobilization of Aspergillus oryzae tannase and properties of the immobilized enzyme

Tannase enzyme from Aspergillus oryzae was immobilized on various carriers by different methods. The immobilized enzyme on chitosan with a bifunctional agent (glutaraldehyde) had the highest activity. The catalytic properties and stability of the immobilized tannase were compared with the corresponding free enzyme. The bound enzyme retained 20·3% of the original specific activity exhibited by the free enzyme. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme. The optimum temperature of the reaction was determined to be 40 °C for the free enzyme and 55 °C for the immobilized form. The stability at low pH, as well as thermal stability, were significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilized enzyme retained about 85% of the initial catalytic activity, even after being used 17 times.     

Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae

We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C₄-C₆ and C₃-C₈ chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.This revised version was published online in February 2005 with corrections to Table 1.     

Production and characterization of a milk-clotting enzyme from Aspergillus oryzae MTCC 5341

Microbial milk-clotting enzymes are valued as calf rennet substitutes in the cheese industry. Aspergillus oryzae MTCC 5341 was identified to produce the highest milk-clotting activity during screening of 16 fungal strains. Solid state fermentation using wheat bran along with 4% defatted soy flour and 2% skim milk powder as substrate was optimal for growth of A. oryzae and production of the enzyme. Nearly 40,000 U/g bran of milk-clotting activity was present at the end of 120 h. The enzyme could be recovered by percolating the bran with 0.1 M sodium chloride for 60 min at 4°C. The decolorized enzyme preparation had high ratio of milk clotting to proteolytic activity. Affinity precipitation with alginate and subsequent elution with 0.5 M sodium chloride containing 0.2 M CaCl₂ resulted in an enzyme preparation with specific activity of 3,500 U/mg and 72% yield. Optimum pH and temperature for activity of the enzyme were characterized as 6.3 and 55°C, respectively. Milk-clotting enzyme showed differential degree of hydrolysis on casein components. High ratio of milk clotting to proteolytic activity coupled with low thermal stability strengthens the potential usefulness of milk-clotting enzyme of A. oryzae MTCC 5341 as a substitute for calf rennet in cheese manufacturing.     

Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity.

Highly branched mutants of two strains of Aspergillus oryzae (IFO4177, which produces alpha-amylase, and a transformant of IFO4177 [AMG#13], which produces heterologous glucoamylase in addition to alpha-amylase) were generated by UV or nitrous acid mutagenesis. Four mutants of the parental strain (IFO4177), which were 10 to 50% more branched than the parental strain, were studied in stirred batch culture and no differences were observed in either the amount or the rate of enzyme production. Five mutants of the transformed parental strain (AMG#13), which were 20 to 58% more branched than the parental strain, were studied in either batch, fed-batch or continuous culture. In batch culture, three of the mutants produced more glucoamylase than the transformed parental strain, although only two mutants produced more glucoamylase and alpha-amylase combined. No increase in enzyme production was observed in either chemostat or fed-batch culture. Cultures of highly branched mutants were less viscous than those of the parental and transformed parental strains. A linear relationship was found between the degree of branching (measured as hyphal growth unit length) and culture viscosity (measured as the torque exerted on the rheometer impeller) for these strains. DOT-controlled fed-batch cultures (in which the medium feed rate was determined by the DOT) were thus inoculated with either the transformed parent or highly branched mutants of the transformed parent to determine whether the reduced viscosity would improve aeration and give higher enzyme yields. The average rate of medium addition was higher for the two highly branched mutants (ca. 8.3 g medium h(-1)) than for the parental strain (5.7 g medium h(-1)). Specific enzyme production in the DOT controlled fed-batch cultures was similar for all three strains (approx. 0.24 g alpha-amylase and glucoamylase [g of biomass](-1)), but one of the highly branched mutants made more total enzyme (24.3 +/- 0.2 g alpha-amylase and glucoamylase) than the parental strain (21.7 +/- 0.4 g alpha-amylase and glucoamylase).     

Trehalase in conidia of Aspergillus oryzae

Horikoshi, Koki (The Institute of Physical and Chemical Research, Bunkyo-ku, Tokyo, Japan), and Yonosuke Ikeda. Trehalase in conidia of Aspergillus oryzae. J. Bacteriol. 91:1883-1887. 1966.-Trehalases (soluble trehalase and coat-bound trehalase) were found in the conidia of Aspergillus oryzae, and the total activity of the trehalases increased during the germination process. The soluble trehalase was purified by diethylaminoethyl-cellulose column chromatography; its optimal pH, Michaelis constant, and heat stability were studied. In vitro, the trehalases were competitively inhibited by d-mannitol, which was also contained in the conidia. Since the trehalose content in the conidia decreased at an early stage of germination, it was assumed that trehalase might begin to hydrolyze trehalose after the inhibitory effect of d-mannitol decreased.     

Expression of Aspergillus hemoglobin domain activities in Aspergillus oryzae grown on solid substrates improves growth rate and enzyme production

DNA fragments coding for hemoglobin domains (HBD) were isolated from Aspergillus oryzae and Aspergillus niger. The HBD activities were expressed in A. oryzae by introduction of HBD gene fragments under the control of the promoter of the constitutively expressed gpdA gene. In the transformants, oxygen uptake was significantly higher, and during growth on solid substrates the developed biomass was at least 1.3 times higher than that of the untransformed wild-type strain. Growth rate of the HBD-activity-producing strains was also significantly higher compared to the wild type. During growth on solid cereal substrates, the amylase and protease activities in the extracts of the HBD-activity-producing strains were 30-150% higher and glucoamylase activities were at least 9 times higher compared to the wild-type strain. These results suggest that the Aspergillus HBD-encoding gene can be used in a self-cloning strategy to improve biomass yield and protein production of Aspergillus species.