A novel milk-clotting enzyme from Aspergillus oryzae and A. luchuensis is an aspartic endopeptidase PepE presumed to be a vacuolar enzyme

Aspergillus oryzae RIB40 has 11 aspartic endopeptidase genes. We searched for milk-clotting enzymes based on the homology of the deduced amino acid sequence with chymosins. As a result, we identified a milk-clotting enzyme in A. oryzae. We expected other Aspergillus species to have a homologous enzyme with milk-clotting activity, and we found the most homologous aspartic endopeptidase from A. luchuensis had milk-clotting activity. Surprisingly, two enzymes were considered as vacuole enzymes according to a study on A. niger proteases. The two enzymes from A. oryzae and A. luchuensis cleaved a peptide between the 105Phe-106Met bond in κ-casein, similar to chymosin. Although both enzymes showed proteolytic activity using casein as a substrate, the optimum pH values for milk-clotting and proteolytic activities were different. Furthermore, the substrate specificities were highly restricted. Therefore, we expected that the Japanese traditional fermentation agent, koji, could be used as an enzyme source for cheese production.

Enhanced l-Malic Acid Production by Aspergillus oryzae DSM 1863 Using Repeated-Batch Cultivation

l-Malic acid is a C4-dicarboxylic acid and a potential key building block for a bio-based economy. At present, malic acid is synthesized petrochemically and its major market is the food and beverages industry. In future, malic acid might also serve as a building block for biopolymers or even replace the commodity chemical maleic anhydride. For a sustainable production of l-malic acid from renewable resources, the microbial synthesis by the mold Aspergillus oryzae is one possible route. As CO2 fixation is involved in the biosynthesis, high yields are possible, and at the same time greenhouse gases can be reduced. In order to enhance the production potential of the wild-type strain Aspergillus oryzae DSM 1863, process characteristics were studied in shake flasks, comparing batch, fed-batch, and repeated-batch cultivations. In the batch process, a prolonged cultivation time led to malic acid consumption. Keeping carbon source concentration on a high level by pulsed feeding could prolong cell viability and cultivation time, however, did not result in significant higher product levels. In contrast, continuous malic acid production could be achieved over six exchange cycles and a total fermentation time of 19 days in repeated-batch cultivations. Up to 178 g/L l-malic acid was produced. The maximum productivity (0.90 ± 0.05 g/L/h) achieved in the repeated-batch cultivation had more than doubled than that achieved in the batch process and also the average productivity (0.42 ± 0.03 g/L/h for five exchange cycles and 16 days) was increased considerably. Further repeated-batch experiments confirmed a positive effect of regular calcium carbonate additions on pH stability and malic acid synthesis. Besides calcium carbonate, nitrogen supplementation proved to be essential for the prolonged malic acid production in repeated-batch. As prolonged malic acid production was only observed in cultivations with product removal, product inhibition seems to be the major limiting factor for malic acid production by the wild-type strain. This study provides a systematic comparison of different process strategies under consideration of major influencing factors and thereby delivers important insights into natural l-malic acid production.

Immobilization of Aspergillus oryzae DSM 1863 for l-Malic Acid Production

Whole-cell immobilization by entrapment in natural polymers can be a tool for morphological control and facilitate biomass retention. In this study, the possibility of immobilizing the filamentous fungus Aspergillus oryzae for l-malic acid production was evaluated with the two carbon sources acetate and glucose. A. oryzae conidia were entrapped in alginate, agar, and κ-carrageenan and production was monitored in batch processes in shake flasks and 2.5-L bioreactors. With glucose, the malic acid concentration after 144 h of cultivation using immobilized particles was mostly similar to the control with free biomass. In acetate medium, production with immobilized conidia of A. oryzae in shake flasks was delayed and titers were generally lower compared to cultures with free mycelium. While all immobilization matrices were stable in glucose medium, disintegration of bead material and biomass detachment in acetate medium was observed in later stages of the fermentation. Still, immobilization proved advantageous in bioreactor cultivations with acetate and resulted in increased malic acid titers. This study is the first to evaluate immobilization of A. oryzae for malic acid production and describes the potential but also challenges regarding the application of different matrices in glucose and acetate media.

Purification and Enzymatic Properties of a New Thermostable Endoglucanase From Aspergillus Oryzae HML366

Aspergillus oryzae HML366 is a newly screened cellulase-producing strain. The endoglucanase HML ED1 from A. oryzae HML366 was quickly purified by two-step method ammonium sulfate precipitation and strong anion exchange column. SDS-PAGE electrophoresis indicated that the molecular weight of the enzyme was 68 kDa. The optimum temperature of the purified endoglucanase was 60 ℃ and the enzyme activity was stable below 70 ℃. The optimum pH was 6.5, and the enzyme activity was stable at pH between 4.5 to 9.0. The analysis indicated that additional Na+, K+, Ca2+, and Zn2+ reduced the catalytic ability of enzyme to the substrate, but Mn2+ enhanced its catalytic ability to the substrate.The Km and Vmax of the purified endoglucanase was 8.75 mg/mL and 60.24 μg/min·mL, respectively. Our study demonstrated that A. oryzae HML366 can produce a heat-resistant and wide pH tolerance endoglucanase HML ED1, which has potential industrial application value in bioethanol, paper, food, textile, detergent and pharmaceutical industries.

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Effect of fermentation on mixed rumen contents and jackfruit peel using Aspergillus oryzae on in vitro gas production and digestibility

The objective of this research was to investigate the effect of Aspergillus oryzae on fermentation of mixture of rumen contents and jackfruit peel on in vitro gas production and digestibility. The method used in this study was an experiment using a randomized block design (RBD) of 4 treatments and 3 replications. The treatments including of T0 = 50% rumen contents + 50% jackfruit peel, T1 = 40 % rumen contents + 60% jackfruit peel + 0,4% Aspergillus oryzae, T2 = 30% rumen contents + 70% jackfruit peel + 0,4% Aspergillus oryzae, T3 = 20% rumen contents + 80% jackfruit peel + 0,4% Aspergillus oryzae. Variables observed were gas production, dry matter digestibility (DMD), and organic matter digestibility (OMD). Data were analyzed by using Analysis of Variance (ANOVA) from Randomized Block Design, if there were significant effect between the treatments then tested with least significant different (LSD). The result showed that fermentation of mixture of rumen contents and jackfruit peel have highly significant effect (P<0.01) on gas production, and significant effect (P<0.05) on DMD and OMD. Gas production, DMD and OMD of fermented mixture of rumen contents and jackfruit peel are higher than control without fermentation. It is concluded that the higher jackfruit peels the higher gas production, DMD and OMD.