Enzymatic properties of the glycine d-alanine aminopeptidase of Aspergillus oryzae and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji)

Abstract

The gdaA gene encoding S12 family glycine-D-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the D-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high D-stereospecificity and efficiently released N-terminal glycine and D-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and D-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and D-alanine aminopeptidase activity detected at a pH range of 6 to 9.

Production and Characterization of a New α-Glucosidase Inhibitory Peptide fromAspergillus oryzaeN159-1

“…The filamentous fungus, especially Aspergillus oryzae , has been used in production of traditional fermented food, such as sake, makgeolli (rice wine), miso, meju (soybean paste), and shoyu (soy sauce) [27-29]. Aspergillus oryzae also produces a variety of enzymes, such as amylases and proteases, and acts on the nutrients of soy to break down carbohydrate and protein, forming koji [30]. A. oryzae is listed as a GRAS (i.e., generally regarded as safe) strain by the U.S. Food and Drug Administration.…”

Characterization of a Glycyl-Specific TET Aminopeptidase Complex from Pyrococcus horikoshii

“…No hydrolytic activity toward any other amino acids could be detected, even with long incubation times. We also tried to investigate whether PhTET4 exhibited high D-stereospecificity with D-alanine, as shown for Aspergillus oryzae glycine aminopeptidase (20). For this, D-Ala-pNA was used as the chromogenic substrate under optimal activity conditions (0.1 mM NiCl 2 , pH 9.5, and 85°C).…”

“…While aminopeptidases from different families are capable of hydrolyzing glycine residues, their activities remain weak in comparison with their preferred substrates. To our knowledge, only three aminopeptidases have been found to exhibit clear preferences for glycine residues; the first is a Zn-dependent metallopeptidase from the M61 family secreted by the Gram-negative bacterium Sphingomonas capsulata (30), the second is a eukaryotic S12-family serine peptidase found in the cytosol of Actinomucor oryzae (20), and the third is the cytosolic GAP of Actinomucor elegans (31), for which the residues implicated in the enzymatic mechanism are still ambiguous. Therefore, PhTET4 is the first GAP enzyme identified in archaea.…”

Acid protease production by Aspergillus oryzae on potato pulp powder with emphasis on glycine releasing activity: A benefit to the food industry

“…In the present study, we have examined glycine-releasing activity during the SSF of potato pulp powder using the industrial fungus, A. oryzae (F6 strain) because this activity is thought to influence the palatability . Also, the proteolytic enzymes of A. oryzae play important roles in the breakdown of protein into a variety of peptides and amino acids that enhance the palatability of fermented foods(Marui et al, 2012).The glycine-releasing activity of the culture from potato pulp powder was 0.57 ± 0.25 U/g.In a previous study, Marui et al, (2012) described GdaA, the glycine aminopeptidase of A. oryzae RIB 40, which utilizes both Gly-pNA and peptides with Gly at the amino terminus as substrates. This aminopeptidase share most of the activity for releasing glycine among intracellular enzymes, as a cell-free extract of gdaA-disrupted strain, derived from RIB 40, showed significantly lower glycine-releasing activity, compared with its control strain.…”