Purification, characterisation and salt-tolerance molecular mechanisms of aspartyl aminopeptidase from Aspergillus oryzae 3.042
Introduction
Fermented soybean products, such as soy sauce, soybean paste, Douchi, Sufu and Tianyou, play a key role in traditional fermented foods industry in China. These products are usually produced with high salinity (5–20%, w/v) using Aspergillus oryaze 3.042 (A. oryzae 3.042) as a main fermentation strain. The annual production of soy sauce exceeded 50% of the total condiment output in China, reaching 9.38 million tons in 2014 (http://www.askci.com/news/chanye/2015/02/04/165020koaw.shtml). Nevertheless, the quality and the raw material utilisation rate of fermented soybean products, especially soy sauce, are low, which result in poor competitiveness in both domestic and foreign markets. The above two parameters of soy sauce, soybean paste, Douchi, Sufu and Tianyou depend greatly upon the salt-tolerance of proteases secreted by A. oryzae 3.042 (Lee et al., 2010, Wang et al., 2013), but salt-tolerance of the dominant proteases, specifically the neutral proteases secreted by A. oryzae 3.042, is poor (Wang et al., 2013). The physicochemical properties and salt-tolerance molecular mechanisms of several known salt-tolerant proteases from A. oryzae remain unclear (Wang et al., 2013).
A. oryzae, especially A. oryzae 3.042 and A. oryzae RIB40, are widely accepted as ‘generally recognised as safe’ (GRAS) due to their long history of safe applications in the manufacture of traditional fermented foods in East Asia and pharmaceutical-grade enzymes (Maeda et al., 2005, Vishwanatha et al., 2009). The entire A. oryzae RIB40 genome had been published, and 130 protease-like genes were identified, but most were not characterised (Machida et al., 2005). Amongst these genes, about 20 encoded proteases had aminopeptidase activity (Kusumoto et al., 2008). Researchers had isolated aspartyl aminopeptidases (AAP) from A. oryzae (Kusumoto et al., 2008), yeast (Yokoyama, Kawasaki, & Hirano, 2006), Escherichia coli (Watanabe, Tanaka, Akagawa, Mogi, & Yamazaki, 2007) and Lactobacillus delbrueckii (Stressler et al., 2016). The aforementioned studies demonstrated that AAPs isolated from different microorganisms were salt-tolerant and included varying numbers of isomeric subunits. They also showed the differential effects of metal ions and inhibitors on the activities of AAPs from various microorganisms. Notably, AAP can specifically release glutamic acid and aspartic acid from peptides that contain glutamic acid and aspartic acid residues in their N-termini. These two amino acids, which impart an umami taste with low thresholds, play a key role in fermented soybean products (Stressler et al., 2016). Currently, little is known about the salt-tolerance mechanisms of AAP, and such a capacity is important to maintain, activate and/or modulate the activity of AAP during manufacture of fermented soybean products in high salinity.
Bioinformatics tools are becoming more powerful and reliable due to the growing number and availability of experimentally determined protein structures (Arnold et al., 2006, Suplatov et al., 2015, Wiltgen and Tilz, 2009). The SWISS-MODEL and MODELLER programs are most frequently used for homology or comparative protein structure modelling (Arnold et al., 2006, Kashlan et al., 2011). Molecular dynamics (MD) simulation (e.g., NAMD and VMD) can also provide atomic-level and time-dependent information for structure and functionality of protein, which are difficult to obtain experimentally (Lin et al., 2012, Sotomayor and Schulten, 2007). Most importantly, MD simulation can also model aspects of protein structures in solution (Amorim, Netz, & Guimarães, 2010). Furthermore, secondary structure and solvent accessibility of proteins have frequently been predicted by the PHD and PROF programs of PredictProtein server (Devos et al., 2004, Rost, 1996, Rost, 2001).
Given the above analyses, it will be of great theoretical importance and potential application to isolate, purify and identify AAP from A. oryzae 3.042, to characterise it and to elucidate its salt-tolerance molecular mechanisms.
Section snippets
Materials and chemicals
A. oryzae 3.042 was purchased from Guangdong Institute of Microbiology (Guangzhou, China). The other chemicals were of the highest commercial grade and were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Assay of AAP activity
The activity of AAP was assayed according to the method of Kusumoto et al. (2008) with minor revisions. Enzyme solution was incubated with 2 mM Asp-pnitroanilide (pNA; Sigma-Aldrich, Tokyo, Japan) in 30 mM Tris–HCl buffer (pH 7.5) containing 10% glycerol (w/v) at 37 °C for
Isolation, purification, molecular weight determination and identification
The precipitations of AAP and NP I at 20%, 30%, 40%, 50%, 60%, 70% and 80% ammonium sulphate saturations were investigated in preliminary experiments (data not shown). Almost all AAP and NP I were precipitated at 80% and 70% of ammonium sulphate saturations, respectively. As shown in Table 1 and Fig. 1A and B, a protein with AAP activity is isolated and purified using phosphate buffer extraction, ammonium sulphate precipitation, HiTrap Q HP column chromatography and Superose 6 column
Acknowledgements
The authors gratefully acknowledge the following programs for their financial support: the National Natural Science Foundation of China (31301538), the China Postdoctoral Science Foundation (2016M600380), the National Key Research and Development Program (2016YFD0400700-05), the Six Talent Peaks Project in Jiangsu Province (2015-NY-16), the Social Development Project of Jiangsu Science and Technology Department (SBE2016740834) and the Start-up Foundation for Advanced Talents of Jiangsu
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