Research Article: Purification and characterization of neutral protease from Aspergillus oryzae Y1 isolated from naturally fermented broad beans

Date Published: June 12, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Xiao-lin Ao, Xi Yu, Ding-tao Wu, Chao Li, Tong Zhang, Shu-liang Liu, Shu-juan Chen, Li He, Kang Zhou, Li-kou Zou.


The strain Y1, with a notably high production of neutral protease, was isolated from naturally fermented broad beans and subsequently identified as Aspergillus oryzae, through the analysis of its morphology characteristics and 18S rDNA sequence. Naturally fermented broad beans are the main raw material in Sichuan broad-bean sauce. The neutral protease from Aspergillus oryzae Y1 was purified using ammonium sulphate precipitation and DEAE-Sepharose Fast Flow chromatography, which resulted in a 10.0-fold increase in the specific activity (2264.3 U/mg) and a recovery rate of 21%. The estimated molecular mass of the purified protease was approximately 45 kDa. The optimal pH and temperature of the purified protease were 7.0 and 55 °C, respectively. The heat resistance of the purified protease was significantly higher than the commercial protease. The effect of metal ions on the activity of the purified protease approximated that of commercial neutral protease. Furthermore, the maximum hydrolysis rate (Vmax) and apparent Michaelis–Menten constant (Km) values of the purified protease were 256.4103 μg/mL min and 20.0769 mg/mL, respectively. The purified protease had a higher affinity for the substrate than the commercial neutral protease. All the results suggest that this neutral protease exhibits the potential for application in industry due to its good resistance to high temperatures and wide range of acids and bases.

Partial Text

Proteases may be classified as either acidic protease, neutral protease or alkaline protease, according to their optimum pH reactions. Proteases, especially the neutral protease, make up the largest proportion of industrial enzymes around the world (Kasana et al. 2011). Neutral proteases are widely applied in the food (Tavano 2013; Yuzuki et al. 2015), feed (Zhang et al. 2014), pharmaceutical (Umeadi et al. 2008) and leather (Asker et al. 2013) industries because of their distinct benefits, including a mild catalysis process, low pollution level and high yield. In the food industry, the neutral protease is generally used for debittering soy sauce (Machida et al. 2005) and brewing beer (Wang et al. 2013). However, the neutral protease is generally poor in thermal stability and is easily deactivated, which limits its application in some industries that require high temperatures. Neutral proteases are produced primarily by microorganisms, animals and plants. Those produced by microorganisms are most commonly applied in industry, because of their strong hydrolytic ability and wide adaptability to catalytic reaction conditions, which make them more adaptable to downstream processing than those proteases obtained from plants or animals (Sandhya et al. 2005; Gupta et al. 2002). Furthermore, neutral fungal proteases present a level of higher peptidase activity compared to other commercial enzymatic preparations (Guerard et al. 2002). The main characteristic of this enzyme is its affinity toward hydrophobic amino acids, which is advantageous for its use as a debittering agent (Sumantha et al. 2005).

A number of comparative benefits of purified protease from A. oryzae Y1 have been ascertained in this study. For one, the molecular mass of purified protease from Y1 was found to be inconsistent, unlike that of other neutral proteases, with a molecular mass of 43 kDa, which is larger than those found in Bacillus megaterium (25, 28 kDa) (Asker et al. 2013), Aeromonas veronii PG01 (33 kDa) (Divakar et al. 2010), Bacillus (34 kDa) (Breite et al. 2010), and A. parasiticus (36 kDa) (Anitha and Palanivelu 2013). The activity of the purified protease was strongly inhibited by 1 mM EDTA and PMSF, and, thus, was identified as a metal-dependent serine protease. A number of previous studies have been conducted on serine protease with molecular masses of approximately 25–37 kDa (Asker et al. 2013; Qiuhong et al. 2006; Cavello et al. 2013), all of which show the proteases concerned to be independent of metal ions. These results, therefore, suggest that the purified protease from A. oryzae Y1 is indeed a distinct protease.




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