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Biochemical, structural and molecular kinetics characteristics of salt-tolerant aspartyl aminopeptidase from Aspergillus oryzae 3.042 by Dr. Gao Xianli
 

1. Introduction

Soy sauce is a traditional fermented soybean condiment widely consumed in China, Japan, Korea and other Asian countries. It is usually produced by two-step fermentation including solid-state and liquid fermentation using Aspergillus oryzae 3.042. Aspergillus oryzae 3.042 produces plentiful enzymes, especially proteases, in the former process, which are used to catalyze soybean protein degradation (the key origin of soy sauce flavor) in the whole latter process (approximate 6 months). Thus proteases play a key role in soy sauce production. Unfortunately, most of the proteases will be inactivated within the first 3 days due to the high salt environment of the latter process (18% NaCl, w/v) and the weak salt-tolerance of the proteases. The insufficient salt-tolerant proteases in the latter process are responsible for the low protein utilization rate and quality of Chinese soy sauce when compared with those of Japan and Korea. Many previous works have done to improve the amount of proteases secreted by Aspergillus oryze 3.042 in the former process. However, the utilization rate of soybean and quality were not improved owing to the weak salt-tolerance of proteases secreted by Aspergillus oryze 3.042. Thus the separation, identification, elucidating the mechanism and elevating the amount of salt-tolerant proteases secreted by Aspergillus oryze 3.042 would be of great importance for soy sauce industry, even other industries, such as soy paste, Douchi, Sufu and leather industry which are manufactured using enzymes under high osmotic pressure. To date, we still know little about the salt-tolerant proteases secreted by Aspergillus oryze 3.042 which is the most widely used strain in soy sauce production. The aims of this work are to (i) separate and identify salt-tolerant protease secreted by Aspergillus oryze 3.042, and (ii) elucidate its preliminary mechanism of salt-tolerance.

2. Results

2.1 Salt-tolerant aspartyl aminopeptidase was prepared based on ion-exchange chromatography and gel chromatograph(Figure 1 and Figure 2).

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Figure 5. The RMSD values of salt-tolerant aspartyl aminopeptidase, alkaline protease (salt-tolerant) and neutral protease I (not salt-tolerant) under the environment of 300 K and 3 M NaCl. Aspartyl aminopeptidase had obvious lower RMSD value than that of alkaline protease and neutral protease I, suggesting that the whole carbon skeleton of aspartyl aminopeptidase was more stable than that of its controls.

 


 

Figure 6. The RMSF values of salt-tolerant aspartyl aminopeptidase, alkaline protease (salt-tolerant) and neutral protease I (not salt-tolerant) under the environment of 300 K and 3 M NaCl. Aspartyl aminopeptidase had obvious lower RMSF value than that of alkaline protease and neutral protease I, suggesting that the individual amino acid of aspartyl aminopeptidase was more stable than that of its controls.

4. Conclusion

We separated and identified a salt-tolerant protease, aspartyl aminopeptidase, from Aspergillus oryzae 3.042. According to the tandem mass spectrometry, the enzyme displayed 44% amino acid identity with the vacuolar aminopeptidase I from yeast (PDB ID: 4R8F, data not shown). The aspartyl aminopeptidase exhibited maximum activity at 50 ¡ãC and pH 7.0¨C8.0 and had a molecular weight of approximate 67 kDa (SDS-PAGE, data not shown). About 30% activity was retained after incubation in 3 M NaCl for 360 h. More stable spatial structure, lower flexibility of individual amino acid (RMSF) and whole carbon skeleton (RMSD) of aspartyl aminopeptidase in high salinity solution were responsible for its higher salt-tolerance when compared with its controls.

 

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