Characterization of a Novel Aspartic Protease from <i>Rhizomucor miehei</i> Expressed in <i>Aspergillus niger</i> and Its Application in Production of ACE-Inhibitory Peptides
<i>Rhizomucor miehei</i> is an important fungus that produces aspartic proteases suitable for cheese processing. In this study, a novel aspartic protease gene (<i>RmproB</i>) was cloned from <i>R. miehei</i> CAU432 and expressed in <i>Aspergillus niger</i...
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MDPI AG
2021-11-01
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| Series: | Foods |
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| Online Access: | https://www.mdpi.com/2304-8158/10/12/2949 |
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| author | Shounan Wang Peng Zhang Yibin Xue Qiaojuan Yan Xue Li Zhengqiang Jiang |
| author_facet | Shounan Wang Peng Zhang Yibin Xue Qiaojuan Yan Xue Li Zhengqiang Jiang |
| author_sort | Shounan Wang |
| collection | DOAJ |
| description | <i>Rhizomucor miehei</i> is an important fungus that produces aspartic proteases suitable for cheese processing. In this study, a novel aspartic protease gene (<i>RmproB</i>) was cloned from <i>R. miehei</i> CAU432 and expressed in <i>Aspergillus niger</i>. The amino acid sequence of RmproB shared the highest identity of 58.2% with the saccharopepsin PEP4 from <i>Saccharomyces cerevisiae</i>. High protease activity of 1242.2 U/mL was obtained through high density fermentation in 5 L fermentor. RmproB showed the optimal activity at pH 2.5 and 40 °C, respectively. It was stable within pH 1.5–6.5 and up to 45 °C. RmproB exhibited broad substrate specificity and had <i>K</i><sub>m</sub> values of 3.16, 5.88, 5.43, and 1.56 mg/mL for casein, hemoglobin, myoglobin, and bovine serum albumin, respectively. RmproB also showed remarkable milk-clotting activity of 3894.1 SU/mg and identified the cleavage of Lys21-Ile22, Leu32-Ser33, Lys63-Pro64, Leu79-Ser80, Phe105-Met106, and Asp148-Ser149 bonds in κ-casein. Moreover, duck hemoglobin was hydrolyzed by RmproB to prepare angiotensin-I-converting enzyme (ACE) inhibitory peptides with high ACE-inhibitory activity (IC<sub>50</sub> of 0.195 mg/mL). The duck hemoglobin peptides were further produced at kilo-scale with a yield of 62.5%. High-level expression and favorable biochemical characterization of RmproB make it a promising candidate for cheese processing and production of ACE-inhibitory peptides. |
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| series | Foods |
| spelling | doaj.art-8cd66796775d49ddb8f88f9ec60969a82023-11-23T08:16:40ZengMDPI AGFoods2304-81582021-11-011012294910.3390/foods10122949Characterization of a Novel Aspartic Protease from <i>Rhizomucor miehei</i> Expressed in <i>Aspergillus niger</i> and Its Application in Production of ACE-Inhibitory PeptidesShounan Wang0Peng Zhang1Yibin Xue2Qiaojuan Yan3Xue Li4Zhengqiang Jiang5Department of Nutrition and Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, ChinaKey Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, Beijing 100083, ChinaDepartment of Nutrition and Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, ChinaKey Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, Beijing 100083, ChinaKey Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, Beijing 100083, ChinaDepartment of Nutrition and Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China<i>Rhizomucor miehei</i> is an important fungus that produces aspartic proteases suitable for cheese processing. In this study, a novel aspartic protease gene (<i>RmproB</i>) was cloned from <i>R. miehei</i> CAU432 and expressed in <i>Aspergillus niger</i>. The amino acid sequence of RmproB shared the highest identity of 58.2% with the saccharopepsin PEP4 from <i>Saccharomyces cerevisiae</i>. High protease activity of 1242.2 U/mL was obtained through high density fermentation in 5 L fermentor. RmproB showed the optimal activity at pH 2.5 and 40 °C, respectively. It was stable within pH 1.5–6.5 and up to 45 °C. RmproB exhibited broad substrate specificity and had <i>K</i><sub>m</sub> values of 3.16, 5.88, 5.43, and 1.56 mg/mL for casein, hemoglobin, myoglobin, and bovine serum albumin, respectively. RmproB also showed remarkable milk-clotting activity of 3894.1 SU/mg and identified the cleavage of Lys21-Ile22, Leu32-Ser33, Lys63-Pro64, Leu79-Ser80, Phe105-Met106, and Asp148-Ser149 bonds in κ-casein. Moreover, duck hemoglobin was hydrolyzed by RmproB to prepare angiotensin-I-converting enzyme (ACE) inhibitory peptides with high ACE-inhibitory activity (IC<sub>50</sub> of 0.195 mg/mL). The duck hemoglobin peptides were further produced at kilo-scale with a yield of 62.5%. High-level expression and favorable biochemical characterization of RmproB make it a promising candidate for cheese processing and production of ACE-inhibitory peptides.https://www.mdpi.com/2304-8158/10/12/2949aspartic proteasemilk-clotting<i>Rhizomucor miehei</i><i>Aspergillus niger</i>ACE-inhibitory peptides |
| spellingShingle | Shounan Wang Peng Zhang Yibin Xue Qiaojuan Yan Xue Li Zhengqiang Jiang Characterization of a Novel Aspartic Protease from <i>Rhizomucor miehei</i> Expressed in <i>Aspergillus niger</i> and Its Application in Production of ACE-Inhibitory Peptides Foods aspartic protease milk-clotting <i>Rhizomucor miehei</i> <i>Aspergillus niger</i> ACE-inhibitory peptides |
| title | Characterization of a Novel Aspartic Protease from <i>Rhizomucor miehei</i> Expressed in <i>Aspergillus niger</i> and Its Application in Production of ACE-Inhibitory Peptides |
| title_full | Characterization of a Novel Aspartic Protease from <i>Rhizomucor miehei</i> Expressed in <i>Aspergillus niger</i> and Its Application in Production of ACE-Inhibitory Peptides |
| title_fullStr | Characterization of a Novel Aspartic Protease from <i>Rhizomucor miehei</i> Expressed in <i>Aspergillus niger</i> and Its Application in Production of ACE-Inhibitory Peptides |
| title_full_unstemmed | Characterization of a Novel Aspartic Protease from <i>Rhizomucor miehei</i> Expressed in <i>Aspergillus niger</i> and Its Application in Production of ACE-Inhibitory Peptides |
| title_short | Characterization of a Novel Aspartic Protease from <i>Rhizomucor miehei</i> Expressed in <i>Aspergillus niger</i> and Its Application in Production of ACE-Inhibitory Peptides |
| title_sort | characterization of a novel aspartic protease from i rhizomucor miehei i expressed in i aspergillus niger i and its application in production of ace inhibitory peptides |
| topic | aspartic protease milk-clotting <i>Rhizomucor miehei</i> <i>Aspergillus niger</i> ACE-inhibitory peptides |
| url | https://www.mdpi.com/2304-8158/10/12/2949 |
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