Abstract
Rhizomucor miehei is an important fungus that produces aspartic proteases suitable for cheese processing. In this study, a novel aspartic protease gene (RmproB) was cloned from R. miehei CAU432 and expressed in Aspergillus niger. The amino acid sequence of RmproB shared the highest identity of 58.2% with the saccharopepsin PEP4 from Saccharomyces cerevisiae. 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 Km 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 (IC50 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.
Highlights
Proteases can hydrolyze proteins through the cleavage of peptide bonds, having an important position in industrial enzymes [1]
The milk-clotting proteases from Rhizomucor miehei, R. pusillus and Mucor mucedo have widely been applied in the industries [7,8,9]
R. miehei CAU432 has been deposited in the China General Microbiological Culture Collection Center (CGMCC No 4967) with the whole genome sequenced [24]
Summary
Proteases can hydrolyze proteins through the cleavage of peptide bonds, having an important position in industrial enzymes [1]. Chymosin is a type of neonatal gastric aspartic proteases. The chymosin from calf stomach has been used to produce various cheeses [4]. It is the best milk coagulant owing to the high hydrolysis specificity against κ-casein at the position of Phe105-Met106 [5]. Many studies have focused on novel proteases as substitutes for bovine chymosin [1]. Microbial proteases are devoted as one of the promising substitutes for the bovine chymosin [6]. The milk-clotting proteases from Rhizomucor miehei, R. pusillus and Mucor mucedo have widely been applied in the industries [7,8,9]. Novel proteases from these fungi and efficient expression have potential application for cheese production
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