Characterization of a Novel Aspartic Protease from Rhizomucor miehei Expressed in Aspergillus niger and Its Application in Production of ACE-Inhibitory Peptides

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 K_m 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.

Keywords: ACE-inhibitory peptides; Aspergillus niger; Rhizomucor miehei; aspartic protease; milk-clotting.

Figure 1

Multiple amino acid sequence alignment of RmproB with other A1 family aspartic proteases. RmproB was the aspartic protease from R. miehei in this study. The sequences, O42630.1 (Aspergillus fumigatus), Q01294.2 (Neurospora crassa), P07267.1 (Saccharomyces cerevisiae), Q03168.2 (Aedes aegypti), and Q05744.1 (Gallus gallus) were downloaded from the NCBI protein database. Identical residues are shaded in red, and conserved residues are shown in blue boxes. The conversed catalytic residues are marked with asterisks.

Figure 2

Time-course of RmproB produced by A. niger in 5 L fermentor (A) and SDS-PAGE analysis of the purified RmproB (B). Symbols are enzyme activity (●), protein concentration (■), and wet cell weight (▲) of high-density fermentation (A). Lane M, protein molecular weight marker; lane 1, A. niger FBL-A for control; lane 2, the crude supernatant; lane 3, RmproB after QSFF; lane 4, RmproB after S-100 (B).

Figure 3

Optimal pH (A, black polyline) and pH stability (A, blue polyline) of RmproB, optimal temperature (B, black polyline) and thermostability (B, blue polyline) of RmproB. The optimal pH of RmproB was determined in 50 mM various buffers at 40 °C. The buffers used are KCl-HCl (■, pH 1.0–2.0); lactate (●, pH 2.0–4.0); citrate (▲, pH 3.5–6.5); phosphate (◆, pH 6.5–8.0). The pH stability was assessed by measuring the residual protease activity after the enzyme was pre-incubated at 40 °C for 30 min in buffers mentioned above. The optimal temperature was tested in 50 mM lactate buffer (pH 2.5) at different temperatures (20–60 °C). The thermostability was evaluated by investigating the residual protease activity after pre-incubation in 50 mM lactate buffer (pH 2.5) at 25–60 °C for 30 min.

Figure 4

SDS-PAGE analysis of α_s-casein (A), β-casein (B), and κ-casein (C) hydrolyzed by RmproB. Lane M, protein molecular weight marker; other lanes, three types of caseins hydrolyzed by RmproB (10 SU/mL) in 50 mM pH 4.0 citrate buffer for 0, 5, 10, and 30 min.

Figure 5

Flow diagram of production of duck hemoglobin peptides at kilo-scale. The solid content was defined as the ratio of the weight of the lyophilized solid to the total solution volume. The yield was defined as the percentage of the weight of the obtained duck hemoglobin peptides to the initial substrate.