Cloning, Expression, and Characterization of a Milk-Clotting Aspartic Protease Gene (Po-Asp) from Pleurotus ostreatus

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.

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Cloning and Characterization of a Novel Aspartic Protease Gene from Marine-Derived Metschnikowia reukaufii and its Expression in E. coli

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-008-8400-3 ◽

2008 ◽

Vol 159 (1) ◽

pp. 119-132 ◽

Cited By ~ 15

Author(s):

Jing Li ◽

Zhenming Chi ◽

Zhiqiang Liu ◽

Lixi Yue ◽

Ying Peng ◽

...

Keyword(s):

Aspartic Protease ◽

Protease Gene ◽

E Coli

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Purification and characterization of milk-clotting enzymes from oyster mushroom (Pleurotus ostreatus (Fr.) Kumm)

Applied Biochemistry and Microbiology ◽

10.1134/s0003683809060088 ◽

2009 ◽

Vol 45 (6) ◽

pp. 623-625 ◽

Cited By ~ 9

Author(s):

G. V. Lebedeva ◽

M. T. Proskuryakov

Keyword(s):

Pleurotus Ostreatus ◽

Oyster Mushroom ◽

Purification And Characterization ◽

Milk Clotting

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Expression and characterization of aspartic protease gene in eggs and larvae stage of Ancylostoma caninum

Parasitology Research ◽

10.1007/s00436-009-1332-1 ◽

2009 ◽

Vol 104 (6) ◽

pp. 1327-1333 ◽

Cited By ~ 7

Author(s):

Yurong Yang ◽

Hua Wei ◽

Weiwen Qin ◽

Jing Zheng

Keyword(s):

Aspartic Protease ◽

Protease Gene ◽

Ancylostoma Caninum ◽

Eggs And Larvae

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Cloning and Expression of an Active Aspartic Proteinase Gene From Aspergillus Oryzae DRDFS13 in Pichia Pastoris

10.21203/rs.3.rs-101507/v1 ◽

2020 ◽

Author(s):

Jermen Mamo ◽

Konstantina Kostadinovska ◽

Martin Kangwa ◽

Marcelo Fernandez-Lahore ◽

Fassil Assefa

Keyword(s):

Pichia Pastoris ◽

Aspergillus Oryzae ◽

Recombinant Proteins ◽

Aspartic Protease ◽

Cloning And Expression ◽

Major Protein ◽

Protease Gene ◽

Recombinant Yeast ◽

Milk Clotting ◽

Cheese Production

Abstract BackgroundPichia pastoris is a yeast widely used in expressing recombinant proteins from eukaryotic organisms. In the present study, the total RNA was extracted from a eukaryotic fungus; Aspergillus oryzae DRDFS13 and reverse transcribed into cDNA using specific primers. The gene for aspartic protease was amplified and sequenced and then cloned into pGAPZαA for further expression in P. pastoris. The recombinant yeast (P. patoris X-33Ap) was cultivated in YPD media at pH 5 and 7 for 6 days and the production of recombinant proteins was checked by total protein determination, milk-clotting activity assay, and SDS-PAGE analysis. ResultsThe gene sequence results showed 98% similarity with aspartic protease gene from A. oryzae RIB40. The aspartic protease gene cloned into pGAPZαA (later pMKAP) was successfully expressed in P. pastoris as an active extracellular protease with the highest MCA (190.47 MCU/mL) of secreted enzyme from the recombinant yeast was obtained at pH 5 and 6 days of incubation time. The major protein expressed by the recombinant P. Pastoris X-33 AP has a molecular mass between 32 and 46 kDa. When analyzed for clotting activity, the protein was able to clot skim-milk in 2 min. The clotting activity was found to be 190.47 U/mL.ConclusionThus, the milk-clotting protease extracted form the recombinant yeast in the present study could be a suitable candidate for cheese production. However, further study of the recombinant proteins need to be carried out and its application in cheese production by analyzing the organoleptic and chemical properties of the cheese produced.

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