Milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits on individual caseins

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 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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Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit

International Journal of Biological Macromolecules ◽

10.1016/j.ijbiomac.2017.02.034 ◽

2017 ◽

Vol 98 ◽

pp. 847-854 ◽

Cited By ~ 18

Author(s):

Mahmoud Salehi ◽

Mahmoud Reza Aghamaali ◽

Reza H. Sajedi ◽

S. Mohsen Asghari ◽

Eisa Jorjani

Keyword(s):

Aspartic Protease ◽

Purification And Characterization ◽

Milk Clotting ◽

Withania Coagulans

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Biochemical characterization of a semi-purified aspartic protease from sea catfish Bagre panamensis with milk-clotting activity

Food Science and Biotechnology ◽

10.1007/s10068-019-00614-8 ◽

2019 ◽

Vol 28 (6) ◽

pp. 1785-1793 ◽

Cited By ~ 1

Author(s):

Idalia Osuna-Ruiz ◽

María Fernanda Espinoza-Marroquin ◽

Jesús Aarón Salazar-Leyva ◽

Emyr Peña ◽

Carlos Alfonso Álvarez-González ◽

...

Keyword(s):

Biochemical Characterization ◽

Aspartic Protease ◽

Milk Clotting ◽

Milk Clotting Activity

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Isolation and Partial Characterisation of Milk-clotting Aspartic Protease from Streblus asper

Food Science and Technology International ◽

10.1177/1082013206063839 ◽

2006 ◽

Vol 12 (2) ◽

pp. 103-109 ◽

Cited By ~ 9

Author(s):

S. Senthilkumar ◽

D. Ramasamy ◽

S. Subramanian

Keyword(s):

Ethylenediaminetetraacetic Acid ◽

Residual Activity ◽

Inhibitory Effect ◽

Aspartic Protease ◽

Enzyme Inactivation ◽

Size Exclusion ◽

Helical Conformation ◽

Milk Clotting ◽

Streblus Asper ◽

Milk Clotting Protease

A rennin-like milk-clotting protease from the twigs of Streblus asper was purified by a factor of 65 times with 36% recovery using ethanol precipitation, ion-exchange and size-exclusion chromatographic techniques. The enzyme was found to be monomeric in nature having a molecular mass of 55kDa. The enzyme acts optimally at 55°C and was stable in the temperature range of 30–40°C. Easy enzyme inactivation by moderate heating, makes this protease extract potentially useful for cheese production. The purified enzyme is an acid protease with an optimum pH of 5.5 and it retained 96% of its residual activity between pH 5.0 and 6.0. Pepstatin A inhibited the proteinase activity, whereas iodoacetamide, phenylmethyl sulphonyl fluoride, β-mercaptoethanol and ethylenediaminetetraacetic acid had no significant inhibitory effect suggesting the presence of aspartic acid residue at the active site. The milkclotting aspartic protease showed predominant α-helical conformation in phosphate buffer as evidenced from circular dichroic spectroscopy.

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The Role of Microbial Aspartic Protease Enzyme in Food and Beverage Industries

Journal of Food Quality ◽

10.1155/2018/7957269 ◽

2018 ◽

Vol 2018 ◽

pp. 1-15 ◽

Cited By ~ 10

Author(s):

Jermen Mamo ◽

Fassil Assefa

Keyword(s):

Wheat Gluten ◽

Aspartic Protease ◽

Aspartic Proteases ◽

Milk Clotting ◽

Protease Enzyme ◽

Plant Protease ◽

Milk Clotting Enzyme ◽

Wide Range ◽

Food And Beverage ◽

Endothia Parasitica

Proteases represent one of the three largest groups of industrial enzymes and account for about 60% of the total global enzymes sale. According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, proteases are classified in enzymes of class 3, the hydrolases, and the subclass 3.4, the peptide hydrolases or peptidase. Proteases are generally grouped into two main classes based on their site of action, that is, exopeptidases and endopeptidases. Protease has also been grouped into four classes based on their catalytic action: aspartic, cysteine, metallo, and serine proteases. However, lately, three new systems have been defined: the threonine-based proteasome system, the glutamate-glutamine system of eqolisin, and the serine-glutamate-aspartate system of sedolisin. Aspartic proteases (EC 3.4.23) are peptidases that display various activities and specificities. It has two aspartic acid residues (Asp32 and Asp215) within their active site which are useful for their catalytic activity. Most of the aspartic proteases display best enzyme activity at low pH (pH 3 to 4) and have isoelectric points in the pH range of 3 to 4.5. They are inhibited by pepstatin. The failure of the plant and animal proteases to meet the present global enzyme demand has directed to an increasing interest in microbial proteases. Microbial proteases are preferred over plant protease because they have most of the characteristics required for their biotechnological applications. Aspartic proteases are found in molds and yeasts but rarely in bacteria. Aspartic protease enzymes from microbial sources are mainly categorized into two groups: (i) the pepsin-like enzymes produced byAspergillus,Penicillium,Rhizopus, andNeurosporaand (ii) the rennin-like enzymes produced byEndothiaandMucorspp., such asMucor miehei,M. pusillus, andEndothia parasitica. Aspartic proteases of microbial origin have a wide range of application in food and beverage industries. These include as milk-clotting enzyme for cheese manufacturing, degradation of protein turbidity complex in fruit juices and alcoholic liquors, and modifying wheat gluten in bread by proteolysis.

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

Applied Biochemistry and Biotechnology ◽

10.1007/s12010-013-0674-4 ◽

2013 ◽

Vol 172 (4) ◽

pp. 2119-2131 ◽

Cited By ~ 15

Author(s):

Chaomin Yin ◽

Liesheng Zheng ◽

Liguo Chen ◽

Qi Tan ◽

Xiaodong Shang ◽

...

Keyword(s):

Pleurotus Ostreatus ◽

Aspartic Protease ◽

Protease Gene ◽

Milk Clotting

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Enzymatic milk clotting activity in artichoke (Cynara scolymus) leaves and alpine thistle (Carduus defloratus) flowers. Immobilization of alpine thistle aspartic protease

Food Chemistry ◽

10.1016/j.foodchem.2016.02.060 ◽

2016 ◽

Vol 204 ◽

pp. 115-121 ◽

Cited By ~ 16

Author(s):

Marilena Esposito ◽

Prospero Di Pierro ◽

Winnie Dejonghe ◽

Loredana Mariniello ◽

Raffaele Porta

Keyword(s):

Aspartic Protease ◽

Cynara Scolymus ◽

Milk Clotting ◽

Milk Clotting Activity

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Memapsin 2, a drug target for Alzheimer's disease

Biochemical Society Symposium ◽

10.1042/bss0700213 ◽

2003 ◽

Vol 70 ◽

pp. 213-220 ◽

Cited By ~ 1

Author(s):

Gerald Koelsch ◽

Robert T. Turner ◽

Lin Hong ◽

Arun K. Ghosh ◽

Jordan Tang

Keyword(s):

Crystal Structure ◽

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Transition State ◽

Amyloid Precursor Protein ◽

Therapeutic Target ◽

Drug Target ◽

Aspartic Protease ◽

Precursor Protein ◽

Memapsin 2

Mempasin 2, a ϐ-secretase, is the membrane-anchored aspartic protease that initiates the cleavage of amyloid precursor protein leading to the production of ϐ-amyloid and the onset of Alzheimer's disease. Thus memapsin 2 is a major therapeutic target for the development of inhibitor drugs for the disease. Many biochemical tools, such as the specificity and crystal structure, have been established and have led to the design of potent and relatively small transition-state inhibitors. Although developing a clinically viable mempasin 2 inhibitor remains challenging, progress to date renders hope that memapsin 2 inhibitors may ultimately be useful for therapeutic reduction of ϐ-amyloid.

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