Three phase partitioning, a scalable method for the purification and recovery of cucumisin, a milk-clotting enzyme, from the juice of Cucumis melo var. reticulatus.

Cucumisin [EC 3.4.21.25] was first purified from Cucumis melo var. reticulatus juice by three-phase partitioning (TPP). Optimum purification parameters of the TPP system were determined as 60% ammonium sulfate saturation with 1.0:1.25 ratio of crude extract: t-butanol at pH and temperature of 8.0 and 20°C, respectively. Cucumisin was purified with 4.61 purification fold and 156% activity recovery. The molecular weight of the recovered cucumisin was determined as 68.4kDa and its isoelectric point is 8.7. Optimum pH and temperature of cucumisin were pH 9.0 and 60-70°C, respectively. The protease was very stable at 20-70°C and a pH range of 2.0-12.0. Km and Vmax constants were 2.24±0.22mgmL-1 and 1048±25μ Mmin-1, respectively. The enzyme was stable against numerous metal ions and its activity was highly enhanced by Ca2+, Mg2+, and Mn+2. Cucumisin activity was 2.35-folds increased in the presence of 5mM of CaCl2. It was inactivated by Co2+, Cd2+, Zn2+ and Fe2+ and dramatically by PMSF. Cucumisin milk-clotting activity was highly stable when stored under freezing (-20°C) compared at 4°C and 25°C. Finally, TPP revealed to be a useful strategy to concentrate and purify cucumisin for its use as a milk-clotting enzyme for cheese-making.

• Publication year

  2017

• Venue

  International Journal of Biological Macromolecules

• Publication date

  2017-09-01

• Fields of study

  Agricultural and Food Sciences, Medicine, Chemistry

• Identifiers
  DOI 10.1016/j.ijbiomac.2017.04.060PMID 28428129
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Data in support of three phase partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes

  2016cited by this paper
• Three Phase Partitioning System, an Emerging Non-ChromatographicTool for Proteolytic Enzymes Recovery and Purification

  2016cited by this paper
• Purification, physico-chemico-kinetic characterization and thermal inactivation thermodynamics of milk clotting enzyme from Bacillus subtilis MTCC 10422

  2016cited by this paper
• The addition of calcium chloride in combination with a lower draining pH to change the microstructure and improve fat retention in Cheddar cheese

  2015cited by this paper
• Three phase partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes.

  2015cited by this paper
• Characterisation of a milk-clotting extract from Balanites aegyptiaca fruit pulp

  2014cited by this paper
• Plant proteases as milk-clotting enzymes in cheesemaking: a review

  2014cited by this paper
• Use of the combined phase partitioning systems for recovery of proteases from hepatopancreas of Pacific white shrimp

  2014cited by this paper
• Three-phase partitioning and proteins hydrolysis patterns of alkaline proteases derived from fish viscera

  2014cited by this paper
• Three-phase partitioning as an efficient method for the purification and recovery of ficin from Mediterranean fig (Ficus carica L.) latex

  2014cited by this paper
• The effect of calcium chloride addition on the microstructure and composition of Cheddar cheese

  2013cited by this paper
• Purification, recovery, and characterization of chick pea (Cicer arietinum) β-galactosidase in single step by three phase partitioning as a rapid and easy technique.

  2013cited by this paper
• Three phase partitioning leads to subtle structural changes in proteins.

  2013cited by this paper
• Chapter 718 – Cucumisin

  2013cited by this paper
• Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression and biochemical characterization

  2013cited by this paper
• Chapter 717 – Plant Subtilisins

  2013cited by this paper
• Caseinolytic and milk-clotting activities from Moringa oleifera flowers.

  2012cited by this paper
• Religiosin B, a milk-clotting serine protease from Ficus religiosa

  2012cited by this paper
• Purification and biochemical characterisation of a novel protease streblin

  2011cited by this paper
• Recent advances in milk clotting enzymes

  2011cited by this paper
• Purification and characterization of manganese-dependent alkaline serine protease from Bacillus pumilus TMS55.

  2011cited by this paper
• Extraction and three-phase partitioning behavior of proteases from papaya peels.

  2010cited by this paper
• Three-phase partitioning of protease from Calotropis procera latex

  2010cited by this paper
• Factors Influencing Enzyme Activity

  2010cited by this paper
• Production and characterization of a milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor indicae-seudaticae N31: (Milk-clotting protease from the newly isolated Thermomucor indicae-seudaticae N31)

  2010cited by this paper
• Milk clotting and proteolytic activity of an enzyme preparation from Bromelia hieronymi fruits

  2010cited by this paper
• Benghalensin, a highly stable serine protease from the latex of medicinal plant Ficus benghalensis.

  2009cited by this paper
• Characterisation of partially purified milk-clotting enzyme from Solanum dubium Fresen seeds

  2009cited by this paper
• Characterization of the milk-clotting properties of extracts from artichoke (Cynara scolymus, L.) flowers

  2007cited by this paper
• A novel serine protease cryptolepain from Cryptolepis buchanani: purification and biochemical characterization.

  2006cited by this paper
• Purification and characterization of a serine protease from Cucumis trigonus Roxburghi.

  2006cited by this paper
• Plant serine proteases: biochemical, physiological and molecular features.

  2005cited by this paper
• Evolutionary families of peptidase inhibitors.

  2004cited by this paper
• Comparative studies on the gelling properties of cardosins extracted from Cynara cardunculus and chymosin on cow's skim milk

  2003cited by this paper
• Stabilization mechanism of MPEG modified trypsin based on thermal inactivation kinetic analysis and molecular modeling computation

  2001cited by this paper
• Cucumisin-like protease from the latex of Euphorbia supina.

  2000cited by this paper
• Cleavage specificity of cucumisin, a serine protease, with synthetic substrates.

  2000cited by this paper
• Substrate Specificity of Cucumisin on Synthetic Peptides

  2000cited by this paper
• Predicting cutting and clotting time of coagulating goat's milk using diffuse reflectance: effect of pH, temperature and enzyme concentration.

  2000cited by this paper
• Cucumisin like protease from the sarcocarp of Benincasa hispida var. Ryukyu.

  1998cited by this paper
• Influence of calcium chloride addition to milk on the cheese yield

  1997cited by this paper
• Purification and Some Properties of a Protease from the Sarcocarp of Musk Melon Fruit.

  1997cited by this paper
• Three phase partitioning: concentration and purification of proteins.

  1997cited by this paper
• Milk-clotting activity of cucumisin, a plant serine protease from melon fruit

  1996cited by this paper
• Cleavage specificity of cucumisin, a plant serine protease.

  1995cited by this paper
• Improved isolation, stability and substrate specificity of cucumisin, a plant serine endopeptidase

  1995cited by this paper
• Purification and Characterization of an Endoprotease from Melon Fruit

  1994cited by this paper
• Cucumisin, a serine protease from melon fruits, shares structural homology with subtilisin and is generated from a large precursor.

  1994cited by this paper
• Substrate-gel electrophoresis for composition and molecular weight of proteinases or proteinaceous proteinase inhibitors.

  1993cited by this paper
• Protease D from the sarcocarp of honeydew melon fruit.

  1993cited by this paper
• Extraction and study of certain physico-chemical properties of a new proteolytic enzyme from the latex of jatropha curcas linn.

  1988cited by this paper
• Evaluation of half‐life of immobilized enzyme during continuous reaction in bioreactors: A theoretical study

  1987cited by this paper
• Amino acid sequence around the reactive serine of cucumisin from melon fruit.

  1984cited by this paper
• A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.

  1976cited by this paper
• Isolation and characterization of a proteinase from the sarcocarp of melon fruit.

  1975cited by this paper
• Factors Influencing Enzyme Activity

  1974cited by this paper
• [30] Milk-clotting enzyme from Mucor pusillus var. Lindt

  1970cited by this paper
• Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4

  1970cited by this paper

• Characteristics of Fermented Milk Product Produced by Coagulating Bovine, Ovine and Caprine Milks with Chickpea (Cicer arietinum L.)

  2025cites this paper
• Thermostable and Solvent-Tolerant Alkaline Protease from Galium aparine: Purification and Industrial Applications.

  2025cites this paper
• Biovaluation of Cucurbitaceae fruit byproducts as potential ruminant meat tenderizers: A scoping review and process conceptual framework

  2025cites this paper
• Single step three phase partitioning for the purification of bromelain enzyme from pineapple (Ananas comosus) fruit

  2025cites this paper
• Protease from banana stem postharvest (Musa x paradisiaca): Extraction, purification, and its potential milk-clotting activity.

  2025cites this paper
• Three Phase Partitioning of Serine Protease from Ash Gourd (Benincasa hispida), Its Characterization and Application on Whey Protein Hydrolysis

  2024influential citation
• Calotropis procera Extract for Wagashi Cheese Making in West Africa: A Review of Applications and Perspectives for Other Plant Coagulants Utilization

  2024cites this paper
• Enhanced production, artificial intelligence optimized three-phase partitioning extraction, and in silico characterization of extracellular neutral Bacillus cereus proteinase

  2024cites this paper
• Unveiling the Potential of Agrifood By-products: a Comprehensive Review of Phytochemicals, Bioactivities and Industrial Applications

  2024cites this paper
• Purification of <i>Moringa oleifera</i> Leaves Protease by Three-Phase Partitioning and Investigation of Its Potential Antibacterial Activity

  2023cites this paper
• Effect of purified Calotropis gigantea latex protease on the cheese quality

  2023cites this paper
• Optimization of Three Phase Partitioning Purification of Papain from Carica papaya Latex by Response Surface Methodology

  2023cites this paper
• Optimization of three-phase partitioning system for the purification of protease from Calotropis gigantea latex using response surface methodology

  2023cites this paper
• Efficient three phase partitioning of actinidin from kiwifruit (Actinidia deliciosa) and its characterization

  2023influential citation
• Simultaneous extraction of chlorophylls, proteins, and carbohydrates from isolated Chlorella thermophila using a triphasic separation technique: A biorefinery approach

  2023cites this paper
• Production of Plant Proteases and New Biotechnological Applications: An Updated Review

  2022cites this paper
• Three-phase partitioning for the separation of proteins, enzymes, biopolymers, oils and pigments: a review

  2022cites this paper
• Milk-Gelling Properties of Proteases Extracted from the Fruits of Solanum Elaeagnifolium Cavanilles

  2022cites this paper
• Three-phase partitioning, a recyclable method for the purification of R-phycoerythrin from a red algae Porphyra yezoensis

  2022cites this paper
• Edible Vitalmelon Fruit Extract Inhibits Adipogenesis and Ameliorates High-Fat Diet-Induced Obesity

  2022cites this paper
• Peptidases used in dairy technology: Current knowledge and relevant applications

  2022influential citation
• Enzymes recovery by three phase partitioning

  2021cites this paper
• Biological protein precipitation: A green process for the extraction of cucumisin from melon (Cucumis melo L. inodorus) by-products

  2021cites this paper
• Valorisation of food agro-industrial by-products: From the past to the present and perspectives.

  2021cites this paper
• Advances in research on calf rennet substitutes and their effects on cheese quality.

  2021cites this paper
• The multiple facets of three-phase partitioning in the purification, concentration, yield and activity of enzymes and proteins

  2021cites this paper
• Three-phase partitioning (TPP) of proteases from parasites, plants, tissue and bacteria for enhanced activity

  2021cites this paper
• Vegan Alternatives to Processed Cheese and Yogurt Launched in the European Market during 2020: A Nutritional Challenge?

  2021cites this paper
• Development of low-cost paper-based biosensor of polyphenol oxidase for detection of phenolic contaminants in water and clinical samples

  2020influential citation
• Melon By-Products: Biopotential in Human Health and Food Processing

  2020cites this paper
• Management of Fruit Industrial By-Products—A Case Study on Circular Economy Approach

  2020cites this paper
• Three phase partitioning for simultaneous extraction of oil, protein and polysaccharide from rice bran

  2020cites this paper
• Valorization of melon fruit (Cucumis melo L.) by-products: Phytochemical and Biofunctional properties with Emphasis on Recent Trends and Advances

  2020cites this paper
• Fermentation conditions of serine/alkaline milk-clotting enzyme production by newly isolated Bacillus licheniformis BL312

  2019cites this paper
• Technology of obtaining milk-clotting enzyme from fungal culture Funalia Sp. for application in cheese production

  2019cites this paper
• Therapeutic proteases from plants: biopharmaceuticals with multiple applications

  2019cites this paper
• Recent Developments and Applications of Three-Phase Partitioning for the Recovery of Proteins

  2019cites this paper
• Enhanced microalgal protein extraction and purification using sustainable microwave-assisted multiphase partitioning technique

  2019cites this paper
• Applying native proteases from melon to hydrolyze kilka fish proteins (Clupeonella cultriventris caspia) compared to commercial enzyme Alcalase.

  2019cites this paper
• TPP Combined with DGUC as an Economic and Universal Process for Large-Scale Purification of AAV Vectors

  2019cites this paper
• Microalgal Protein Extraction From Chlorella vulgaris FSP-E Using Triphasic Partitioning Technique With Sonication

  2019cites this paper
• One-step recovery of latex papain from Carica papaya using Three Phase Partitioning and its use as milk-clotting and meat-tenderizing agent.

  2019cites this paper
• Three phase partitioning to concentrate milk clotting proteases from Wrightia tinctoria R. Br and its characterization.

  2018cites this paper
• Partial Purification and Characterization of Polyphenol Oxidase from the Wild Edible Mushroom Lepiota Procera Using Three-Phase Partitioning

  2018cites this paper
• Milk-Clotting Plant Proteases for Cheesemaking

  2018cites this paper
• Aqueous Methods for Extraction/Recovery of Macromolecules From Microorganisms of Atypical Environments: A Focus on Three Phase Partitioning

  2018cites this paper
• Biochemical properties of a new thermo- and solvent-stable xylanase recovered using three phase partitioning from the extract of Bacillus oceanisediminis strain SJ3

  2017cites this paper