# Actinidain
**Actinidain** (also written actinidin; EC 3.4.22.14) is a cysteine protease, an enzyme that cleaves protein chains, found in high amounts in kiwifruit of the genus *Actinidia*. It accounts for about 50–60% of the soluble proteins in kiwifruit and belongs to the papain subfamily of cysteine proteases, alongside papain from papaya, bromelain from pineapple, and ficin from fig.[1](https://www.sciencedirect.com/science/article/pii/S0023643816307897) Its practical uses follow from this abundance: the enzyme tenderizes meat, clots milk for cheese making, hydrolyzes milk proteins, and can produce bioactive peptides.[1](https://www.sciencedirect.com/science/article/pii/S0023643816307897)
| Key fact | Detail |
|---|---|
| Classification | Cysteine protease, EC 3.4.22.14, member of the papain subfamily (family C1)[1](https://www.sciencedirect.com/science/article/pii/S0023643816307897) |
| Source | Kiwifruit (*Actinidia* spp.), where it makes up about 50–60% of soluble protein[1](https://www.sciencedirect.com/science/article/pii/S0023643816307897) |
| Molecular properties | Protein of 24 kDa (24,221 by sequence analysis); pI values of 9.4 and 9.5 for two isolated forms[2](https://www.sciencedirect.com/science/article/pii/B9780123942944000043) |
| Optimum conditions | Optimal pH 3.5 and optimal temperature 40 °C for the purified enzyme from the Chinese cultivar Xuxiang[1](https://www.sciencedirect.com/science/article/pii/S0023643816307897) |
| Meat tenderization | Shear force reduced by more than half in pork and rabbit muscle at 0.5 mg enzyme per 100 g muscle[1](https://www.sciencedirect.com/science/article/pii/S0023643816307897) |
| Peptide production | ACE-inhibitory peptide yields of 7.2% to 14.2% from five plant proteins, with ACE inhibitory rates of 71.1% to 88.3%[1](https://www.sciencedirect.com/science/article/pii/S0023643816307897) |
| Allergen designation | Act d 1, the name used for the enzyme's immunological properties[2](https://www.sciencedirect.com/science/article/pii/B9780123942944000043) |
## Name and history
The name actinidin derives from *Actinidia*, the genus of the source plant, and was first proposed by Arcus in 1959.[2](https://www.sciencedirect.com/science/article/pii/B9780123942944000043) Arcus, working with *Actinidia chinensis*, then known as Chinese gooseberry, was prompted to search for an enzyme by the observation that incorporating raw fruit into table jelly prevents it from setting.[3](https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/actinidia-deliciosa) The fruit itself was new to the West: seeds of *A. chinensis*, native to China, were brought to New Zealand in 1904 and developed into the kiwifruit cultivars known today.[3](https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/actinidia-deliciosa)
The enzyme has carried several names. It has been referred to as [Actinidia](https://www.edgepedia.org/actinidia) anionic protease, and the spelling actinidain was adopted to keep the suffix '-ain' used for cysteine peptidases, although some authors continue to use actinidin.[3](https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/actinidia-deliciosa) McDowall published the first work on the enzyme purified to crystallinity, and work at Massey University over the following decade produced detailed studies of the primary sequence, the X-ray crystal structure, and the specificity and kinetics of its esterolytic activity.[3](https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/actinidia-deliciosa) In immunological contexts the protein is designated Act d 1.[2](https://www.sciencedirect.com/science/article/pii/B9780123942944000043)
## Molecular and catalytic properties
Actinidain is a protein of 24 kDa, with a calculated mass of 24,221 from sequence analysis, and two isolated forms have pI values of 9.4 and 9.5.[2](https://www.sciencedirect.com/science/article/pii/B9780123942944000043) Both acidic and basic forms of the enzyme occur in kiwifruit, and expression is highest in fruit at harvest, decreasing as the fruit ripens.[4](https://www.researchgate.net/profile/Roswitha-Schroeder-2/publication/248888147_Identification_and_characterisation_of_acidic_and_novel_basic_forms_of_actinidin_the_highly_abundant_cysteine_protease_from_kiwifruit/links/56c53a4608ae736e7047170a/Identification-and-characterisation-of-acidic-and-novel-basic-forms-of-actinidin-the-highly-abundant-cysteine-protease-from-kiwifruit.pdf)
Like papain, the enzyme uses a catalytically active cysteine residue, positioned as Cys-25, in forming the enzyme-substrate complex.[5](https://ir.canterbury.ac.nz/bitstream/handle/10092/9970/thesis_fulltext.pdf?sequence=1) Specificity studies indicate a strong preference for a basic side chain in the S1 site of the enzyme and a preference for an aromatic group in the S2 site.[6](https://mro.massey.ac.nz/items/1f4feab6-9686-411c-9431-df5196cfb579) Kinetic comparisons with papain show that substrates with aromatic N-substituents have lower kcat/Km values for actinidin, while the difference is much smaller for substrates with aliphatic substituents; the lower values correspond mainly to higher Km, indicating that substrate binding differs between the two enzymes, a difference explained by their distinct substrate binding sites seen in X-ray crystallographic studies.[7](https://pubmed.ncbi.nlm.nih.gov/7002215/) In one direct comparison of stereochemical selectivity toward enantiomeric N-acetylphenylalanylglycine 4-nitroanilides, the ratio (kcat/Km)D was 375 for papain, 93 for ficin, and 39 for actinidin.[8](https://pmc.ncbi.nlm.nih.gov/articles/PMC1130721/?page=1) Structural work has also identified features unique to actinidin, including a highly anionic surface and an Asp142-Lys145 salt bridge that forms a cavity constraining diffusion of methanol away from the catalytic site.[9](https://pmc.ncbi.nlm.nih.gov/articles/PMC1221960/)
## Meat tenderization
Actinidin hydrolyzes both collagen and myofibrillar proteins, the two protein systems that determine meat texture, and it is applied as a meat tenderizer on this basis.[10](https://pmc.ncbi.nlm.nih.gov/articles/PMC12441743/) In a comparison of cysteine proteases, actinidin and bromelain were found to be particularly effective in collagen decomposition, and are expected to have a high connective tissue breakdown effect when applied to meat.[11](https://pmc.ncbi.nlm.nih.gov/articles/PMC10119465/)
A 1988 study by Deborah A. Lewis and B. S. Luh at the [University of California, Davis](https://www.edgepedia.org/university-of-california-davis) tested actinidin on broiled bovine semitendinosus steaks: an actinidin activity of 400 U/mL produced Kramer shear values and sensory tenderness scores equivalent to those from Adolph's papain-based meat tenderizer at 18 U/mL, and both treatments gave significantly more tender steaks than no treatment.[12](https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1745-4514.1988.tb00368.x) Notably, actinidin did not over-tenderize the steak surface as the papain-based tenderizer did, and hydrolysis of myofibrillar proteins before broiling was considerably less for actinidin than for papain at activities giving equal tenderness.[12](https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1745-4514.1988.tb00368.x) This matters because papain's broad specificity can indiscriminately decompose connective tissue and myofibrillar proteins, producing undesirable "mushy" meat that limits commercial use.[1](https://www.sciencedirect.com/science/article/pii/S0023643816307897) In later work with purified actinidin from the Chinese cultivar Xuxiang, shear force was reduced by more than half in pork and rabbit muscle at a dosage of 0.5 mg per 100 g of muscle.[1](https://www.sciencedirect.com/science/article/pii/S0023643816307897)
## Dairy applications and comparison with papain and bromelain
Actinidin shows milk-clotting ability, a property more common in acidic proteases such as chymosin, and has been used as a plant-derived milk coagulant in cheese manufacture.[1](https://www.sciencedirect.com/science/article/pii/S0023643816307897) It has also been employed to reduce the antigenicity of milk proteins by modifying protein conformation and by cleaving and masking epitopes of β-lactoglobulin and αs1-casein.[10](https://pmc.ncbi.nlm.nih.gov/articles/PMC12441743/)
Kinetic studies of milk protein hydrolysis place actinidin between its relatives. For whey protein concentrate and milk protein concentrate suspensions, the Michaelis constant Km for actinidin ranged from 2.13 × 104 to 3.18 × 104, equivalent to 21.3 to 31.8 mg mL−1, close to the papain range of 18.7 to 30.5 mg mL−1.[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC10706065/) In degree-of-hydrolysis measurements, papain showed proteolytic activity similar to actinidin, while bromelain behaved very differently: whey protein hydrolysates made with bromelain reached only around 9% degree of hydrolysis at 60 °C, whereas papain reached around 18% after 5 hours of incubation.[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC10706065/) Part of this difference is mechanistic: cleavage of disulphide bonds is not supported by bromelain, and bromelain leaves a higher remaining β-lactoglobulin fraction than papain or actinidin.[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC10706065/) Of the caseins, the αs-caseins are more susceptible to hydrolysis by actinidin and papain than β- or κ-casein.[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC10706065/)
## Allergenicity
The enzyme is a recognized kiwifruit allergen under the designation Act d 1, the name used when describing its immunological properties.[2](https://www.sciencedirect.com/science/article/pii/B9780123942944000043) This dual character, a food-processing enzyme that is itself a major allergen of its source fruit, is a practical consideration in products containing raw or minimally processed kiwifruit.
## References
1. [Zhang, B. et al. (2017). Characterization of actinidin from Chinese kiwifruit cultivars and its applications in meat tenderization and production of ACE inhibitory peptides. LWT 78.](https://www.sciencedirect.com/science/article/pii/S0023643816307897)
2. [Kiwifruit Proteins and Enzymes: Actinidin and Other Significant Proteins, ScienceDirect](https://www.sciencedirect.com/science/article/pii/B9780123942944000043)
3. [Actinidain, Handbook of Proteolytic Enzymes (Third Edition), ScienceDirect Topics](https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/actinidia-deliciosa)
4. [Schröder, R. et al. Identification and characterisation of acidic and novel basic forms of actinidin, the highly abundant cysteine protease from kiwifruit. Functional Plant Biology.](https://www.researchgate.net/profile/Roswitha-Schroeder-2/publication/248888147_Identification_and_characterisation_of_acidic_and_novel_basic_forms_of_actinidin_the_highly_abundant_cysteine_protease_from_kiwifruit/links/56c53a4608ae736e7047170a/Identification-and-characterisation-of-acidic-and-novel-basic-forms-of-actinidin-the-highly-abundant-cysteine-protease-from-kiwifruit.pdf)
5. [Characterisation of bioactive proteins, University of Canterbury thesis](https://ir.canterbury.ac.nz/bitstream/handle/10092/9970/thesis_fulltext.pdf?sequence=1)
6. [Kinetics and mechanism of proteolytic enzyme catalysed reactions, Massey University PhD thesis](https://mro.massey.ac.nz/items/1f4feab6-9686-411c-9431-df5196cfb579)
7. [Baker, E. N. et al. (1980). The specificity of actinidin and its relationship to the structure of the enzyme. Biochimica et Biophysica Acta 616: 30–34.](https://pubmed.ncbi.nlm.nih.gov/7002215/)
8. [Variation in the P2-S2 stereochemical selectivity towards the enantiomeric N-acetylphenylalanylglycine 4-nitroanilides among the cysteine proteinases papain, ficin and actinidin, Biochemical Journal](https://pmc.ncbi.nlm.nih.gov/articles/PMC1130721/?page=1)
9. [Variation in aspects of cysteine proteinase catalytic mechanism deduced by spectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations, Biochemical Journal](https://pmc.ncbi.nlm.nih.gov/articles/PMC1221960/)
10. [Enzyme Technology in the Food Industry: Molecular Mechanisms, Applications, and Sustainable Innovations, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC12441743/)
11. [Methods for improving meat protein digestibility in older adults, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC10119465/)
12. [Lewis, D. A. and Luh, B. S. (1988). Application of actinidin from kiwifruit to meat tenderization and characterization of beef muscle protein hydrolysis. Journal of Food Biochemistry 12: 147–158.](https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1745-4514.1988.tb00368.x)
13. [Milk Protein Hydrolysis by Actinidin—Kinetic and Thermodynamic Characterisation and Comparison to Bromelain and Papain, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC10706065/)
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*Topic: Encyclopedia › Life and health › Biological foundations › Biochemistry and metabolism › Enzyme classes and activities › Proteolytic and peptidase enzymes › Proteases by catalytic mechanism › Cysteine proteases › Papain family (C1) › Plant papain-like proteases*
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