# Actinidain

Actinidain (EC 3.4.22.14, actinidin, Actinidia anionic protease, proteinase A2 of Actinidia chinensis) is a type of cysteine protease enzyme found in fruits including kiwifruit (genus Actinidia), pineapple, mango, banana, figs, and papaya. This enzyme is part of the peptidase C1 family of papain-like proteases.
As a known allergen in kiwifruit, the enzyme is under preliminary research for its effect on tight junction proteins of intestinal epithelial cells.
Actinidain is commercially useful as a meat tenderiser and in coagulating milk for dairy products, like yogurt and cheese. The denaturation temperature of actinidain is 60 °C (140 °F), lower than that of similar meat tenderising enzymes bromelain from pineapple and papain from papaya.

## History

Actinidain was first identified in 1959 when A.C. Arcus looked into why jellies made with kiwifruit don’t solidify. They went on to show that this phenomenon was caused by a proteolytic enzyme attacking gelatin. This enzyme would go on to be named actinidin as it was identified in a fruit in the genus Actinidia. While similar proteins have been found in other fruits, this cysteine protease is unique to the kiwifruit. A thiol group was identified to be essential for enzyme activity, which is why it was grouped with enzymes like papain and bromelain.

## Function

While no clear function has been identified, the enzyme begins to accumulate in the fruit early on and is suspected to be important for fruit development. Actinidain has been found to have a detrimental effect on the larvae of Spodoptera litura, however not enough research has been done into whether the enzyme can be used as a pesticide. It may also be used as a storage protein.

## Sequence and structure

Actinidain has an enzyme classification number (EC) of 3.4.22.14. The 3 classifies it as a hydrolase. It is further classified as acting on peptide bonds, also known as a peptidase (3.4). The .22 represents the cysteine endopeptidases and then the .14 is actinidain’s unique identifier within that group. Actinidain is first produced in the kiwi when it is about half its size and then increases in both protease activity and enzyme production until the fruit is fully matured. The enzyme is encoded by a large gene family and is expressed in most tissues of the kiwifruit plant, not just the fruit itself.
Actinidain is similar to papain in size, shape, active site location and conformation, as well as in kinetic studies, which is especially interesting as they only share 48% amino acid similarity. Electron density mapping shows similar α-helices and overall polypeptide folding. While the electron density map indicates 218 amino acids, further sequencing work suggests 220 amino acids with the extra two being found at the C-terminus. The active site includes cysteine and histidine residues that are conserved across several other proteins in the fruit peptidase family. Electron density mapping indicates a double crossover with domain 1 being made up of AA 19-115 and 214-218 and domain II composing of AA 1-18 and 116-213, with both the N-terminal and the C-terminal ends crossing over into both domains. Domain 1 has several α-helices whereas domain 2 is primarily made up of one anti-parallel β-sheet. Actinidain comprises up to 50% of the kiwifruit’s soluble protein content at harvest. Actinidain is active over a wide range of pH, including very acidic conditions, with a pH optimum from 5-7. At least ten different isoforms that all have the same molecular weight and cysteine protease activity as actinidain have been identified but they vary in isoelectric point from acidic (pI 3.9) to basic (pI 9.3).

## Human health impacts

Actinidain is able to function at low acidities (pH 1-2) that are found in the human GI tract and therefore is found to assist with protein digestion in the stomach and small intestine. Actinidain enhances the human body’s ability to digest food, particularly when working together with pepsin and pancreatin, by hydrolyzing food proteins more efficiently than human digestive enzymes. Further work is being done into the usefulness of kiwifruit as a digestive aid.
Actinidain is the major allergen in kiwifruit. There does not appear to be any trend when looking at who is allergic to kiwi as it varies within age, geographical differences, and other characteristics clinicians use to track allergens, although the allergy often presents itself as mild symptoms in the mouth. Actinidain provokes both IgG and IgE responses antibody responses, with the IgE binding activity being associated with the severe (anaphylaxis) responses.

## Potential applications

Actinidain is used as a high-quality meat tenderizer. When marinating with pork, actinidain was found to tenderize it by affecting the myofibrils and the connective tissue, which are similar to the tissues that are broken down through mechanical tenderization.
Studies have shown that actinidain might be a good alternative milk coagulant, replacing chymosin, a common coagulant used in cheese making.