Zingibain, zingipain, or ginger protease () is a
cysteine protease
Cysteine proteases, also known as thiol proteases, are hydrolase enzymes that degrade proteins. These proteases share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad.
Discovered by Gopal Chund ...
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
found in
ginger
Ginger (''Zingiber officinale'') is a flowering plant whose rhizome, ginger root or ginger, is widely used as a spice
A spice is a seed, fruit, root, bark, or other plant substance primarily used for flavoring or coloring food. Spices ...
(''
Zingiber officinale
Ginger (''Zingiber officinale'') is a flowering plant whose rhizome, ginger root or ginger, is widely used as a spice and a folk medicine. It is a herbaceous perennial which grows annual pseudostems (false stems made of the rolled bases of lea ...
'')
rhizome
In botany and dendrology, a rhizome (; , ) is a modified subterranean plant stem that sends out roots and shoots from its nodes. Rhizomes are also called creeping rootstalks or just rootstalks. Rhizomes develop from axillary buds and grow hori ...
s.
It
catalyses the preferential
cleavage of
peptide
Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides.
A ...
s with a
proline
Proline (symbol Pro or P) is an organic acid classed as a proteinogenic amino acid (used in the biosynthesis of proteins), although it does not contain the amino group but is rather a secondary amine. The secondary amine nitrogen is in the prot ...
residue at the P2 position. It has two distinct
forms
Form is the shape, visual appearance, or configuration of an object. In a wider sense, the form is the way something happens.
Form also refers to:
*Form (document), a document (printed or electronic) with spaces in which to write or enter data
* ...
, ginger protease I (GP-I) and ginger protease II (GP-II).
As a member of the
papain-like protease
Papain-like proteases (or papain-like (cysteine) peptidases; abbreviated PLP or PLCP) are a large protein family of cysteine protease enzymes that share structural and enzymatic properties with the group's namesake member, papain. They are found i ...
family of cysteine proteases, zingibain shares several structural and functional similarities with more well-studied enzymes such as
papain
Papain, also known as papaya proteinase I, is a cysteine protease () enzyme present in papaya (''Carica papaya'') and mountain papaya (''Vasconcellea cundinamarcensis''). It is the namesake member of the papain-like protease family.
It has wide ...
,
bromelain
Bromelain is an enzyme extract derived from the stems of pineapples, although it exists in all parts of the fresh pineapple. The extract has a history of folk medicine use. As an ingredient, it is used in cosmetics, as a topical medication, and as ...
, and
actinidin
Actinidain (, ''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. Th ...
. These peptidases contain an active cysteine residue in their centers that catalyzes the hydrolytic cleavage of peptide bonds. Zingibain is noted for its activity as a
proteinase
A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalyzes (increases reaction rate or "speeds up") proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the for ...
and a
collagenase
Collagenases are enzymes that break the peptide bonds in collagen. They assist in destroying extracellular structures in the pathogenesis of bacteria such as ''Clostridium''. They are considered a virulence factor, facilitating the spread of ga ...
.
It was first isolated, purified, and reported in 1973 by Ichikawa et al. at
Japan Women's University
is the oldest and largest of private Japanese women's universities. The university was established on 20 April 1901 by education reformist .
The university has around 6000 students and 200 faculty. It has two campuses, named after the neighbo ...
.
Recently, zingibain was found to exist as two
isozyme In biochemistry, isozymes (also known as isoenzymes or more generally as multiple forms of enzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. Isozymes usually have different kinetic parameters (e.g. dif ...
s, GP-I and GP-II, which were isolated by chromatography, with molecular weights of approximately 22,500 Da.
Mechanism
Zingibain utilizes a catalytic triad of Cys, His, and Asn residues in its active site in order to cleave peptide bonds hydrolytically. The presence of Asn175 stabilizes the
imidazole
Imidazole (ImH) is an organic compound with the formula C3N2H4. It is a white or colourless solid that is soluble in water, producing a mildly alkaline solution. In chemistry, it is an aromatic heterocycle, classified as a diazole Diazole refers ...
ring of His, ensuring it is positioned optimally to catalyze hydrolysis.
The mechanism begins with a proton transfer from Cys25 to His159.
The sulfide anion then attacks the amino acid's
alpha carbon
In the nomenclature of organic chemistry, a locant is a term to indicate the position of a functional group or substituent within a molecule.
Numeric locants
The International Union of Pure and Applied Chemistry (IUPAC) recommends the use of ...
, displacing the
amine group
In chemistry, amines (, ) are compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are formally derivatives of ammonia (), wherein one or more hydrogen atoms have been replaced by a substituent such a ...
, which attaches to His159.
The alpha carbon on the stabilized amino acid is then attacked by a water molecule, which displaces the sulfide of Cys25 to convert the amino acid to a
carboxylic acid
In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an R-group. The general formula of a carboxylic acid is or , with R referring to the alkyl, alkenyl, aryl, or other group. Carboxylic ...
, which is released from the enzyme active site.
The experimental introduction of
dithiothreitol
Dithiothreitol (DTT) is the common name for a small-molecule redox reagent also known as Cleland's reagent, after W. Wallace Cleland. DTT's formula is C4H10O2S2 and the chemical structure of one of its enantiomers in its reduced form is shown on ...
, a known
thiol group
In organic chemistry, a thiol (; ), or thiol derivative, is any organosulfur compound of the form , where R represents an alkyl or other organic substituent. The functional group itself is referred to as either a thiol group or a sulfhydryl gro ...
protector, improves proteolytic activity, providing further verification of the importance of the central cysteine residue to enzymatic activity.
Zingibain exhibits maximum turnover rate at 60 °C and rapidly denatures at 70 °C.
Proteolysis is largely unhampered during cooking with ginger. Optimal temperature ranges of papain and ficin are elevated relative to zingibain, whereas bromelain operates at a slightly lower range.
Maximum proteolytic activity of zingibain occurs at pH of 6.0, although the enzyme is still active in pH ranges from 4.5 to 6.0 (optimal pH for
meat marinades).
GP-II, the more acidic of the two isozymes, exhibits a
pI of 4.82, and GP-I exhibits pI values at 5.05 or 5.16.
These multiple pI values lend support to a theory that GP-I may be a mixture of two proteins.
Structure
Zingibain was first purified and characterized with X-ray crystallography in 2000 by researchers at
Boston University
Boston University (BU) is a private research university in Boston, Massachusetts. The university is nonsectarian, but has a historical affiliation with the United Methodist Church. It was founded in 1839 by Methodists with its original campu ...
.
The enzyme is 221 amino acids long and glycosylated with 2
N-linked oligosaccharide chains at Asn96 and Asn154.
The polypeptide chain of zingibain folds into two polar domains of roughly equal size, divided by a central neutral cleft.
The first domain contains
alpha helices
The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...
, and the second has
antiparallel beta sheet
The beta sheet, (β-sheet) (also β-pleated sheet) is a common motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone hydrogen bonds, forming a g ...
s.
This separation of polar and non-polar regions facilitates protein-protein interactions between the enzyme and a large range of substrates.
The active site of zingibain, located in the central cleft, is 5.5 Å deep and 9.5 Å long.
The active site contains the catalytic triad of Cys25, His159, and Asn175, which both cooperatively enable acid/base catalysis.
Zingibain exhibits binding specificity to peptide substrates with proline in the P2 position.
The S2 subsite of zingibain contains the amino acid chain Trp67-Met68-Asn69-Thr133-Ala157, which makes the site too compact to accommodate larger hydrophobic aromatic substrate residues favored by other enzymes in the papain family.
Proline, however, is stabilized by multiple non-covalent interactions with this region.
The enzyme structure is stabilized by
hydrogen bond
In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing a ...
s, as well as
crosslinking Cross-linking may refer to
*Cross-link
In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers ca ...
sulfide bonds between three pairs of cysteine residues (Cys22-Cys63, Cys56-Cys95, and Cys153-Cys200), analogous to many other papains.
While the enzyme exists as a monomer in solution, crystallized zingibain forms
tetramer
A tetramer () (''tetra-'', "four" + '' -mer'', "parts") is an oligomer formed from four monomers or subunits. The associated property is called ''tetramery''. An example from inorganic chemistry is titanium methoxide with the empirical formula Ti ...
s, or dimers of dimers, linked by glycosylation chains on each subunit. Zingibain exhibits complex-type N-linked oligosaccharide chains at two residues.
Chains are between 5-13 glycosyl units long, and composed of
N-acetylglucosamine
''N''-Acetylglucosamine (GlcNAc) is an amide derivative of the monosaccharide glucose. It is a secondary amide between glucosamine and acetic acid. It is significant in several biological systems.
It is part of a biopolymer in the bacterial ...
,
fucose
Fucose is a hexose deoxy sugar with the chemical formula C6H12O5. It is found on ''N''-linked glycans on the mammalian, insect and plant cell surface. Fucose is the fundamental sub-unit of the seaweed polysaccharide fucoidan. The α(1→3) link ...
,
mannose
Mannose is a sugar monomer of the aldohexose series of carbohydrates. It is a C-2 epimer of glucose. Mannose is important in human metabolism, especially in the glycosylation of certain proteins. Several congenital disorders of glycosylation ...
, and
xylose
Xylose ( grc, ξύλον, , "wood") is a sugar first isolated from wood, and named for it. Xylose is classified as a monosaccharide of the aldopentose type, which means that it contains five carbon atoms and includes an aldehyde functional gro ...
. Zingibain sugar sequences are almost identical to oligosaccharides seen in lectins from
Japanese pagoda tree seeds,
laccase a from
sycamore
Sycamore is a name which has been applied to several types of trees, but with somewhat similar leaf forms. The name derives from the ancient Greek ' (''sūkomoros'') meaning "fig-mulberry".
Species of trees known as sycamore:
* ''Acer pseudoplata ...
cells, and S-glycoproteins from ''
Brassica campestris''.
Biological significance
Within ginger rhizomes, ginger protease participates in multiple functional roles for maintenance and upkeep of
plant cell
Plant cells are the cells present in green plants, photosynthetic eukaryotes of the kingdom Plantae. Their distinctive features include primary cell walls containing cellulose, hemicelluloses and pectin, the presence of plastids with the capabi ...
s.
Zingibain, like most cysteine proteases, is synthesized as a 40-50 kDa
proprotein
A protein precursor, also called a pro-protein or pro-peptide, is an inactive protein (or peptide) that can be turned into an active form by post-translational modification, such as breaking off a piece of the molecule or adding on another molecule ...
within cytoplasmic
polysome
A polyribosome (or polysome or ergosome) is a group of ribosomes bound to an mRNA molecule like “beads” on a “thread”. It consists of a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into po ...
s bound to
cell membrane
The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...
s.
Within the
endoplasmic reticulum
The endoplasmic reticulum (ER) is, in essence, the transportation system of the eukaryotic cell, and has many other important functions such as protein folding. It is a type of organelle made up of two subunits – rough endoplasmic reticulum ( ...
, these elongated chains are tagged with a
KDEL ER retention signal and placed into large KDEL
vesicles
Vesicle may refer to:
; In cellular biology or chemistry
* Vesicle (biology and chemistry), a supramolecular assembly of lipid molecules, like a cell membrane
* Synaptic vesicle
; In human embryology
* Vesicle (embryology), bulge-like features o ...
that move from the ER to protein storage
vacuole
A vacuole () is a membrane-bound organelle which is present in plant and fungal cells and some protist, animal, and bacterial cells. Vacuoles are essentially enclosed compartments which are filled with water containing inorganic and organic mo ...
s in
cell wall
A cell wall is a structural layer surrounding some types of cells, just outside the cell membrane. It can be tough, flexible, and sometimes rigid. It provides the cell with both structural support and protection, and also acts as a filtering mech ...
s.
Zingibain likely participates in protein storage (within
seed
A seed is an embryonic plant enclosed in a protective outer covering, along with a food reserve. The formation of the seed is a part of the process of reproduction in seed plants, the spermatophytes, including the gymnosperm and angiospe ...
s or
plant tissue
In biology, tissue is a biological organizational level between cells and a complete organ. A tissue is an ensemble of similar cells and their extracellular matrix from the same origin that together carry out a specific function. Organs are ...
), but predominantly degrades and mobilizes storage proteins. It can also respond to
abiotic
In biology and ecology, abiotic components or abiotic factors are non-living chemical and physical parts of the environment that affect living organisms and the functioning of ecosystems. Abiotic factors and the phenomena associated with them under ...
and
biotic stresses, such as
heat shock
The heat shock response (HSR) is a cell stress response that increases the number of molecular chaperones to combat the negative effects on proteins caused by stressors such as increased temperatures, oxidative stress, and heavy metals. In a normal ...
, cold temperatures, and
dehydration
In physiology, dehydration is a lack of total body water, with an accompanying disruption of metabolic processes. It occurs when free water loss exceeds free water intake, usually due to exercise, disease, or high environmental temperature. Mil ...
, to eliminate any resulting misfolded or
denatured proteins.
Uses
Meat tenderizer
Like
papain
Papain, also known as papaya proteinase I, is a cysteine protease () enzyme present in papaya (''Carica papaya'') and mountain papaya (''Vasconcellea cundinamarcensis''). It is the namesake member of the papain-like protease family.
It has wide ...
from
papaya
The papaya (, ), papaw, () or pawpaw () is the plant species ''Carica papaya'', one of the 21 accepted species in the genus ''Carica'' of the family Caricaceae. It was first domesticated in Mesoamerica, within modern-day southern Mexico and ...
s and
bromelain
Bromelain is an enzyme extract derived from the stems of pineapples, although it exists in all parts of the fresh pineapple. The extract has a history of folk medicine use. As an ingredient, it is used in cosmetics, as a topical medication, and as ...
from
pineapple
The pineapple (''Ananas comosus'') is a tropical plant with an edible fruit; it is the most economically significant plant in the family Bromeliaceae. The pineapple is indigenous to South America, where it has been cultivated for many centuri ...
s, it is used as a
meat tenderizer
A meat tenderizer, or meat pounder is a hand-powered tool used to tenderize slabs of meat in the preparation for cooking. Although a meat tenderizer can be made out of virtually any object, there are three types manufactured specifically for ...
.
When added to cooking meat, usually within raw or dried ginger, zingibain has been shown to increase the tenderness of meat.
Meat tenderization occurs due to zingibain's rapid proteolysis of major muscle proteins within meat, especially
actomyosin
Myofilaments are the three protein filaments of myofibrils in muscle cells. The main proteins involved are myosin, actin, and titin. Myosin and actin are the ''contractile proteins'' and titin is an elastic protein. The myofilaments act toget ...
and
Type I collagen
Type I collagen is the most abundant collagen of the human body. It forms large, eosinophilic fibers known as collagen fibers.
It is present in scar tissue, the end product when tissue heals by repair, as well as tendons, ligaments, the endomys ...
, which is found in
muscle joints.
While other papain enzymes, including papain, ficin, and bromelain, are more commonly used to tenderize meat, zingibain shows similar or elevated proteolytic activity.
In fact, zingibain is the only catalogued plant protease with collagenolytic activity. Zingibain may be a preferable meat tenderizer to papain due to the resulting texture of meat produced. While papain can hydrolyze actomyosin, it also breaks down other major tissue proteins, that lead to a mushy meat texture.
The specificity of zingibain's binding ensures predominant hydrolyzation of actomyosin and Type I collagen.
Zingibain is also used to flavor
sausage
A sausage is a type of meat product usually made from ground meat—often pork, beef, or poultry—along with salt, spices and other flavourings. Other ingredients, such as grains or breadcrumbs may be included as fillers or extenders.
...
s and
baked products.
Rennet substitute
For over the past 100 years, ginger protease has traditionally been used to curdle
milk
Milk is a white liquid food produced by the mammary glands of mammals. It is the primary source of nutrition for young mammals (including breastfed human infants) before they are able to digestion, digest solid food. Immune factors and immune ...
to create
ginger milk curd
Ginger milk curd, also known as ginger-juice milk curd, ginger milk pudding or simply ginger milk, is a Chinese hot dessert originated in Shawan Ancient Town, Panyu District, Guangzhou in the Guangdong Province in southern China. The main ingr ...
, a gel-like
Cantonese dish made from hot milk and
ginger juice. The milk clotting ability and specificity of ginger protease to proteolysis of
κ-casein
Κ-casein, or kappa casein, is a mammalian milk protein involved in several important physiological processes. Chymosin splits K-casein into an insoluble peptide (para kappa-casein) and water-soluble glycomacropeptide (GMP). GMP is responsible f ...
make the enzyme a potential vegetable
rennet
Rennet () is a complex set of enzymes produced in the stomachs of ruminant mammals. Chymosin, its key component, is a protease enzyme that curdles the casein in milk. In addition to chymosin, rennet contains other enzymes, such as pepsin and a ...
substitute for
cheese production.
Milk coagulation is traditionally accomplished by coagulating enzymes extracted from sources such as rennet. In rennet, three
chymosin
Chymosin or rennin is a protease found in rennet. It is an aspartic endopeptidase belonging to MEROPS A1 family. It is produced by newborn ruminant animals in the lining of the abomasum to curdle the milk they ingest, allowing a longer residen ...
isozymes hydrolyze κ-casein, a major protein fraction within milk, between
Phe105 and
Met106. Hydrophilic sub-regions of κ-casein are cleaved off, leaving behind largely hydrophobic aggregate. The enzymes thus destabilize κ-casein
micelle
A micelle () or micella () (plural micelles or micellae, respectively) is an aggregate (or supramolecular assembly) of surfactant amphipathic lipid molecules dispersed in a liquid, forming a colloidal suspension (also known as associated collo ...
s and encourage clumping of hydrophobic protein residues, causing milk to curdle.
Major industrial drawbacks of rennet include its limited supply and high cost, its inaccessibility to
vegetarians
Vegetarianism is the practice of abstaining from the consumption of meat (red meat, poultry, seafood, insects, and the flesh of any other animal). It may also include abstaining from eating all by-products of animal slaughter.
Vegetarianism may ...
and practicing members of certain
religious groups, and recent European national bans on utilization of
recombinant calf rennet.
Fungal proteases are largely unsuitable as rennet substitutes, and enzymes from many plant extracts have been shown to produce low yields, poor textures, and bitter flavors of cheese.
Commercial drawbacks
However, crude ginger protease extracted from ginger extract is unstable, with a
half-life
Half-life (symbol ) is the time required for a quantity (of substance) to reduce to half of its initial value. The term is commonly used in nuclear physics to describe how quickly unstable atoms undergo radioactive decay or how long stable ato ...
of about 2 days at 5 °C, making it problematic for commercial applications.
While the enzyme's half-life does not impede its efficacy during cooking, this low storage stability requires improvement for commercialization.
Commercial attempts to stabilize the enzyme for large-scale production have investigated potential methods to inactivate the free
sulfhydryl group
In organic chemistry, a thiol (; ), or thiol derivative, is any organosulfur compound of the form , where R represents an alkyl or other organic substituent. The functional group itself is referred to as either a thiol group or a sulfhydryl gro ...
within the enzyme's active site. Mechanistic possibilities include oxidizing the sulfhydryl, exchanging it with
disulfide bridges
In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
, forming
quinone
The quinones are a class of organic compounds that are formally "derived from aromatic compounds uch as benzene or naphthalene
Uch ( pa, ;
ur, ), frequently referred to as Uch Sharīf ( pa, ;
ur, ; ''"Noble Uch"''), is a historic city in the southern part of Pakistan's Punjab province. Uch may have been founded as Alexandria on the Indus, a town founded by Alexand ...
by conversion of an even number of –CH= groups into –C(=O)– groups with any necessary rearrangement of double ...
-thiol adducts, or binding the sulfhydryl to a
heavy metal ion.
0.2%
sodium ascorbate
Sodium ascorbate is one of a number of mineral salts of ascorbic acid (vitamin C). The molecular formula of this chemical compound is C6H7NaO6. As the sodium salt of ascorbic acid, it is known as a mineral ascorbate. It has not been demonstrate ...
was found to stabilize zingibain for up to 14 days at 5 °C, whereas comparable concentrations of
EDTA
Ethylenediaminetetraacetic acid (EDTA) is an aminopolycarboxylic acid with the formula H2N(CH2CO2H)2sub>2. This white, water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-soluble complexes eve ...
and
CaCl2 had minimal impact on stability.
Zingibain has been observed to deactivate itself through
autolysis, which can be pre-empted by reacting the active sulfhydryl group with
cystine
Cystine is the oxidized derivative of the amino acid cysteine and has the formula (SCH2CH(NH2)CO2H)2. It is a white solid that is poorly soluble in water. As a residue in proteins, cystine serves two functions: a site of redox reactions and a me ...
or PCMB.
Acetone
Acetone (2-propanone or dimethyl ketone), is an organic compound with the formula . It is the simplest and smallest ketone (). It is a colorless, highly volatile and flammable liquid with a characteristic pungent odour.
Acetone is miscib ...
powders are a viable commercial method of stabilization of zingibain. After hydrophobic plant
polyphenol
Polyphenols () are a large family of naturally occurring organic compounds characterized by multiples of phenol units. They are abundant in plants and structurally diverse. Polyphenols include flavonoids, tannic acid, and ellagitannin, some of ...
s are removed from crude ginger, acetone powder is introduced at low temperatures in order to dehydrate the root pulp.
The enzyme is stabilized due to reduced water activity, lower concentrations of plant pigments, and more rigid 3D structures at lower temperatures.
See also
*
Gingerol
Gingerol ( gingerol) is a phenolic phytochemical compound found in fresh ginger that activates spice receptors on the tongue. It is normally found as a pungent yellow oil in the ginger rhizome, but can also form a low-melting crystalline solid. T ...
References
External links
*
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