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TANNASE = TANNIN ACYL HYDROLASE
CAS Number: 9025-71-2
EC Number: 232-804-4
MDL number: MFCD00212735
Tannase is an enzyme that hydrolyzes Tannin containing Depside bonds such as Tannic acid and Chlorogenic acid.
Tannase is chemically identified as Tannin acylhydrolase with the following registry number: EC 3.1.1.20 and CAS RN: 9025-71-2.
Tannase is produced by a non-genetically modified Aspergillus oryzae strains, with the criteria laid out in Regulation (EC) 1332/2008 on food enzymes.
In the food industry, Tannase gives particularly excellent effects for improving tea quality (reduce bitterness) and preventing white clouding due to the coacervation of tea tannin.
Main enzyme reactions involving Tannase is shown below.
Apart from the main enzymatic activities, Tannase does not contain significant levels of subsidiary activities.
Tannase is intended for use in the production of tea based beverages (ready to drink teas, tea extracts) and botanical extracts.
When using Tannase for other kinds of beverage, try with concentration between 0.01% and 0.2%, at 30 to 40℃ for a certain time period, to find out the optimum conditions.
Tannase should be noted that ferric ions as inhibitor may affect the result.
Tannase decomposes the tea gallated polyphenols into gallic acid and polyphenols to prevent combining with caffeine which is the cause of tea turbidity.
Tannase can clear any kinds of teas but does not change the taste.
Tannase catalyzes the hydrolysis of tannic acid to produce gallic acid and glucose.
The tannase is produced by submerged fermentation of a selected fungal strain followed by purification, formulation and drying.
The properties of tannase vary from species to species.
Tannase has a molecular weight of 46.5-90 kDa and exists as a monomer, while tannase from Rhodococcus sp. and L. plantarum contains two subunits.
So far, all tannases from yeast and fungi are glycoproteins, but there seems to be no such post-translational modification in bacteria.
Tannase is an acidic protein with an optimum pH range of 4.5-7.0.
The optimal temperature of different kinds of Tannase is different, and the optimum temperature of most bacterial tannase is between 30 and 40 °C.
When methyl gallate was used as a substrate and the reaction temperature was 30-40 °C, the bacterial tannase substrate affinity (Km) from Selenomonas ruminantium and Enterobacter sp. was 1.6 and 3.7, respectively.
More than 28% of bacterial tannase requires metal ions as a cofactor to stimulate its maximum catalytic efficiency.
Tannase has also been found that the activity of B. subtilis tannase is increased in polar protic solvents such as glycerol, isopropanol, ethanol, methanol and isoamyl alcohol, while butanol, acetic acid and acetone reduce the activity of tannase.
Tannase is Light yellow powder.
Color may vary from batch to batch.
Color intensity is not an indication of enzyme activity.
Tannase has two known domains and one known active site.
Tannase can be found in plants, bacteria, and fungi and has different purposes depending on the organism Tannase is found in.
Tannase also has many purposes for human use.
The crystal structure of Tannase varies slightly depending on the strain being observed, in this case we are looking at the tannase SN35N strain produced in Lactobacillus plantarum.
On average, Tannase's molecular weight is in the range of 50-320 kDa.
Natural B. subtilis tannase consists of 9.3% α-helix, 33.6% parallel β-sheet, 17.2% β-turn and 39.9% random coil.
The β conformation plays a dominant role in tannase activity, and the secondary structure of tannase is stringently dependent on its microenvironment (temperature and pH).
B. Subtilis tannase scanning probe microscopyic analysis (SPM) showed that tannase showed different degrees of aggregation, the structure is similar to round or oval, the size is different, and the average diameter is 44 nm.
The crystal structure of tannase is a small plate-like crystal.
Three-dimensional structural analysis of L. plantarum tannase revealed that it exhibited α/β structure with 18 α-helices and 13 β-strands.
Tannase (EC 3.1.1.20) belongs to the class of hydrolases.
Tannase catalyzes the hydrolysis of digallate to gallate.
The systematic name of this enzyme class is tannin acylhydrolase.
Other names in common use include tannase S, and tannin acetylhydrolase.
Tannase is a natural adaptive intracellular/extracellular inducible hydrolase and placed in the esterase superfamily.
Tannase can be obtained from plants, animals and microorganisms, but the microbial-derived tannase is more extensive because Tannase's stability is higher than that of plant and animal sources.
The enzyme tannase (EC 3.1.1.20) catalyzes the following reaction:
digallate + H2O = 2 gallate
Tannase is a key enzyme in the degradation of gallotannins and ellagicitannins, two types of hydrolysable tannins.
Specifically, tannase catalyzes the hydrolysis of ester and depside bonds of hydrolysable tannins to release glucose and gallic or ellagic acid.
Tannase belongs to the family of hydrolases, specifically those acting on carboxylic ester bonds.
The systematic name is tannin acylhydrolase.
Other names in common use include tannase S, and tannin acetylhydrolase.
One way in which the structure of tannase is tied with its function involves a loop structure, called the flap.
The flap connects β8 and β9 sheets and is located under the catalytic triad.
As a result of weak electron densities, this structure is very flexible.
Due to Tannase's flexibility, the flap is better able to guide the substrate in entering the enzyme and helps to strengthen the overall binding of the complex by forming additional interactions with other parts of the substrate.
Tannase is present in microorganisms, plants and animals.
However, microorganisms are mainly used for commercial production.
A complete list of tannase-producing microorganisms is provided by Chávez-González et al., (2012).
From the list of species of tannase producers given by them one can infer that interestingly fungi, yeasts and bacteria are the dominant groups among the microorganisms.
Among fungi, Aspergilli and Penicillia are the major groups although 20 different genera of fungi are known as tannase producers.
So far 27 species of Aspergillus, 24 species of Penicillium, 4 species of Trichoderma and 3 species of Fusarium are reported as tannase producers.
Among bacteria about 21 different genera are known as tannase producers and among them Lactobacilli are the dominant groups (13 species) followed by Pediococcus (4 species), Serratia, (3 species), Leuconostoc (2 species), Pantonea (2 species) Streptococcus (2 species) among others.
Early study focused on screening of microorganisms available as stock culture from culture collection centers which primarily derived microorganism from soil.
However, later investigators designed media specific for screening tannase producers from natural environment such as forest litter, human faeces, fermented foods, sheep excreta, tannery effluents, olive mill waste water, etc.
Tannase cleaves ester and depside linkages in such hydrolyzable tannins as tannic acid and chebulinic acid.
Tannase also acts on the ester and depside linkages in methylgallate and m-digallic acid, respectively.
Tannase hydrolyzes only those substrates that contain at least two phenolic OH groups in the acid component.
The esterified COOH group must be on the oxidized benzene ring and must not be ortho to one of the OH groups
USES and APPLICATIONS of TANNASE:
-The production of gallic acid is important in the pharmaceutical industry as it's needed to create trimethoprim, an antibacterial drug.
-Tannase also has many applications in the food and beverage industry.
-Specifically, Tannase's used to make food and drinks taste better, either by removing turbidity from juices or wines, or removing the bitter taste of tannins in some food and drinks, such as acorn wine.
-Additionally, because tannase can break ester bonds of glucose with various acids (chebulinic, gallic, and hexahydrophenic), Tannase can be used in the process of fruit ripening.
-Tannase is also used in the food, feed, beverages, pharmaceutical, and chemical industries to produce gallic acid, instant tea, coffee flavored refreshing drinks and acron wines.
-In addition, tannase is used to clarify beer and juice, improve the flavor of the wine and make animal feed.
-Tannase participates in fruit ripening by breaking the ester bonds of glucose with chebulinic, gallic and hexahydrophenic acid.
-In the chemical industry, tannase can be used to analytical probe preparation, determine the structure of naturally occurring gallic acid esters, detect cancer cells, and treat tannins-containing wastewater in the olive oil and leather industries.
-Tannase is used to improve taste and damage the haze sensitivity of phenols in tea, remove anti-nutritional effect of tannins in feed, and make tannin a better tanning agent for leather, and so on.
-Tannase can also be used to produce non-galloylated catechins.
-Tannase finds wide applications in various industries including food and beverage, feed, cosmetics and leather, where the enzyme plays Tannase's role by modifying tannins in desirable ways.
FUNCTION of TANNASE:
PLANTS:
Tannase functions differently in the cell depending on the organism being observed.
In many plants, tannase is used to produce tannins, which are found in leaves, wood, and bark.
The production of tannins in plants is essential for defense against herbivory, as they cause a strong unpalatable flavor.
Tannins are considered secondary metabolites in plants.
Therefore, their production by tannase plays no direct role in plant primary metabolism.
MICROORGANISMS:
On the other hand, tannase serves a different purpose in many microorganisms.
In the cell, tannase is a key enzyme in the degradation of gallotannins.
This is important, because some microorganisms use tannase to breakdown hydrolysable tannins, such as gallotannins, to form glucose and gallic acid.
These byproducts are created from the hydroxylation of the aromatic nucleus of the tannin, followed by ring cleavage.
Glucose and gallic acid can then be readily converted to metabolites (i.e. pyruvate, succinate, and acetyl coenzyme A) that can be used in the Krebs cycle.
Specific microorganisms that utilize tannase in this way include Pseudomonas species.
SPECIES DISTRIBUTION:
Tannase is present in a diverse group of microorganisms, including rumen bacteria. Many other bacterial species have been found to produce tannase by being isolated from different types of media such as soil, wastewater, compost, forest litter, feces, beverages, pickles, etc.
Bacteria and archaea species with tannase activity have been found in the genera: Achromobacter, Atopobium, Azotobacter, Bacillus, Citrobacter, Corynebacterium, Enterobacter, Enterococcus, Fusobacterium, Gluconoacetobacter, Klebsiella, Lactobacillus, Lonepinella, Methanobrevibacter, Microbacterium, Oenococcus, Pantoea, Pediococcus, Providencia, Pseudomonas, Selenomonad, and Serratia. In addition, some fungal species are dominant tannase producers, such as Aspergilli species.
MECHANISM of TANNASE:
Tannin acyl hydrolase, commonly called tannase, catalyzes the hydrolysis of ester and depside bonds in such hydrolysable tannins as tannic acid, thereby releasing glucose and gallic acid in the end.
By the catalysis with tannase, gallic acid glucose ester with tannic acid is successively hydrolyzed into 1,2,3,4,6-gallic acid tannin, 2,3,4,6-tetrapalic acid tannin and two monomers gallic acid-glucose, and finally gallic acid and glucose are produced.
In addition to catalyzing the hydrolysis of the central ester bond between the two aromatic rings of digallate (depsidase activity), tannase may also have an esterase activity (hydrolysis of terminal ester functional groups that are attached to only one of the two aromatic rings).
Digallate is the conjugate base of digallic acid, but are often used synonymously.
Similarly, gallate and gallic acid are used interchangeably. B
oth digallic and gallic acid are organic acids that are seen in gallotannins and are usually esterified to a glucose molecule.
In other words, tannins (which contain digallate/digallic acid) are the natural substrate of tannase.
When tannins, specifically gallotannins, are broken down by tannase through the hydrolysis of ester bonds, gallic acid and glucose are formed.
CATALYTIC MECHANISM of TANNASE:
Tannase is the most studied enzyme in tannin biodegradation.
Tannase catalyzes the hydrolysis of esters and depside bonds of various substrates, including gallanonins, gallic acid esters, epigallocatechin gallate and epicatechin gallate, releasing gallic acid and glucose.
L. plantarum tannase consists of two domains, one α/β-hydrolase domain (residues 4-204 and 396-469) and a "lid" domain (residues 205-395).
The glycerol molecule in the cryoprotectant solution binds to the active site of the tannase, which mimick the binding of the galloyl moiety to its substrate.
The active site of tannase is located in the α/β-hydrolase domain, of which three amino acid residues Ser163, Asp419 and His451 are catalytic triads.
L. plantarum tannase contains 18 α-helices and 13 β-strands, and Ser163 is present in the Gly161-X-Ser163-X-Gly165 pentapeptide motif between β6 and α6.
In the active site, Ser163 forms a hydrogen bond with the NE2 atom of the His451 imidazole ring.
During the reaction, His451 deprotonated the hydroxyl group of Ser163, and Ser163 was stabilized by hydrogen bonding with Asp419.
PHYSICAL and CHEMICAL PROPERTIES of TANNASE:
Physical state: powder
Color: No data available
Odor: No data available
Melting point/freezing point: No data available
Initial boiling point and boiling range: No data available
Flammability (solid, gas): No data available
Upper/lower flammability or explosive limits: No data available
Flash point: No data available
Autoignition temperature: No data available
Decomposition temperature: No data available
pH: No data available
Viscosity
Viscosity, kinematic: No data available
Viscosity, dynamic: No data available
Water solubility: No data available
Partition coefficient: n-octanol/water: No data available
Vapor pressure: No data available
Density: No data available
Relative density: No data available
Relative vapor density: No data available
Particle characteristics: No data available
FIRST AID MEASURES of TANNASE:
-Description of first-aid measures:
*General advice:
First aiders need to protect themselves.
Show this material safety data sheet to the doctor in attendance.
*If inhaled:
After inhalation:
Fresh air.
Call in physician.
*In case of skin contact:
Take off immediately all contaminated clothing.
Rinse skin with water/ shower.
*In case of eye contact
After eye contact:
Rinse out with plenty of water.
Remove contact lenses.
*If swallowed:
After swallowing:
Immediately make victim drink water (two glasses at most).
Consult a physician.
-Indication of any immediate medical attention and special treatment needed:
No data available
ACCIDENTAL RELEASE MEASURES of TANNASE:
-Personal precautions, protective equipment and emergency procedures:
*Advice for non-emergency personnel:
Ensure adequate ventilation.
-Environmental precautions:
Do not let product enter drains.
-Methods and materials for containment and cleaning up:
Cover drains.
Collect, bind, and pump off spills.
Take up carefully.
Dispose of properly.
Clean up affected area.
FIRE FIGHTING MEASURES of TANNASE:
-Extinguishing media:
*Suitable extinguishing media:
Water
Foam
Carbon dioxide (CO2)
Dry powder
*Unsuitable extinguishing media:
For this substance/mixture no limitations of extinguishing agents are given.
-Further information:
Prevent fire extinguishing water from contaminating surface water or the ground water system.
EXPOSURE CONTROLS/PERSONAL PROTECTION of TANNASE:
-Control parameters:
--Ingredients with workplace control parameters:
-Exposure controls:
--Personal protective equipment:
*Eye/face protection:
Use Safety glasses.
*Skin protection:
Full contact:
Material: Nitrile rubber
Minimum layer thickness: 0,11 mm
Break through time: 480 min
Splash contact:
Material: Nitrile rubber
Minimum layer thickness: 0,11 mm
Break through time: 480 min
-Body Protection:
protective clothing
-Control of environmental exposure
Do not let product enter drains.
HANDLING and STORAGE of TANNASE:
-Precautions for safe handling:
*Advice on safe handling:
Work under hood.
*Hygiene measures:
Change contaminated clothing.
Wash hands after working with substance.
-Conditions for safe storage, including any incompatibilities:
*Storage conditions:
Tightly closed.
Dry.
Keep locked up or in an area accessible only to qualified or authorized persons.
*Storage stability:
Recommended storage temperature: 2 - 8 °C
STABILITY and REACTIVITY of TANNASE:
-Chemical stability:
The product is chemically stable under standard ambient conditions (room temperature) .
-Possibility of hazardous reactions:
No data available
-Conditions to avoid:
no information available
SYNONYMS:
Tannin acyl Hydrolase
