2. CHARACTERIZATION, PRODUCTION AND MASS
CALCULATION OF INVERTASE ENZYME
Assigned by: Dr. Muhammad Suleman
HoD Department of Chemistry
Project by: Asghar Ali
Roll No. 21535
Class M Phill Chemistry 1st Semester
3. INTRODUCTION:
Also nkown as D-fructofuranosidfructohydrolase
It is sucrose degrading enzyme
Contrast with acid hydrolysis method
Chemical methods were extensively used before identification of invertase enzyme. However, acid
hydrolysis have many disadvantages such as byproduct generation and low efficiency which restrict
its use in the industry
Produced in plants, bacteria, fungi, and some animal
4. History and Discovery
Invert sugar production was the chemical conversion
Mitscherlich (1842) presented as invertase could change dextrorotatory cane sugar
into a levorotatory sugar
In 1847 this mixture was known as a glucose and fructose by dubrunfaut
Berthelot (1860) isolated invertase from the yeast
5. Enzyme Classification
Formal names are β-fructofuranosidase fructosidase, D-fructofuranoside
fructohydrolase, sucrase, invertin, saccharase, etc and the systematic name of
invertase are sucrose glycosidase,.
Invertase is a glycoprotein with great quantity of mannose residues which belongs
to a glycoside hydrolase (GH) family consisting of 370 enzymes.
These enzymes have 32 common amino acids likes GH32 family.
Based upon pH
a) Acidic invertases (pH 4.5–5.5), b) neutral and/or alkaline (pH 6.5–8.0)
Bacterial invertase is active in wide pH range
Exteracelluar invertases, usually don’t have a carbohydrate group unlike
intracellular invertases
10. Recombinant Process
In a study, an optimal expression of recombinant invertase
was achieved by integrating of cassette made of formate
dehydrogenase gene promoter and methanol oxidase (NOX)
gene terminator, into a methylotrophic yeast, Hansenula
polymorpha, genome.
Three fermentation approaches, including solid-state
fermentation (SSF), semi-solid state and submerged
fermentation (SmF) have been applied for the production of
microbial enzymes.
11. Purification
A conventional three-step procedure for invertase purification is:
1. Precipitation with sulfate salts (especially ammonium sulfate)
or miscible organic solvents (such as ethanol or acetone),
2. Anion-exchange chromatography or affinity chromatography
3. Gel filtration/Size-exclusion chromatography.
12. Relative Activities
The activity of invertase produced by yeasts was reported between 0.48 IU/ ml to
107.4 IU/ml
The Emericela nidulans ,a fungus was introduced as fungal strains with maximum
invertase activity (361.9 IU/ml) when grown on rye flour under SmF
Invertase activity of A. niger EM77 in the presence of sucrose as an energy source
and SSF conditions was found 144.39 IU/g
Different studies were shown 0.35–1399 IU/ml as rang of invertase activity
produced from bacteria under SmF conditions
Lactobacillus brevis Mm-6 produce invertase with high activity of 1399 IU/ml
13. Mass of Invertase Enzyme
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of invertase from Baker's
yeast [Lane 1; molecular weight markers (14–66 kDa) (20 g), lane 2; crude yeast extract (20 g), and lane 3;TPP
purified invertase (aqueous phase) (25 g)]
14. CHARACTERIZATION
Sucrose determining and invertase activity measuring assay
Invertase enzyme activity can be measured by several analytical approaches.
These approaches are either based on natural substrates or based on artificial
substrates.
Invertases catalyze the breakdown of sucrose to free fructose and glucose. So, the
invertase assay determines the amount of glucose derived from sucrose hydrolysis
15. CHARACTERIZATION
Sucrose determining and invertase activity measuring assay
NATURAL SUBSTRATE METHODS
Natural substrate methods measure the glucose (reducing sugar) by
spectrophotometry of colored reactions using
dinitrosalicylic acid (DNSA, red color, absorbance ¼ 540–570 nm)
OxiRed probe (570 nm) or
o-dianisidine (brown color, absorbance 425–475 nm).
Although HPLC, in-gel assays, ultrasound, polarimetry and enzyme assay kits
are also described for glucose measuring
16. CHARACTERIZATION
Sucrose determining and invertase activity measuring assay
The invertase enzyme should be incubated with reducing sugars (sucrose) in
acetate buffer solution. After that, the reaction mixture is incubated at 20–40 oC,
pH 5 for 10–15 min. For stopping of the enzymatic reaction, dinitrosalicylic acid
reagent is used. After addition of acid reagents, the reaction mixture is heated for
5–10 min in a water bath at 100 oC. Finally, the absorbance of the reaction mixture
should be read in 540 nm with using a spectrophotometer. An IU (unite of
invertase) is the quantity of enzyme that releases 1 μmol glucose per minute in
each mg of total protein under the assay condition. The total protein quantity is
measured by Bradford’s method
17. Optimum invertase activity
Thermal Stability:
• Invertase is stable at 30–50 oC
• As temperature arise gradually decrease the activity of the enzyme probably due to
the destruction of the enzyme structure.
• Finally, at 70 oC enzyme inactivated completely.
• Carbohydrate group in the structure of invertase led to increase the thermostability.
This property makes themideal for industrial application
• 65 oC is the optimum temperature of invertase activity produced by Aspergillus
niger
The most production of the enzyme was gained at 30 oC, but in 60 oC the lowest
production of the enzyme was discovered
18. Optimum invertase activity
pH and Temperature
There is a relation between pH and temperature.
pH 3.0, temperature 60 oC and pH 4.6, temperature 55 oC
Invertases act in a wide range of pH (3.0–5.0). But various invertases, including acid, natural
and alkaline have maximum activity in its optimum pH
Effect of Substrate
The enzyme sensitivity of invertase toward substrate is decreased in the presence of
cations such as Hg+2, Ag+2, Ca+2 and Cu+2.
But addition of Co ions and ethylene diaminetrichloroacetic acid (EDTA) remove this
inhibitory effect.
Increasing and decreasing of invertase activity in front of various ions reveals that invertase
is a metalloenzyme
19. APPLICATIONS
Health benefits of invertase
Honey Production: The bees use the invertase enzyme to produce honey, so the
sugar in the natural nectar is used to produce honey
Metabolism: Honey enzymes such as invertase can be investigated for their
metabolic activity. A high level of metabolism occurs in the top portion of
asparagus spears which can be related to high invertase activity in this portion.
Antioxidant properties: Invertase enzyme has a significant antioxidant activity and
antibacterial effect. These characteristics of the invertase enzyme enable it to
prevent bacterial infections and oxidative gut fermentation.
Ulcer preventing: Invertase will reduce the stomach toxicity because it will produce
simple sugars which easily digested and not persist a long time in the stomach to
generate toxic fermentation
20. APPLICATIONS
Health benefits of invertase
Defense against bacterial infections: Enzymes such as invertase in honey has the
capability to convert glucose into hydrogen peroxide and lead to kill bacteria
Natural respiratory support: It has been shown that invertase sources can reduce
colds, flu and other respiratory diseases such as bronchitis, asthma, and allergies.
Cancer treatment: Some researches indicated that invertase might show some
chemotherapeutic properties. Researchers have found that the invertase enzymes
in honey can help to treat patients with advanced bone and stomach cancer to the
extent that it may cause a tumor to regress
21. APPLICATIONS
Industrial applications
Enzymes have been extensively used in industrial processes because of their gentle
reaction conditions, for example pH and temperature, simply degradable, no
toxicity, minimum production of by-products and easy purification protocol
INVERT SYRUP: Yeast invertase is a β-fructosidase whereas the fungus produces an
α-glucosidase type of invertase. These two types of invertase are different in their
mode of action. The β-fructosidase hydrolyzes the sucrose from fructose end while
the α-glucosidase hydrolyzes sucrose from the glucose end. Both of reaction yields
a glucose and fructose mixture, known as invert syrup. Invert syrup is sweeter than
sucrose because of the high sweetness of fructose
22. APPLICATIONS
Industrial applications
Bakery Products: one of the considerable applications of invertase is the
manufacture of syrup with non-crystallizable sugar from sucrose. It is commonly
used to avoid the sugar crystal aggregation in marzipan, gingerbread dough or
praline filling
As a humectant: Due to its hygroscopic nature, invert syrup is used as a humectant
in the manufacture of soft centered candies and fondants.
Fermentation: Whenever sucrose is exposed to fermentation for production of
lactic acid, alcoholic drinks, glycerol, as well as in bakery products the presence of
invertase is mandatory
Inulinase activity: Invertase also has inulinase activity, so it can be used for
hydrolysis of poly fructose compounds (inulin) to fructose
23. Production and Preparation: Other common applications of invertase include
production and preparation of artificial honey, cookies, marshmallows, creams,
candies, jams, soft-centered chocolates, chocolate concealed cherries, truffles
powder milk for infants, and many other uses
Microbial invertases can be used for the production of cattle feed and honey bees
food.
Invertase is a useful enzyme for baking, because it provide fructose or glucose for
gas production in fermentation processes.
APPLICATIONS
Industrial applications
24. Pharmaceutical Industry: In addition to the food industry, invertase is also widely
used in the pharmaceutical industry. Invertases has been largely employed in the
formulation of drugs, digestive tablets, cough syrup, infant foods and
nutraceuticals
Invertase is able to produce fructooligosaccharides (FOS) such as nystose,
fructofuranosylnystose and kestose.
FOS is an important non-digestible polar carbohydrate compose of short fructose
chains, which has a variety of applications in prebiotic food, beverage and bread
products. The FOS is excellent for diabetic patients because of the lower caloric
value of FOS with similar sweetness. In addition, FOS have been employed in the
production of short-chain fatty acids. FOS can regulate sugar and lipid metabolism
and mineral absorption, as well as prevent constipation and colon cancer.
APPLICATIONS
Industrial applications
25. Manufacture of Biofuels: Invertase has a great feature to be used for the
manufacture of biofuels as a good alternative for gasoline. Invertase tends to
increase the glucose amount of molasses which is a great ethanol feedstock.
Cosmetics: Invertase has application in production of cosmetics plasticizing
materials and paper industries.
Invertase plays a major role in hydrolysing cane molasses as cheap carbon sources
into fructose and glucose
Invertase has a main role in the production of glycerol, lactic acid, and ethanol
Invertase can serves as a biosensor for easy and rapid detection of sucrose for
commercial applications
APPLICATIONS
Industrial applications
