INVERTASE.pptx

INVERTASE ENZYME
STRUCTURE
PROPERTIES
PRODUCTION
CHARCTERIZATION
SIGNIFICANCE
USES

1. 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 1 st Semester

2. 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

3. 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

4. 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 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

5. Enzyme Structure Invertase shows an unusual octameric quaternary structure composed of two types of dimers.

6. Sources FUNGAL SOURCES YEAST INSECTS PLANT SOURCES Insect Sources Aspergillus Cladosporium Fusarium Aureobasidium Eremothecium Ceratocystis Penicillium Rhizopus Sclerotinia Trichoderma Verticillium Yarrowia lipolytica Paecylomices Metarhizium Saccharomyces Zygosaccharomyces Candida Cryptococcus Xanthophyllomyces Schwanniomyces Bacillus Bifidobacterium Enterobacter Leishmania Lactobacillus Microbulbifer Pseudobutyrivibrio Thermotoga Xanthomonas Penicillium Anabaena Leishmania Agrilus Bombyx Bombyx Sphenophorus Arabidopsis Rumex Solanum Saccharum Nicotiana Camellia Agave Bamboo Manihot Cichorium Dendrobium Elsholtzia Gossypium Hevea Kummerowia Eriobotrya Zea Pisum   Rosa Sorghum

7. Isolation of Invertase Enzyme from natural sources cannot be accountable.

8. 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.

9. Purification A conventional three-step procedure for invertase purification is: Precipitation with sulfate salts (especially ammonium sulfate) or miscible organic solvents (such as ethanol or acetone), Anion-exchange chromatography or affinity chromatography Gel filtration/Size-exclusion chromatography.

10. Relative Activities The activity of invertase produced by yeasts was reported between 0.48 IU/ ml to 107.4 IU/ml T he 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

11. 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 )]

12. 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

13. 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

14. 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

15. Optimum invertase activity Thermal Stability: Invertase is stable at 30–50 o C As temperature arise gradually decrease the activity of the enzyme probably due to the destruction of the enzyme structure. Finally , at 70 o C enzyme inactivated completely. Carbohydrate group in the structure of invertase led to increase the thermostability . This property makes themideal for industrial application 65 o C is the optimum temperature of invertase activity produced by Aspergillus niger The most production of the enzyme was gained at 30 o C , but in 60 o C the lowest production of the enzyme was discovered

16. Optimum invertase activity pH and Temperature There is a relation between pH and temperature . pH 3.0, temperature 60 o C and pH 4.6, temperature 55 o C 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. Questions (if any)