Polysaccharides Biopolymers ( polyhydroxyalkanoates )

1. Polysaccharides Biopolymers (polyhydroxyalkanoates)

2. POLYSACCHARIDES Microbial polysaccharides are used for food, pharmaceutical, and medical applications: this wide range of usefulness derives from the great diversity in structural and functional properties. Applications of commercially available microbial polysaccharides including xanthan, xylinan, gellan, curdlan, pullulan, dextran, scleroglucan, schizophyllan, and cyanobacterialpolysaccharides The commercial value of polysaccharides is based on its ability to modify the flow characteristics of solutions (Rheology). They can incr viscosity and hence used as thickening and gelling agents.

3. Polysaccharides made by microrganisms are secreted from the cell to form a layer over the surface of the organism, often of substantial depth in comparison with the cell dimensions (Figure 1). • Because of their position they are characterized as exopolysaccharides, to distinguish them from any polysaccharides that might be found within the cell. • The functions are thought to be mainly protective, either as a general physical barrier preventing access of harmful substances, or more specific as a way of binding and neutralizing bacteriophage. In appropriate environments they may prevent dehydration. Exocellular or ‘capsular’ polysaccharide layer (labeled P) from Streptococcus pneumoniae

4. Prevent phagocytosis by other micro- organisms or the cells of the immune system. The capsular polysaccharides (CPSs) are often highly immunogenic, and may have evolved their unusual diversity as a way of avoiding antibody responses: advantage of this feature can be taken in the development of vaccines. • adhesion and penetration of the host; Plant lectins (glycoproteins) that have specific binding properties with respect to carbohydrate structures;general defense of plants against bacterial infection. • Polysaccharides can be involved in pathogenicity. Pseudomonas aeruginosa, commonly found in respiratory tract infections, produces alginate which contributes to blockage in the respiratory tract • polysaccharide biotechnology: xanthan, priced at about US$14 per kg, used mainly in food applications, to cyclic dextrans, valued at about US$50perkg and used in high-value applications in research and pharmaceuticals • Certain microbes are known to produce nearly all the major plant polysaccharides such as glucans, alginate-like materials, and even cellulose – as well as the complex bacteria-specific materials.

5. Production Batch culture Excess of carbon supply Carbon/nitrogen ratio of 10:1 When acidic PS limited polysaccharides are synthesized 90% oxygen saturation Biosynthesis Starting with glucose or appropriate sugar and several enzymatic reactions, Recovery Marked increase in viscosity of culture broth Can be precipitated by salts, acids or organic solvents

6. Microbial PS Plant PS 20 PS of commercial adv and value Dependent of rheological property Cheap Uncontrolled Occurs for a short duration Only microbial polysaccharide currently produced on a Large scale: XANTHAN

7. Production: 20,000 tonnes per year E415 XANTHAN: polysaccharide polymer produced by Xanthomonascampestris Long chain polysaccharide composed of the sugars glucose, mannose, and glucuronic acid. The backbone is similar to cellulose, with added sidechains of trisaccharides (3 sugars in a chain) Gram negative bacteria, plant pathogen causes black rot in Brassica (cauliflower and broccoli) produces a slimy gel that protects bacteria from viruses and prevents it from drying out. Has very high viscosity and is stable at extreme physical and chemical environments, shows physical and chemical properties like plastic, used in many ways: eg. oil drilling Pentasaccharide: Glu-man-glcA-Ac-Pyr Branched polymer with b-1,4 linked glucan (glucose polymer) backbone bound to trisaccharide (Man, GlcA, Man). Man has Acetate or Pyruvate groups (number is variable dependent on bacterial strain) Viscosity is denpendent on contents of pyruvate and acetate

8. Glucan backbone B-glc (1-4)-B-Glc (1-4) 1-3 Pyr 6 4 b-Man-(1,4)-b-GlcA- (1-2)-a-Man-6-O-Ac Trisaccharide chain

9. Commercial production of Xanthan Gum X. Campestris should be grown on a cheap and abundant source of carbon Wild type can use glucose, sucrose and starch but not lactose Whey is a waste product of cheese making industry used as filler in several foods 95% water, 3.5-4% lactose and small amounts of proteins, minerals LMW organic cpds Only advantage is that it can be used as a carbon source for growing industrial MO Due to lactose intolerance alternatives are required Disposal of whey in rivers can lead to depletion of dissolved O2 Transporation of whey to landfill sites is exceptionally expensive Groundwater leaching and contamination a concern

10. Genetic engineering of X. campestris to grow on WHEY E.coli lacZY (b-galactosidase and lac permease)cloned to broad host range plasmid under transcriptional control of X. campestris bacteriphase promoter. Introduced in E.coli and transformed to X. campestris by tripartite mating Transformants wgich maintained the plasmid expresses lacZY and used lactose as sole carbon, produced hih levels of xanthan gum with glucose, lactose or whey as carbon source. Compared to wild type which produced xanthan only when grown on glucose Helped to convert a nuisance waste product into a substrate for production of economically valuable bopolymer.

11. They are glucans (polymers of glucose) DEXTRANS A(1-6) linkages some have a-1,2 and a1,3, and 1,4 MW is 15,000-500,000 Applications: Blood plasma expanders for prevention of thrombosis Wound healing and dressing purification of biomolecules Production Microorganisms like Leuconostocmesentroides They are produced by extracellular enzymes like dextransucrase which act on sucrose and bring polymerization of glucose residues and liberates free fructose in medium Batch fermentation

12. Multi-celled Algae (divided into several groups across classification) ␣ Have a similar function as plants do on land ␣ Photosynthesize, base of food chain, provide habitat for other organisms ␣ Most live attached to rocks ␣ Structures are different from plants Division (Phylum) Chlorophyta Green algae Division (Phylum) RhodophytaRedAlgae Division (Phylum) Phaeophyta Brown algae

13. Algae Structures Photosynthesis, takes up water and nutrients Buoyancy: air filled, allows algae to float in water Photosynthesis, takes up water and nutrients Attaches algae to rock, doesn’t grow into rock, only for attachment

15. Seaweed and marine algae have been valued for centuries in Asia and the Pacific Islands for their nutritional and healing properties as they are packed with potassium, vitamins A, B, C, D, and E. They are very high in iodine content and are used to treat some thyroid conditions. A brown seaweed (kelp), is found to have antibacterial and antiviral properties and has been extensively used in clinical trials to lower blood pressure in heart patients.

16. Several marine seaweeds and submerged vegetation in seawater are reported to have antagonistic activity and have been found effective against various viruses. Polysaccacharides from marine environment Purple layer contains a sulphated polysaccharide called Porphyran, which is a complex galactan, and has shown higher gelling capacity reported to inhibit the growth of Sarcoma 180 tumors in mice. A substance named porphyosin isolated from Porphyra exhibited anti ulcer activity. 3-linked 6-O-methyl-β-D-galactopyranose residue 4-linked 6-O-sulfo-α-L-galactopyranose residue(the biological precursor of the 3,6-anhydro residue (agarose) red alga Porphyra umbilicalis

17. Agar: well known gelling agent. Gelidium spp. Gracilaria (Red seaweeds (Rhodophyceae) Agarose Agaropectin RED ALGAE (1 4)-3,6-anhydro-α-L-galactopyranosyl-(1 3)-β-D-galactopyranan Agarose is purified form of agar. Both have variety of uses in biochemistry, molecular biology, microbiology. Used as gelling agent for meats, confectionery, icing stabilizer in baked goods in food industry Used as laxative (as it cannot be metabolized) in medicinal applications Also as flexible moulds in dentistry and criminology Agar polysaccharides are effective against poliovirus, herpes simplex, dengue viruses, etc.

18. Alginates soluble in water and form gels with addition of calcium or bivalent ions Phaeophyceae Calcium ions form a link b/w G molecules If ions removed gel will break BROWN ALGAE Laminiaria spp.Sargassam spp. Alginate gels cannot be reversed and are not stable at low pH. But high viscosity and hydration make them useful for salad dressings, frozen foods, icings and film formation. Textile and paper industry uses to thicken inks, coat papers and reduce staining Enzyme immobilization

19. Calcium alginates provide a moist healing environment by converting the exudate into a gel. A reaction between the calcium in the dressing and the sodium in the wound exudate results in a chemical ion exchange, which forms a gel-like substance. The gel conforms to the wound, providing a soft, moist healing environment. Due to it’s dehydrating effect, this dressing should not be used with dry wounds. Also shows hemostatic activity Sorbsan can be easily separated and fluffed up before placing it into a wound bed Sorbsan is made from seaweed into delicate fibers and cut into unwoven sheets 30000 tonnes per year and supply much more than this

20. First in Ireland Rhodophyceae Carrageenans RED ALGAE Carrageenans are ideal food additives: they have a range of gelling and emulsifying properties ranging from a soft slime to a brittle gel that one could nearly walk upon. They also have a high reactivity with a range of materials including, most importantly, milk proteins, being widely used at low concentrations in dairy products to prevent fractionation of milk constituents.  In fact, a major application is found in chocolate milk, a very popular daily drink in the USA and Europe, and now spreading elsewhere. Carrageenans are also very good at keeping chocolate in suspension. Best quality from Philippines

22. GLUCOSE POLYMER (glucomer) SCLEROGLUCAN Neutral polysaccharide with b-1,3 glucan backbone and single glucose residue branches b (1,6) Branching occurs at a regular sequence at every 3rd glucose unit in the polymer backbone chain Scleroglucan is a fungal hexopolysaccharide Useful for stabilizing latex paints, printing inks and drilling muds GELLAN Linear heteopolysaccharide 2glu, 1GlcA and one rhamnose Pseudomonas elodea A deacetylatedgellan forms fim brittle gels : GELRITE (US company) Used in food industry as low temperature thickner

23. POLLULAN A GLUCOSE POLYMER (a-glucan) with a-1,4 and few a 1,6 70% of glucose is converted to pollulan Fungus: Aurobasidiumpollulans CURDLAN A GLUCOSE POLYMER (b-glucan) glucose hed by b-1,3 Alcaligenesfaecalis, Rhizoiumtrifolli, Agarobacteriumrhizogenes Forms strong gels ehen heated to 55oC, gelling agent, immuno modulator and immobilization of enzymes

24. Pectin α-(1-4)-linked D-galacturonic acid Pectin is a long chain of pectic acid and pectinic acid molecules. Because these acids are sugars, pectin is a polysaccharide. It is prepared from citrus peels and the remains of apples (pomace) after they are squeezed for juice. In the plant, pectin is the material that joins the plant cells together. When fungus enzymes break down the pectin in fruit, the fruit gets soft and mushy. During ripening, pectin is broken down by the enzymes pectinase and pectinesterase; in this process the fruit becomes softer as the cell walls break down.

25. Uses • Pectin is a thickener in many products. If there is sufficient sugar in the mixture, pectin forms a firm gel. • Jams and jellies are thickened with pectin. Pectin binds water, and thus keeps products from drying out. It stabilizes emulsions. • Pectin combines with the calcium and whey proteins of milk, stabilizing foams and gels made with cream or milk. • In the cigar industry, pectin is considered an excellent substitute for vegetable glue and many cigar smokers and collectors will use pectin for repairing damaged tobacco wrapper leaves on their cigars. • Pectin is also used in throat lozenges as a demulcent. In cosmetic products, pectin acts as stabilizer. Pectin is also used in wound healing preparations and specialty medical adhesives, such as colostomy devices • Pectin is not digested, and is considered a beneficial dietary fiber. In human digestion, pectin passes through the small intestine more or less intact. In the large intestine and colon, microorganisms degrade pectin and liberate short-chain fatty acids that have positive influence on health (prebiotic effect)

26. Ghum or Gum Materials Natural gums are polysaccharides of natural origin, capable of causing a large viscosity increase in solution, even at small concentrations. In the food industry they are used as thickening agents, gelling agents, emulsifiers and stabilisers. E numbers are number codes for food additives and are usually found on food labels throughout the European Union Agar (E406), obtained from seaweed Alginic acid (E400), from seaweed Beta-glucan, from oat or barley bran Carrageenan (E407), from seaweed Chicle gum, an older base for chewing gum obtained from the chicle tree Dammar gum, from the sap of Dipterocarpaceae trees Gellan gum (E418), produced by bacterial fermentation Glucomannan (E425), from the konjac plant Guar gum (E412), from guar beans Gum arabic (E414), from the sap of Acacia trees Gum ghatti, from the sap of Anogeissus trees Gum tragacanth (E413), from the sap of Astragalus shrubs Karaya gum (E416), from the sap of Sterculia trees Locust bean gum (E410), from the seeds of the carob tree Mastic gum, a chewing gum from ancient Greece obtained from the mastic tree Psyllium seed husks, from the Plantago plant Sodium alginate (E401), from seaweed Spruce gum, a chewing gum of American Indians obtained from spruce trees Tara gum (E417), from the seeds of the tara tree Xanthan gum (E415), produced by bacterial fermentation

27. Beta-glucan, from oat or barley bran polysaccharides occurring in the bran of cereal grains, the cell wall of baker's yeast, certain types of fungi, and many kinds of mushrooms. The cereal based beta-glucans occur most abundantly in barley and oats and to a much lesser degree in rye and wheat. They are useful in human nutrition as texturing agents and as soluble fiber supplements, but problematic in brewing as excessive levels make the wort too viscous. Yeast derived beta glucans are notable for their immunomodulatory function. The differences between soluble and insoluble beta glucans are significant in regards to application, mode of action, and overall biological activity.

28. Beta 1,3-D glucans are being referred to as biological response modifiers because of their ability to activate the immune system. However, it should be noted that the activity of Beta 1, 3-D glucan is different from agents that stimulate the immune system. Agents that stimulate the immune system can push the system to over-stimulation, and hence are contraindicated in individuals with autoimmune diseases, allergies, or yeast infections. Beta 1, 3-D glucans seem to make the immune system work better without becoming overactive. They accomplish this by activating phagocytes, which are immune system cells whose function is to trap and destroy foreign substances in our bodies such as bacteria, viruses, fungi, and parasites. In addition to enhancing the activity of phagocytes, beta-1,3 glucans also reportedly lower elevated levels of LDL cholesterol, aid in wound healing, help prevent infections, enhance NK cell function, and help in the prevention and treatment of cancer

29. Chicle gum is the natural gum from Manilkara chicle, Wrigley Company was a prominent user of this material, today there are only a few companies that still make chewing gum from natural chicle. This is because by the 1960s chicle was replaced by butadiene-based synthetic rubber which was cheaper to manufacture. Gellan gum: is a water-soluble polysaccharide produced by Sphingomonas elodea Also known commercially as Phytagel™ or Gelrite®, is used primarily as a gelling agent, alternative to agar, in microbiological culture. It is able to withstand 120 °C heat, making it especially useful in culturing thermophilic organisms. One needs only approximately half the amount of gellan gum as agar to reach an equivalent gel strength, though the exact texture and quality depends on the concentration of divalent cations present

30. Dammar Gum or Jhuna used as room freshner, control of mosquitoes and in worshiping Glucomannanis mainly a straight-chain polymer, with a small amount of branching. The component sugars are β-(1→4)-linked D-mannose and D-glucose in a ratio of 1.6:1. The degree of branching is about 8% through β-(1→6)-glucosyl linkages. Konjac

31. Guar gum, also called guaran, is a galactomannan. It is primarily the ground endosperm of guar beans. The guar seeds are dehusked, milled and screened to obtain the guar gum. It is typically produced as a free flowing, pale, off-white colored, coarse to fine ground powder. Guar gum retards ice crystal growth non-specifically by slowing mass transfer across the solid/liquid interface. It shows good stability during freeze-thaw cycles

32. Psyllium seed husks, plantago Isabgol

33. Animal Biomass: Chitin

34. b-1,4 linkage N-acetylglucosamine Chitin is a long-chain polymer of a N-acetylglucosamine, a derivative of glucose. It is the main component of the cell walls of fungi, the exoskeletons of arthropods, such as crustaceans (like the crab, lobster and shrimp) and the insects, including ants, beetles and butterflies, the radula of mollusksand the beaks of the cephalopods, including squid and octopuses. Chitin has also proven useful for several medical and industrial purposes. Chitin is a biological substance which may be compared to the polysaccharide cellulose and to the protein keratin. Although keratin is a protein, and not a carbohydrate like chitin, both keratin and chitin have similar structural functions.

35. Chitin is the second most abundant polysaccharide in nature (after cellulose). At least 10 gigatons of chitin are synthesized and degraded each year in the biosphere. Chitin is translucent, pliable, resilient and quite tough. In arthropods, however, it is often modified, becoming embedded in a hardened proteinaceous matrix, which forms much of the exoskeleton. In its pure form it is leathery, but when encrusted in calcium carbonate it becomes much harder

36. Chitin is one of many naturally occurring polymers. Its breakdown may be catalyzed by enzymes called chitinases, secreted by microorganisms such as bacteria and fungi, and produced by some plants. Some of these microorganisms have receptors to simple sugars from the decomposition of chitin. If chitin is detected, they then produce enzymes to digest it by cleaving the glycosidic bonds in order to convert it to simple sugars and ammonia.

37. Chitosan (a more water-soluble derivative of chitin). It is also closely related to cellulose in that it is a long unbranched chain of glucose derivatives. Commercially derived from shrimps

38. Preparation of chitin and chitosan

39. Biotechnological Uses of Chitinolytic enzymes Chitin has a broad range of applications in biochemical, food, and various chemical industries. It has antimicrobial, anticholesterol and antitumor activities Chitin and its related materials are also used in wastewater treatment drug, wound healing, and dietary fiber Industrial (water purification, stabilizer, pharmaceuticals) Medicinal (surgical threads, wound healing, role in immune response to allergic diseases) Agricultural (inducer of plant immune system and hence enhance defense mech.; fertilizer

40. Chitinases In vertebrates, chitinases are usually part of the digestive tract. In insects and crustaceans, chitinases are associated with the need for partial degradation of old cuticle. Implicated in plant resistance against fungal pathogens because of their inducible nature and antifungal activities invitro (Chitinase in fungi is thought to have autolytic, nutritional, and morphogenetic roles. In viruses, chitinases are involved in pathogenesis In bacteria, chitinases play a role in nutrition and parasitism. In addition to the above potential applications, chitinases can be used for the production of chitooligosaccharides, which have been found to function as antibacterial agents, elicitors of lysozyme inducers, and immunoenhancers Chitinases can also be used in agriculture to control plant pathogens