CUBT402 - PRODUCTION OF ORGANIC ACIDS AND ENZYMES.pdf

Food Biotechnology CUBT402 Production Of Organic Acids And Enzymes Chinhoyi University of Technology - Department of Biotechnology  Content Citric acid Gluconic acid Fumaric acid Enzymes  Food Biotechnology CUBT402 Production Of Organic A...

Food Biotechnology CUBT402 Production Of Organic Acids And Enzymes Chinhoyi University of Technology - Department of Biotechnology  Content Citric acid Gluconic acid Fumaric acid Enzymes  Food Biotechnology CUBT402 Production Of Organic Acids Chinhoyi University of Technology - Department of Biotechnology  Introduction A large number of organic acids with actual or potential uses are produced by microorganisms These include: citric, itaconic, lactic, malic, tartaric, gluconic, mevalonic, salicyclic, gibberelic, diamino-pimelic and propionic acids  Citric Acid A weak organic acid It is a natural In biochemistry, the conjugate base of citric acid, citrate, is important as an intermediate in the citric acid cycle About1.5 million tonnes of citric acid are produced every year by fermentation  Structure and properties of citric acid Citric acid is a tribasic acid Crystallises with the large rhombic crystals containing one molecule of water of crystallisation, which is lost when it is heated to 130°C At high temperatures of about 175°C, it is converted to itaconic acid, aconitic acid, and other compounds H2C - COOH OH – C – COOH H2C - COOH  Biochemistry of citric acid production Citric acid is an intermediate in the citric acid cycle (TCA) It is caused to accumulate by one of the following methods: (a) Mutation – giving rise to mutant organisms which may only use part of a metabolic pathway, or regulatory mutants; that is using a mutant lacking an enzyme of the cycle (b) Inhibiting the free-flow of the cycle through altering the environmental conditions, e.g. temperature, pH, medium composition (especially the elimination of ions and cofactors considered essential for particular enzymes) The Tricarboxylic Acid Cycle (TCA)  Fermentation for citric acid production Citric acid accumulates during the controlled fermentation by some species of Penicillium and Aspergilius - A.niger, A.clavatus, A. wenti, P. leutum, P. citrinum, etc Commercial production based on carbohydrate medium e.g. molasses and various strains of A. niger and occasionally A. wenti A. niger can accumulate high amounts of citric acid on carbohydrate medium Penicillium not commercially used because of low productivity Some yeasts e.g. Candida spp. (including C. quillermondi now promising Paraffins used as substrate from around 1970s Fermentation of molasses and other sugars can be either surface or submerged Fermentation with paraffins is submerged  Choice of fungal strain: It is important to choose a stable fungal strain A. niger - commercially used strain Great variation in terms of morphology and physiology in A. niger strains Strains carefully selected for positive characteristics: Efficient strains (high yield) High sporulation activity Posses uniform biochemical properties and highly stable Produce small amounts of oxalic acid Easily cultivable  Choice of medium: Selection of favourable fermentation medium- most critical factor in obtaining high-level accumulation of citric acid by A. niger Nutrient deficiency in terms of trace metals or phosphate is required This may vary with the strain used Medium should be slightly deficient in phosphates or one or more of the metals Mn, Fe, Zn and Cu  Surface fermentation: Culture is inoculated on the surface of the production media Stationary fermentation Old method but still practiced Inoculation preparation: spores of A. niger inoculated in shallow pans at 25 C for 4 -14 days In Japan, it is performed on rice bran or in liquid solution in flat aluminium or stainless steel pans using A. niger Special strains of A. niger which can produce citric acid despite the high content of trace metals in rice bran required  Cont.. Sucrose (>15%) is considered the best source for citric acid production – excess sucrose remains unconverted Essential amounts of inorganic salts: N, P, S, Mg salts are added The type and quantity of ions depends on the fungal strain e.g., A.niger 62 - 0.1mg/L of Fe while A. niger 59 requires 10 mg/L of Fe for optimum productivity pH maintained at 3.4-3.5 using HCl Proper aeration is required to maintain continuous citric acid production  Cont.. Fermentation lasts for 7-10 days Yields: 60-80 gms/ 100 gms of sugar incorporated Citric acid is extracted from the bran by leaching It is then precipitated from the resulting solution as calcium citrate  Submerged fermentation: Fermenter made of acid-resistant materials e.g. stainless steel Fungus is grown dispersed in liquid medium Pre-treated beetroot molasses, sucrose or glucose used as carbon source MgSO4, 7H2O and KH2PO4 at about 1% and 0.05-2%, respectively are added The pH is maintained at less than 3.5 Cu (500 ppm) is used as an antagonist of the enzyme aconitase which requires Fe  Cont.. 1-5% of methanol, isopranol or ethanol added to unpurified substrates to increase yield Yields are reduced in media with unpurified materials Mechanical agitation not required - high aeration is deleterious to citric acid production - Air may be bubbled through and anti-form is added Fungus occurs as a uniform dispersal of pellets in the medium C. quillermondii utilises sugar medium while C. lipolytica uses paraffin Fermentation lasts for 5 to 14 days and yield up to 110 g/L  Extraction of citric acid Broth is filtered until clear Calcium citrate is precipitated by the addition of magnesium-free (Ca(OH)2) Since Mg is more soluble than Ca, some acid may be lost in the solution as magnesium citrate if Mg is added Calcium citrate is filtered and the filter cake is treated with sulphuric acid to precipitate the Ca Dilute solution containing citric acid is purified by treatment with activated carbon and passing through iron exchange beds Purified dilute acid is evaporated to yield crystals of citric acid Further purification may be required to meet pharmaceutical stipulations  Uses of citric acid Used in the food industry, in medicine, pharmacy and in various other industries Food industry: Food acidulant in the manufacture of jellies, jams, sweets and soft drinks Artificial flavouring in various foods including soft drinks Used in processed cheese manufacture Cosmetic industry Used in astringent lotions (e.g. aftershave lotions), hair rinses and hair wig setting fluids  Cont.. Medicine and pharmacy: Sodium citrate is used in blood transfusion and bacteriology to prevent blood clotting Used in efferverscent powers which depend for their efferverscence on CO2 produced from the reaction between citric acid and sodium bicarbonate Rapidly and completely metabolised in the human body and can therefore serve as a source of energy  Cont.. Other uses in industry: Widely used in electroplating, leather tanning and in removal of Fe clogging the pores of the sand face in old oil wells Recently used to replace phosphates in the manufacture of detergents Phosphates in effluents leads to eutrophication  Fumaric Acid It is a naturally occuring organic acid also known as (E)-2-butenedioic acid or trans-1,2-ethylenedicarboxylic acid Produced chemically from maleic anhydride, a product butane Rising costs and climate changes issues of petro-based fumaric acid Fermentation by filamentous fungi an alternative source Most microbes produce fumaric acid in small amounts as an intermediate of TCA cycle Only Mucorales and Rhizopus accumulate fumaric acid Rhizopus nigricans used for commercial production  Cont.. Strain selection is very important: Some strains of Rhizopus don’t produce fumaric acid Others produce mixture of fumaric and lactic acid Some species of Rhizopus e.g. R. nigricans lack invertase activity- cannot utilise sucrose directly- molasses can be used as C-source  Biochemical pathway of fumaric acid Citrate cycle reductive carboxylation pathways leading to fumaric acid accumulation  Fermentation process of fumaric acid Highly aerobic surface or submerged fermentation Deficiency of oxygen leads to accumulation of ethanol Fermentation occurs at 28 to 30 ºC Medium contains hexoses, salts and ammonia or urea C/N ratio of compounds in the medium controls the yields Amounts of trace elements is critical - Zn should be limiting Excess of Zn leads to formation of other organic acids  Cont.. Flowchart for fumaric acid production via fermentation. a) Formulation tanks containing glucose and nutrients; b) Seed fermentor; c) Production fermentor; d) Filter; e) Acidification tank; f) Filter; g) Rotary dryer  Recovery of fumaric acid Fumaric acid is poorly soluble in water and saturation occurs at 0.7 g/100mL Fumaric acid crystallises in medium to form a gel This gel thickens the media and coats the mycelium: slows or stops fermentation R. nigricans cannot withstand high acidic conditions Na or KCO3 is added to neutralise and maintain pH between 5-6, and prevent crystallisation CaCO3 cannot be used as Ca fumarate also crystallises forming gel The harvested broth containing sodium fumarate is filtered to remove cells and acidified by sulphuric acid so that the acid crystallises Fumaric acid will then precipitates out of the solution and sent to a rotary dryer to be completely recovered  Applications of fumaric acid Used in the polymer industry for the manufacture of resins Can be converted maleic acid by heating in acid solution Maleic acid used in manufacture of alkyd resins, unsaturated polyester coating compounds and plasticisers  Cont.. Engel et al., 2008  Gluconic Acid Gluconic acid is a noncorrosive, nonvolatile, non-toxic, mild organic acid Produced by physico-chemical (catalytic oxidation, chemical oxidation, electrolytic oxidation) and biological methods (fermentation) Fermentation is a widely accepted method of gluconic acid production Gluconic acid is produced by fermentation technology using enzyme glucose oxidase, that converts glucose to gluconic acid, or the whole organisms that produce this enzyme (several fungi and bacteria) Microbial production is the preferred method The most studied and widely used fermentation process involves A. niger  Cont.. Formula of gluconic acid (A) and glucono--lactone (B) Metabolic pathway of gluconic acid  Production Of Gluconic Acid Gluconic acid (pentahydroxycaproic acid) is produced from glucose through a simple dehydrogenation reaction catalysed by glucose oxidase Oxidation of the aldehyde group on the C-1 of -D-glucose to a carboxyl group results in the production of glucono-- lactone (C6H10O6) and hydrogen peroxide (H2O2) Glucono- -lactone is further hydrolysed to gluconic acid either spontaneously or by hydrolysing enzyme H2O2 is decomposed to water and oxygen by peroxidase  Cont.. Steps of reaction involved:  Microorganisms in gluconic acid production Hyperproducer strains preferred to enhance yield and purity of gluconic acid Gluconic acid is a major metabolite produced from glucose by fungi like Aspergillus, Penicillium, Mucor, Fusarium and Pullularia  Bacterial genera like Gluconobacter, Pseudomonas and Achromobacter A. niger is currently the most widely used Read on gluconic acid production from Gluconobacter  Process conditions for A. niger Organisms were cultivated by submerged fermentation Glucose is the C-source at 110 – 250 g/L Nitrogen and phosphorus sources at a very low concentration (20 mM) Very high aeration and agitation rates Stirred, baffled, stainless steel or glass tanks result in higher productivity Optimum temperature and pH for A. niger are 28-30°C and 4.5-6.5 Gluconic acid production is neutralised by addition of NaOH or CaCO3 Addition of these compound results in production of Na or Ca gluconate  Reactions of A. niger A. niger produces all the enzymes required for the conversion of glucose into gluconic acid: glucose oxidase, catalase, lactonase and mutarotase  Mutarotase accelerates the conversion of -glucose monohydrate to -form in the solution Glucose oxidase undergoes self-reduction by the removal of two hydrogens  This reduced enzyme is oxidised by molecular O2 to form H2O2 as by-product  Catalase breaks H2O2 to release water and oxygen  Hydrolysis of glucono--lactone to gluconic acid is facilitated by lactonase The reaction occur spontaneously as the cleavage of lactone occurs rapidly at pH near neutral brought about by the addition of CaCO3 or NaOH  Lactone to be removed from medium is as its accumulation adversely affects the rate of glucose oxidation into its products  Gluconolactonase can be present in A. niger 54 to increase the rate of conversion of glucono--lactone to gluconic acid  Cont.. Oxidation of glucose by A. niger  Recovery of gluconic acid Recovery depends on the method followed for broth neutralisation and the nature of C-sources Gluconic acid, glucono--lactone, Ca gluconate, and Na gluconate are some of the important products Generally, the downstream process is similar for the fermentation processes of both fungal and bacterial species  Cont.. Free gluconic acid as a product: For the recovery of free gluconic acid from Ca gluconate the broth is clarified, decolorised, concentrated and exposed to –10°C in the presence or absence of alcohol Ca salt of gluconic acid crystallises, then recovered and further purified Gluconic acid can be obtained by precipitating the Ca gluconate from hypersaturated solutions in the cold and released by adding sulphuric acid This removes the Ca as calcium sulphate Passing the solution through a column containing a strong cation exchanger is also practised where Ca ions are absorbed  Cont.. Calcium gluconate as a product: Calcium hydroxide or calcium carbonate is used as the neutralising agent Added to the nutritive broth accompanied by heating and vigorous stirring  The broth is concentrated to a hot supersaturated solution of calcium gluconate followed by cooling at 20 °C  Water miscible solvents added to crystallise the compound Treatment with activated carbon facilitates the crystallisation process  Finally they are centrifuged, washed several times and dried at 80 °C  Cont.. Sodium gluconate as product: Sodium gluconate is the principal manufactured form of gluconic acid Prepared by ion exchange Sodium gluconate from the filtered fermented broth is concentrated to 45 % followed by the addition of sodium hydroxide solution raising the pH to 7.5, and drum drying Carbon treatment of the hot solution before drying process is performed to obtain a refined product  Cont.. Glucono--lactone as product: Recovery is very simple  Aqueous solutions of gluconic acid are an equilibrium mixture of glucono--lactone, glucono--lactone and gluconic acid  Glucono--lactone seperated from supersaturated solution at 30– 70°C Gluconic acid obtained at temperatures below 30°C At above 70°C, the resulting product is glucono--lactone  Applications of gluconic acid and its derivatives Components Applications Gluconic acid Prevention of milkstone in dairy industry Cleaning of aluminium cans Glucono--lactone Latent acid in baking powders for use in dry cakes and instantly leavened bread mixes Slow acting acidulant in meat processing such as sausages Coagulation of soybean protein in the manufacture of tofu For cheese curd formation and for improvement of heat stability of milk Sodium salt of Detergent in bottle washing gluconic acid Metallurgy (alkaline derusting) Additive in cement Derusting agent Textile (iron deposits prevention) Paper industry Calcium salt of Calcium therapy gluconic acid Animal nutrition Iron salt of Treatment of anaemia gluconic acid Foliar feed formulations in horticulture  Food Biotechnology CUBT402 Production Of Enzymes Chinhoyi University of Technology - Department of Biotechnology  Overview Enzymes are macromolecular biological catalysts Accelerate, or catalyse the rate of chemical reactions Molecules at the beginning of a process are substrates and the enzyme converts these into different molecules called products Microbial enzymes are the biological catalysts for the biochemical reactions leading to microbial growth and respiration, and formation of fermentation products  Types of enzymes (a) Adaptive Produced only when the need arises e.g. when a cell is deficient of a particular nutrient (b) Constitutive Always produced irrespective of the amount of a particular substrate  Location of enzymes Intra/endocellular enzymes Enzymes which are produced within the cell or at the cytoplasmic membrane Etra/exocellular enzymes Enzymes which are secreted into the fermentation medium which can attack large polymeric substances e.g. amylases, proteases and cellulolytic enzymes  Methods of enzyme production Enzymes are produced via two routes: (a) Solid-state fermentation (SSF) (b) Submerged fermentation (SmF)  Solid-state fermentation The enzyme producing culture is grown on the surface of a suitable semi-solid substrate (e.g. moistened wheat or rice bran with nutrients) Bran is mixed with solution containing nutrient salt pH is maintained at a neutral level Medium is steam sterilised in an autoclave while stirring Medium spread on metal trays up to a depth of 1-10 cm Culture is inoculated either in the autoclave after cooling or in trays High enzyme concentration in a crude fermented material  Cont.. Enzymes produced by SSF Enzyme Microorganism a- Amylase A. oryzae Glucoamylase Rhizopus spp. Lactase A. oryzae Pectinase A. niger Protease A. Niger & A. oryzae Rennet Mucor pusillus  Cont.. Advantages of SSF Low investment cost Allows the use of substrate with high dry matter content thus yields a high enzyme concentration in the crude fermented material Cultivates moulds which cannot grow in the fermenters due to wall growth Moulds allowed to develop into their natural state  Cont.. Disadvantages of SSF More space and more labour is needed Involves greater risk of infection Such systems cannot be easily automated  Submerged fermentation (SmF) Fermentation equipment used is the same as in the manufacture of antibiotics Cylindrical stainless steel tanks equipped with agitators, aerating devices, cooling systems and various ancillary equipment (foam control, pH probes, temperature, oxygen tension, etc) Good growth is not enough to obtain a higher enzyme yield Presence of inhibitors or inducers should also be checked in the medium o e.g. presence of lactose induces the production of ß- galactosidase o Inducers are expensive therefore constitutive mutants are used which do not require inducers  Cont.. Glucose represses formation of some enzymes (-amylases) oGlucose is kept at low concentration oEither added in an incremental manner or as slow metabolisable sugar (lactose or metabolised starch) Certain surfactants in the production medium increases the yield of certain enzymes oNon- ionic detergents (eg. Tween 80, Triton) are frequently used  Cont.. Advantages of SmF Minimum labour and space required Low risk of infection Can be easily automated Disdvantages of SmF Initial investment cost is very high  Enzyme recovery and purification The liquor is rapidly cooled to about 5°C so as to reduce deterioration Microorganisms removed either by filtration or by centrifugation of the refrigerated broth with adjusted pH Precipitation with acetone, alcohols or inorganic salts (ammonium or sodium sulfate) for higher purity of the enzyme In large-scale operations, salts are preferred to solvents because of explosion hazards  Enzyme packaging and finishing Packing of enzymes has become extremely important since the experience of the allergic effect of enzyme dust inhalation by detergent works Nowadays, enzymes are packaged preferably in liquid form but where solids are used, the enzyme is mixed with a filler and it is now common practice to coat the particles with wax so that enzyme dusts are not formed Enzyme propagation steps  Amylase Amylase is an enzyme that catalyses the hydrolysis of starch into sugars Found in human saliva Hydrolysis of starch with amylase first result in formation of a short polymer dextrin and then to disaccharide maltose and finally to glucose Glucose is not as sweet as fructose Glucose converted to fructose by the enzyme glucose isomerase  Types of amylase There are three types:- 1. -Amylase 2. -Amylase 3. -Amylase  -Amylase Also called as 1,4--D-glucan glucanohydrolase Ca metalloenzymes which cannot function in absence of Ca ions Enzyme that catalyses the hydrolysis of internal -1,4-glycosidic bonds in starch to yield products like glucose and maltose Amylose broken down to yield maltotriose and maltose molecules Amylopectin broken down to produce Limit dextrin and glucose molecules Found in saliva and pancreas of animals - major digestive enzyme Optimum pH in animals is 6.7–7.0 Found in plants, fungi (ascomycetes and basidiomycetes) and bacteria (Bacillus)  Cont.. Effects of -Amylases:- Liquefaction - Break down the starch polymer but does not give free sugar Saccharification - Gives free sugars Producing strains:- Bacteria – Bacillus cereus, B.subtilis, B. amyloliquefaciens, B. polymyxa, B. licheniformis, etc Fungi – A. oryzae, A. niger, Penicillum, Cephalosporin, Mucor, Candida, etc  Cont.. Applications:- Production of sweeteners for the food industry Removal of starch sizing from woven cloth Liquefaction of starch pastes which are formed during the heating steps in the manufacture of corn and chocolate syrups Production of bread and removal of food spots in the dry cleaning industry where amylase works in conjunction with protease enzymes  -Amylase Also called as 1,4--D-glucan maltohydrolase Synthesised by bacteria, fungi, and plants Primary sources of ß-Amylase are the seeds of higher plants and sweet potatoes An exo-hydrolase enzyme that acts from the non-reducing end of a polysaccharide chain by hydrolysis of -1,4-glucan linkages to yield successive maltose units During the ripening of fruit, ß-amylase breaks starch into maltose, resulting in the sweet flavor of ripe fruit Optimum pH is 4.0-5.0  -Amylase Cleaves -1,6 glycosidic bonds and the last -1,4 glycosidic bonds at the non-reducing end of amylose and amylopectin to produce glucose Most efficient in acidic conditions at an optimum pH of 3  Lipases Also called as glycerol ester hydrolases Subclass of esterases Splits fats into mono or di-glycerides and fatty acids Extracellular enzymes Mainly produced by fungi e.g. Aspergillus, Mucor, Rhizopus, Penicillium Bacteria-producing lipases include Pseudomonas, Achromobacter and Staphylococcus ssp. Yeasts like Torulopsis and Candida are also commercially used Mode of action of lipases  Cont.. Lipase production induced by adding oils and fats In some cases, fats may have adverse effects on the lipase production Glycerol, a product of lipases action, inhibits lipase formation Lipases are generally bound to the cells and hence inhibit an overproduction Addition of a cation such as Mg ion liberates the lipase and leads to a higher enzyme titer in the production medium  Applications of lipase Industry Action Product/application Detergents Hydrolysis of fats Removal of oil stains from fabrics Dairy foods Hydrolysis of milk fat, cheese ripening, Development of flavoring agents in milk, modification of butter fat cheese, and butter Bakery foods Flavor improvement Shelf-life prolongation Beverages Improved aroma Beverages Food dressings Quality improvement Mayonnaise, dressings, and whippings Health foods Transesterification Health foods Meat and fish Flavor development Meat and fish products; fat removal Fats and oils Hydrolysis Cocoa butter, margarine, fatty acids, glycerol, mono- and diglycerides Chemicals Enantioselectivity, synthesis Chiral building blocks, chemicals Pharmaceuticals Hydrolysis Specialty lipids, digestive aids Cosmetics Synthesis Emulsifiers, moisturizers Leather Hydrolysis Leather products Paper Hydrolysis Paper with improved quality Cleaning Hydrolysis Removal of fats  Pectinases Pectic enzymes include Pectolyase, Pectozyme and Polygalacturonase Breaks down pectin Pectin is a polysaccharide found in plant cell walls Pectin is the jelly-like matrix which helps cement plant cells together and in which other cell wall components such as cellulose fibrils are embedded Basic structure of a pectin consists of -1,4-linked Galactouronic acid with up to 95% of it’s carboxyl groups esterified with methanol Pectinase activated at 45-55 °C at pH range of 3.0- 6.5 Pectinase mode of action  Pectinase production strains Aspergillus niger, A. wentii, Rhizopus, etc Fermentation with A. niger: Lasts for 60-80 hours in fed batch cultures pH 3-4 Temperature 37°C Substrates: 2% sucrose and 2% pectin  Applications of pectinases Commonly used in processes involving the degradation of plant materials e.g. speeding up the extraction of fruit juice from fruits such as apples Can be used in wine production Clarification of fruit juices and grape must Maceration of vegetables and fruits Extraction of olive oil  Proteases Also called as proteolytic enzymes Protease is a mixture of peptidases and proteinases Enzymes that hydrolyse of peptide linkages into primary structure of proteins Peptide bonds links amino acids to form the final structure of a protein Proteinases are extracellular and peptidases are endocellular Second most important enzyme produced on a large-scale after amylase Mode of action of proteases  Types of proteases Based on catalytic residue:- Based on optimal pH:-  Serine proteases Acid proteases  Cysteine proteases Neutral proteases  Threonine proteases Basic proteases (or alkaline  Aspartic proteases proteases)  Glutamic proteases  Metalloproteases  Asparagine peptide lyases  Peptide lyases  Industrial production of proteases Commercially produced microbial proteases contribute to approximately 2/3 of all enzyme sales Bacteria: Bacillus, Pseudomonas, Clostridium, Proteus, and Serratia Fungi: A. niger, A. oryzae, A. flavus and Penicillium roquefortii Bacillus sp are mostly used in the commercial production of proteases Fungal proteases present a wider pH activity range- wider range of uses. Two types of proteases: (a) alkaline serine proteases and (b) acid proteases Alkaline serine proteases - Bacillus licheniformis by submerged culture method Acid proteases - fungi by either semisolid culture or submerged culture method  Applications of proteases Textile industry to remove Baking and brewing industry proteinaceous sizing Soy sauce production Degumming of silk in silk industry Synthesis of aspartame Tenderising of meat Pharmaceutical industry Used in food and dairy Therapeutics industries Photography industry Detergent industry Management of industrial Leather industry wastes --END--