Production of Industrial Enzymes & Their Applications PDF

SITI NUR HAZWANI OSLAN, PHD FACULTY OF FOOD SCIENCE AND NUTRITION, UMS PRODUCTION OF INDUSTRIAL ENZYMES AND THEIR APPLICATIONS  INTRODUCTION ▪ Enzymes are proteins specialized to catalyze biological reactions. TIME EVENTS 2000 BC Egyptian and Sumerians developed...

SITI NUR HAZWANI OSLAN, PHD FACULTY OF FOOD SCIENCE AND NUTRITION, UMS PRODUCTION OF INDUSTRIAL ENZYMES AND THEIR APPLICATIONS  INTRODUCTION ▪ Enzymes are proteins specialized to catalyze biological reactions. TIME EVENTS 2000 BC Egyptian and Sumerians developed fermentation for use in brewing, bread making and cheese making. 800 BC Calves’ stomach and the enzyme, chymosin, used for cheese making. 1878 Yeast cells which cause fermentation were identified and the term ‘enzyme’ has first named meaning ‘in yeast’ in Greek. 1926 Enzymes were first shown to be protein. 1980 Enzyme preparation was developed to improved the digestibility and nutrient availability for animal feed. 2  Microbial sources are preferred ▪ Over 2000 different enzymes are available and only few are used commercially. ▪ Commercial enzymes include enzymes from plant, animal and microbial sources. ▪ From 1960s microbial source is preferred for several reasons, o Economical – produced on large scale within limited space and time. o Can be easily extracted and purified. o Can grow in a wide range of environmental conditions o Capable of producing a wide variety of enzymes. o They can be genetically manipulated to increase the yield of enzymes 3 Sources of enzymes Enzymes are obtained from Plant source – Papain, ficin Animal source – Rennet (chymosin), trypsin Microbial source – amylase, proteases, cellulase, xylanase etc. 4  Microbial Enzymes vs Chemical Advantages of using microbial enzymes over traditional chemical methods for industrial applications: ü Enzymes did not produce hazardous byproducts. ü Enzymes work under reasonable conditions, specific enzyme activity towards specific substrate. ü Enzymes are biodegradable. ü Enzymes are not toxic.  Industrial Enzymes Industry Environment Agriculture Leather tanning Degradation Animal feed Starch processing of residual additive waste Detergent Feed Detoxification of processing Textile toxic substances Chemical/ Agroprocessing Pharmaceutical Food processing Protein hydrolysis Biofuel  Where enzymes are Application Enzymes Uses Food processing Amylase, protease To produce sugars and to digest the proteins in flour Baby foods Trypsin To predigest baby foods Brewing industry Amylase, glucanases, To degrade proteins and proteases, polysaccharides, improve the wort Acetolactatedecarboxylase and fermentation process, increase the (ALDC) flavour. Fruit juices Cellulases, pectinases Clarify fruit juices Dairy industry Rennin, lipase, lactase Production of cheese and other diary products Meat tenderizers papain To soften meat for cooking. Starch industry Amylase, glucoisomerases Convert starch into glucose and simple sugars Paper industry Amylase, cellulase, xylanase, ligninases Degrade starch, aid in sizing, decolorizing, soften the paper Biofuel industry Cellulase, xylanase, ligniniase, lipase Production of ethanol and biodiesel Detergent industry Amylase, protease, lipase, cellulase Remove starch, protein and lipid stains and as fabric conditioner Photographic industry ficin Dissolve gelatin off scrap film, allowing recovery of its silver 5 content. Role of Enzymes in Food Fermentation Enzymes are biological catalysts that speed up biochemical reactions. In food fermentation, enzymes are critical for breaking down complex molecules, facilitating the fermentation process, and contributing to the final product's characteristics. How Enzymes Function In Food Fermentation 1.Breakdown of Carbohydrates: 1. Amylases: These enzymes break down starches into simple sugars (maltose, glucose) that can be fermented by yeast or bacteria. In the brewing industry, for instance, amylases convert starch in grains into fermentable sugars to produce alcohol. 2. Invertase: This enzyme hydrolyzes sucrose into glucose and fructose. It’s crucial in processes like brewing and in the production of certain confections. 2.Proteolysis (Breakdown of Proteins): 1. Proteases: These enzymes break down proteins into smaller peptides or amino acids. In dairy fermentation, proteases like rennet are used to coagulate milk proteins, forming curds in cheese- making. Proteases also tenderize meat products during fermentation and improve their texture. 3.Lipid Breakdown: 1. Lipases: These enzymes break down fats into glycerol and free fatty acids. In dairy products like cheese, lipases enhance the development of flavor, especially in aged cheeses. Lipases are also used in the production of fermented fish sauces. 4.Fermentation and Alcohol Production: 1. Enzymes are integral to alcoholic fermentation. Yeast ferments sugars into alcohol, with enzymes like glucokinase and alcohol dehydrogenase facilitating the conversion of glucose into ethanol and carbon dioxide. In the production of alcoholic beverages such as wine, beer, and spirits, enzymes like amylase and glucose oxidase play a key role in converting starch into fermentable sugars, while alcohol dehydrogenase helps convert these sugars into alcohol.  How the yields can be increased? The yield must be increased in order to minimize the production cost. This can be done by; (i) developing a suitable medium for fermentation (ii)refining the fermentation process and (iii) improving the strain for higher production. 1 1 Strain improvement □ The potential productivity of the organisms is controlled by its genes and hence their genome must be altered for the maximum production of enzymes. □ The techniques involved are ❖ Mutations ❖ Recombination – Protoplast fusion ❖ Recombinant DNA technology 1 2  Mutations □ One of the most successful approaches for strain improvement. □ A mutation is any change in the base sequence of DNA - deletion, insertion, inversion, substitution. □ The types include -Spontaneous mutation -Induced mutation -Site directed mutation 1 3  Types of mutations 1.Spontaneous mutation: Occur spontaneously at the rate of 10-10 and 10-15 per generation and per gene. Occur at low frequency and hence not used much in industrial strain improvement. 2. Induced mutation: The rate of mutation can be increased by various factors and agents called mutagens. ionizing radiations (e.g. X-rays, gamma rays) non-ionizing radiations (e.g. ultraviolet radiations) various chemicals (e.g. mustard gas, benzene, ethidium bromide, Nitrosoguanidine-NTG) 9  Types of mutations 3. Site directed mutations(SDM) (site-specific mutagenesis): □ Change in the base sequence of DNA □ changing the codon in the gene coding for that amino acid. □ Can be done by protein engineering method □ Desired improvements might be increased thermostability altered substrate range reduction in negative feedback inhibition altered pH range, etc., 15  Production of enzymes Stages involved in commercial production of enzymes: I. Isolation of microbes. II. Screening of microbes. III. Fermentation. IV. Increase the yield of the enzymes. 16 Enzyme production Cell integration: Process of breaking cell wall and/or membrane to release intracellular fluids containing molecules or particles of interest 1 2 Process of Enzyme Production 1 8  Enzyme production Cell integration: Process of breaking cell wall and/or membrane to release intracellular fluids containing molecules or particles of interest 1 3 Products of enzyme technology Micro-organisms have been used for thousands of years for making products such as wine, beer, vinegar, soy sauce, bread and cheese The micro-organisms (such as yeast) are really used as a source of enzymes during the manufacture of these products of biotechnology 1 4 1 5  Large Scale Production of Enzymes ✔ The large scale production of enzymes involves culturing microorganisms in chambers called FERMENTERS or BIOREACTORS ✔ Micro-organisms are suitable for use in the large scale production of enzymes in fermenters because: They have rapid growth rates and are able to produce larger numbers of enzyme molecules per body mass than many other organisms Micro-organisms can be genetically engineered to improve the strain and enhance yields Micro-organisms are found in a wide variety of different habitats such that their enzymes are able to function across a range of temperatures and pH Micro-organisms have simple growth requirements and these can be precisely controlled within the fermenter Micro-organisms can utilise waste products such as agricultural waste as substrates 1 6 1 7  Commercial enzyme production Production of Pectinase Pectin is an insoluble substance found in the cell walls of plants In the drinks industry, juice extracted from fruits appears cloudy due to the presence of pectin Pectinase is an enzyme that is used in the industry to break down the pectin The effect of pectinase is to clarify the fruit juice and to make it flow more freely Pectinase is obtained from the fungus Aspergillus niger Aspergillus niger produces pectinase as an extracellular enzyme 1 9 2 0  Enzymes in Biotechnology ∙ Enzymes are used in industrial processes and as analytical reagents in medicine ∙ Thermostability and an ability to withstand extremes of pH are essential properties for enzymes used in many industrial processes ∙ Immobilisation of enzymes is an important technique used in industry as it enables economical operation of a process and protection of enzymes during their use ∙ Because of their sensitivity and specificity, enzymes are used as analytical reagents in systems such as the detection of glucose in human blood and urine 21  Immobilized Enzymes ∙ For some biocatalytic processes, it is desirable to fix soluble enzymes onto a solid surface. These are called immobilized enzymes ∙ The costs associated with the use of enzymes for industrial purposes can also be reduced by immobilising the enzymes ∙ Enzymes for industrial processes are more valuable when they are able to act in an insolubilized state rather than in solution ∙ Enzymes are immobilised by binding them to, or trapping them in a solid support ∙ Various methods for immobilising enzymes are available 22 Methods for Immobilizing 23  Advantages of immobilizing enzymes ✔ Compared with free enzymes in solution, immobilised enzymes have a number of advantages for use in industrial processes ✔ The stability of many enzymes is increased when they are in an immobilised state; they are less susceptible to changes in environmental conditions such as temperature and pH fluctuations ✔ Immobilised enzymes can be recovered and re- used, reducing overall costs ✔ The products of the reaction are not contaminated with enzyme eliminating the need to undertake costly separation of the enzyme from the product ✔ Immobilising enzymes allows for continuous production of a substance with greater automation 24  Enzyme immobilization in production of High Fructose Syrup (HFS) ∙ This industrial process involves the conversion of cheap corn starch into a high fructose syrup for use as a sweetener in confectionary and drinks ∙ Starch paste is incubated with the thermostable enzyme alpha amylase at 90oC for a couple of hours ∙ The temperature is raised to 140oC to denature the amylase and then lowered to around 55oC before adding the fungal enzyme amyloglucosidase Enzyme Immobilisation in Production of HFS....contd ∙ The final stage involves the conversion of glucose syrup into the much sweeter fructose syrup using the enzyme glucose isomerase ∙ Glucose isomerase is immobilised in rigid granules and packed into a column ∙ Glucose syrup is poured into the top of the column and is hydrolysed as it contacts the immobilised enzyme  Enzymes as Industrial Products ∙ Microorganisms are ideal for the large-scale production of enzymes. Many enzymes are used in the laundry industry to remove stains from clothing, and thermostable and alkalistable enzymes have many advantages in these markets. ∙ Certain enzymes are produced in large amounts by some organisms, and instead of being held within the cell, they are excreted into the medium. ∙ These extracellular enzymes, called exoenzymes, can digest insoluble polymers such as cellulose, protein, and starch. The products of digestion are then transported into the cell where they are used as nutrients for growth. ∙ The term extremozyme has been coined to describe enzymes that function at some environmental extreme, such as high temperatures or low pH. Enzymes from extremophiles are desirable for biocatalyses under extreme conditions. 32  EnzymesEnzymes are used in biological are used in biological washingwashing powderspowders ¨Proteases break down the coloured, insoluble proteins that cause stains (e.g. blood and egg) to smaller, colourless soluble polypeptides. ¨Lipases break down fats in grease stains (e.g. Butter and mayonnaise) ¨Can wash at lower temperatures ¨Enzymes do not harm the environment  Enzymes as Industrial Products ∙ Microorganisms are ideal for the large-scale production of enzymes. Many enzymes are used in the laundry industry to remove stains from clothing, and thermostable and alkalistable enzymes have many advantages in these markets. ∙ Major Industrial Products for the Food and Beverage Industries - Alcohol and Alcoholic Beverages ∙ Alcoholic beverages are produced by yeast from the fermentation of sugar to ethyl alcohol and CO2. ∙ Wine is produced from grape juice, beer from fermentation of malted grain (brewing), and distilled beverages from the distillation of fermented solutions. ∙ Commodity alcohol is used as a gasoline additive and industrial solvent. ∙ The active ingredient in vinegar is acetic acid, which is produced by acetic acid bacteria oxidizing an alcohol-containing fruit juice 34 4 2 Yeast as a Food and Food Supplement 45 Thank you 47