Dr Diptendu Sarkardiptendu81@gmail.com

Enzymes Production

Source: Fermentation Microbiology and Biotechnology By EMT Mansi et al

Industrial Microbiology : An Introduction By MJ Waites

Industrial Microbiology By HS Patel

Food and Industrial Microbiology By Raveendra Reddy

Biologically active enzymes may be extracted from any living organism:

Of the hundred enzymes being used industrially,

- over a half are from fungi

- rest are from bacteria , animal (8%) and plant (4%) sources .

Sources of Enzymes

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Enzyme Production

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Microbes are preferred than plants and animals as sources of enzymes because:

- They are generally cheaper to produce.

- Their enzyme contents are more predictable and controllable.

- Plant and animal tissues contain more potentially harmful materials than microbes, including phenolic compounds (from plants).

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Enzyme EC Sources Application

a-Amylase 3.2.1.1 Aspergillus E Baking

Catalase 1.11.1.6 Aspergillus I Food

Cellulase 3.2.1.4 Trichoderma E Waste

Dextranase 3.2.1.11 Penicillium E Food

Glucose oxidase 1.1.3.4 Aspergillus I Food

Lactase 3.2.1.23 Aspergillus E Dairy

Lipase 3.1.1.3 Rhizopus E Food

Rennet 3.4.23.6 Mucor miehei E Cheese

Pectinase 3.2.1.15 Aspergillus E Drinks

Protease 3.4.23.6 Aspergillus E Baking

E: extracellular enzyme; I: intracellular enzyme

Fungal Enzymes

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Enzyme Sources Application

a-Amylase 3.2.1.1 Bacillus E Starch

b-Amylase 3.2.1.2 Bacillus E Starch

Asparaginase 3.5.1.1 Escherichia coli I Health

Glucose

isomerase5.3.1.5 Bacillus I

Fructose

syrup

Penicillin amidase 3.5.1.11 Bacillus IPharmaceutic

al

Protease 3.4.21.14 Bacillus E Detergent

Bacterial Enzymes

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1. Solid substrate cultivation : Adopted for the extraction of enzymes from fungi,such as Penicillum, aspergillus sp.,.

• This is also called Tray cultivation or thin layer culture or Koji fermentation.

• Microbes are culture in a tray which are 2x 40 cm in size.

• Trays are made up of wood or metal.

• Trays are filled with raw materials/ nutrients (1/4 th of the depth).

• Organisms are inoculated on the solid medium and incubated on the soild medium.Incubated at an air-conditioned room or in incubator for few days.

• Dense mat of microbes is formed over the surface of solid medium.

• Mat is then separated and used for the extraction of enzymes.

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Enzyme production

2. Deep-Bad cultivation: microbes are cultivated in Rectangular culture vessels,with the size of 18x200’’.

• Wheat bran, rice bran, potato flakes etc., are used as substrate.

• Any one substrate is added into vessel upto 2’’ hight.

• Microbial inoculum poured over medium.

3. Solid state cultivation: microbes are cultured in a fermenter in order toestablish a continuous culture.

• Media is used rare in semi-solid form.

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The maximum enzyme production is usually in stationary phase of microbe

growth, so a batch or fed-batch process are usually used.

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• The medium must be chosen to stimulate the microbe into synthesizing the correct enzyme.

• For example to stimulate a microbe to synthesise amylase enzymes, a medium with starch but no sugars is used.

• What type of medium would you use to stimulate a microbe to synthesize a protease?

I. A medium with proteins but no amino acids is used.

II. Microbes are encouraged into the log phase initially with a medium with a lot of protein

III. This encourages rapid increase in the number of cells, but not much protease is produced.

IV. Cells are then introduced into the fermentation vessel and allowed to grow for a further 1-8 days.

The large scale production of enzymes involves culturing micro-organisms in chambers calledFERMENTERS 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

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MODIFICATION – possibleapplication of genetic

engineering to improvethe microbial strain

LABORATORY SCALE PILOT– to determine the optimumconditions for growth of the

Micro-organism

PILOT PLANT – small scalefermenter to clarify optimum

operating conditions

SCREENING – choosing anappropriate micro-organism

for the desired enzyme

INDUSTRIAL SCALEFERMENTATION

The Biotechnological Process of Enzyme Production

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Down stream processing

The remaining mixture contains enzymes, waste materials, nutrients and cells

The enzyme is extracted by downstream processing The nature of the downstream processing depends on two considerations:

Whether enzyme is intracellular or extracellular How pure the final product needs to be.

Industrial enzymes can be quite crude, but medicinal enzymes must be extremely pure.

The purer the enzyme, the more complex the downstream processing, and the more expensive it is.

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Considerations when selecting a strain:• Does it do what is required

• Is it safe

• Is it cost effective

Enzymes may be intracellular or extracellular.

What is the advantage of extracellular production?• Already outside cell

• Limited number secreted so easier to isolate

• More robust so less likely to be broken down by heat of chemicals

FEW FACTS

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Why are intracellular enzymes more difficult to isolate than extracellular ones?

• Because they are inside the cell, first the cell has to be broken open then the enzyme separated from the mixture of all the cellular contents.

Why is it more efficient to use isolated enzymes than whole cells ?

• Isolated enzymes are usually more efficient in biotechnology than whole cells, because enzyme concentration is higher and no unwanted enzymes are present.

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Extracellular enzyme

Intracellular enzymeBreak open cells by

grinding or ultra-sonics

Filter

Enzyme in solution

Concentrate by evaporation at low temperature and pressure or by

osmosis

Crude enzyme in solution eg protease in chemical industry

Precipitate Powdered crude enzyme eg pectinase

ChromatographyPure enzyme for medicine eg

glucose oxidase

Cell biomass (useful waste product)

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Uses of enzymes

• commercial uses- e.g. biological washing powders

• medical uses- e.g. biosensors, therapeutic enzymes, drugs

• industrial uses - e.g. bioconversion, fruit juice extraction, sweeteners

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Enzymes are being used more and more for industrial bioconversion i.e. making a chemical product using purified enzymes rather by pure chemical methods (e.g. citric acid production) or using whole cells (e.g. yeast in brewing).

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Large scale enzyme applications

Detergents

• Detergents were the first large scale application for microbial enzymes.

Bacterial proteinases are still the most important detergent enzymes.

Some products have been genetically engineered to be more stable in the hostile environment of washing machines with several different chemicals present.

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• Lipid degrading enzymes, lipase, were used in powder and liquid detergents to decompose fats.

• Lipase is produced in large scale by Aspergillusoryzae host after cloning the Humicola gene into this organism.

• Amylases are used in detergents to remove starch based stains.

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• Cellulases have been part of detergents since early 90s. Cellulase is actually an enzyme complex capable of degrading crystalline cellulose to glucose.

• In textile , cellulases remove cellulose microfibrils, which are formed during washing.

• Alkaline cellulases are produced by Bacillus strains and neutral and acidic cellulases by Trichoderma and Humicola fungi.

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Large scale enzyme applicationsFoods/Beverages produced by Microbial Activity

• Yogurt, cheese, chocolate, butter, pickles, sauerkraut, soy sauce, food supplements (such as vitamins and amino acids), food thickeners (produced from microbial polysaccharides), alcohol (beer, whiskeys, wines) and

silage for animals are all products of microbial activity.

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• The industrial microbiologist/ biotechnologist may be involved in producing concentrated microbial inocula for fermentations or the maintenance of fermentation systems utilized in production

facilities.

• The use of starch degrading enzymes, amylase, was the first large-scale application of microbial enzymes

in food industry.

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• Enzymes have many applications in drink industry. The use of chymosin in cheese making to coagulate

milk protein.

• Another enzyme used in milk industry is beta-galactosidase or lactase, which splits milk-sugar lactose into glucose and galactose.

This process is used for milk products that are

consumed by lactose intolerant consumers.

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• Enzymes are used also in fruit juice manufacturing. Pectins are substances in fruit lamella and cell walls. The cell wall contains also hemicelluloses and cellulose. Pectinase, xylanase and cellulase improve

the liberation of the juice from the pulp.

• Pectinases and amylases are used in juice

clarification.

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• Brewing is an enzymatic process. Malting is a process, which increases the enzyme levels in the grain. In the mashing process the enzymes, amylase, are liberated and they hydrolyse (Break down) the starch into soluble fermentable sugars like maltose, which is a glucose disaccharide.

• Similarly enzymes are widely used in wine productionto obtain a better extraction of the necessary components and thus improving the yield.

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Foods/Beverages produced by Microbial Activity

Foods/Beverages cured or improved by microbial activity

• Production of coffee, tea, cocoa, summer sausage, vanilla, cheese, olives and tobacco all require microbial

activity.

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Food flavoring agents and preservatives

• Organic acids such as citric, malic and ascorbic acids and monosodium glutamate are microbial products

commonly used in foods.

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