Large scale production of

enzymes
Gaurav Sharma (2015A1PS0667P)
Shreshtha Dhankar (2015A1PS0706P)
Introduction
 Enzymes have been used ever since mankind discovered ways to process
food. Food processing steps like milk acidification, milk clotting, alcohol
fermentation and soy bean fermentation are enzyme mediated processes
carried out by microorganisms. However, it was not until the late 19th century
that purified enzymes were used for food processing.
 History: 1901: Eduard Bucher won the Nobel prize in biochemistry for proving
the existence of enzymes. Approximately 20 years before, Christian Hansen
invented and commercialised a process to purify rennet from calf stomach
which revolutionized the dairy industry. Marks beginning of global food
enzyme industry.
 Global sales: US$ 8.18 billion in 2015
 Market leaders: Novozymes, Danisco, Dsm
Metabolic Process

Metabolic Conversion

Substrates,
Physical Enzyme synthesis, Biomass,
parameters Transcription, by product
Translation,
Post translational
processes

INPUT PARAMETERS STRUCTURAL OUTPUT PARAMETERS

COMPONENTS
Biotechnological process of enzyme production
1 - Screening
- Choosing an appropriate micro-organism for the desired enzyme

2 - Modification
- Possible application of genetic engineering to improve the microbial strain

3 - Laboratory scale pilot

- To determine the optimum conditions for growth of micro-organisms

4 - Pilot plant
- Small scale fermenter to clarify optimum conditions

5 - Industrial scale fermenter

Species selection
 High yield
 Short fermentation period
 Utilizes inexpensive substrate
 Reproducibility
 Safe and non-toxic
 Cost effective

 FUNGI (Aspergillus and Tricoderma)

 YEAST (Saccharomyces, Hansenula and Kluyveromuces)
 BACTERIA (E.coli, Bacillus)
Metabolic Engineering
 Classical mutagenesis
 Screening for improved production
 Targeted deletion of unwanted genes
 Chemical agents and UV radiations are used.
Optimization
 Optimization means finding highest specific synthesis rate for a given amount
of biomass. As enzyme synthesis depends on primary metabolism, conditions
favouring enzyme synthesis favour growth. Thus, we need to determine
conditions favouring growth and the relationship between growth rate and
enzyme synthesis rate.
A. Growth coupled synthesis
B. Saturated synthesis
C. Saturated synthesis with CR
D. Repressed synthesis
Fermentation
1) Solid Surface Fermentation
 Media: Wheat bran, whose high content of nutrients includes minerals and
salts.
 Applications: production of amylase, protease, and lipase from Aspergillus
and Mucor species, as well as for pectinase and cellulose from Aspergillus
and Penicillum species.
 Tray process: substrate is spread in a thin layer in incubation rooms
 Drum process: horizontal rotating drums
 After cultivation of fungi with spores, the mycelia are extracted with water or
salt solution in countercurrent mode, and the concentrated enzyme solution is
precipitated.
 Disadvantages: handling costs and control of infection, temperature, humidity
and aeration.
2) Submerged Culture Method
 Less risks of infection and offer reduced handling costs and higher yields
 Mechanical stirred reactors in batch of fed-batch mode
 Capacity: 10,000 to 100,000 litres for 30-150 hours
 Equipment and techniques are most often adapted from antibiotic
fermentations.
 Continuous mode not preferred because of risk of enzyme inactivation of
media sterilization (successfully used for Glucose isomerase)
 The formation of enzymes and many secondary metabolites is often subject
to catabolite repression by high concentrations of glucose.
 In addition to influences of nutrient medium and size and age of inoculum,
operational parameters such as pH, aeration and agitation must be taken
into account to optimize the production of enzymes.
 Addition of surface active agents may lead to increased excretion of
extracellular enzymes.
 Is most important parameter affecting oxygen and nutrient distribution.
 Optimal scales of production process depends on technical and strain
specific considerations, but also on the balance between economy and rosk
assessment.
Submerged culture method
Enzyme Formulation
 The purpose of formulation is to get the enzyme to the customer in a usable
format
 We already know the enzyme performs well, based on application tests and
molecule optimization. So we can now put it into a form that provides stability,
improves the aesthetics or targets delivery.
 Enzyme products may be exposed to harsh environments – such as high
temperatures or pH values – in both storage and use. It is the formulation that
helps protect the enzymes
 For example, enzymes are blended into powder detergents that contain a
bleaching component. Both the enzyme and bleach are critical for the
overall cleaning performance, but if the enzyme is unprotected the bleach
can damage the enzyme during storage and its benefits may be lost. By
developing a solid granulated product that contains protective barriers, we
can keep the enzyme and bleach separate during storage and optimize
effective performance in the wash.
Why do we need formulation?
 The formulation of an enzyme is not limited to the delivery mode but has a far
important principle which deals with the very quality and performance of the
enzyme.
 Enzymes being Protein have a three dimensional structure and are prone to
denaturing. Thus an enzyme concentrate is required to be blended along
with additives which stabilizes its structure, thus preserving its biological
activity.
 Addition of these Stabilizers are custom tailored keeping in mind the best
stability and performance of the enzyme.
 Formulation also allows incorporation of the enzyme concentrate at specific
percentages, thus enabling for a wide spectrum of products depending upon
the application and end use.
 A process may require the enzyme component to be at dilute levels. Hence
an optimized formulation can be worked out for the end user depending
upon their exact requirements allowing for robust performance along with
economic feasibility.
 An important component of the formulation are Preservatives which exhibit
anti-microbial action.
 Other materials which are added to enzymes before sale may consist of
substrates, thiols to create a reducing environment, antibiotics, benzoic acid
esters as preservatives for liquid enzyme preparations, inhibitors of
contaminating enzyme activities and chelating agents.
 Formulation is an art and often the precise details of the methods used to
stabilize enzyme preparations are kept secret or revealed to customers only
under the cover of a confidentiality agreement.
 Sometimes it is only the formulation of an enzyme that gives a manufacturer
the competitive edge over rival companies.
 The key to maintaining enzyme activity is maintenance of conformation, so
preventing unfolding, aggregation and changes in the covalent structure.
Three approaches are possible:
1. use of additives,
2. the controlled use of covalent modification, and
3. enzyme immobilization
 Proteins are stabilized by increasing their concentration and the ionic strength
of their environment. Neutral salts compete with proteins for water and bind
to charged groups or dipoles. This may result in the interactions between an
enzyme's hydrophobic areas being strengthened causing the enzyme
molecules to compress and making them more resistant to thermal unfolding
reactions.
 Not all salts are equally effective in stabilizing hydrophobic interactions, some
are much more effective at their destabilization by binding to them and
disrupting the localized structure of water.
 From this it can be seen why ammonium sulphate and potassium hydrogen
phosphate are a powerful enzyme stabilizers whereas sodium thiosulphate
and calcium chloride destabilize enzymes.
 Many enzymes are specifically stabilised by low concentrations of cations
which may or may not form part of the active site, for example
Ca2+ stabilizes a-amylases and Co2+ stabilizes glucose isomerases.
 At high concentrations (e.g. 20% NaCl), salt discourages microbial growth due
to its osmotic effect. In addition ions can offer some protection against
oxidation to groups such as thiols by salting-out the dissolved oxygen from
solution.
 Enzymes are very much more stable in the dry state than in solution. Solid
enzyme preparations sometimes consist of freeze-dried protein. More usually
they are bulked out with inert materials such as starch, lactose,
carboxymethylcellulose and other poly-electrolytes which protect the enzyme
during a cheaper spray-drying stage.
 Enzymes released onto the market should conform to a number of quality
procedures including regulatory requirements, which are legal and
mandatory.
 This is provided by the quality assurance (QA) within the company. Enzyme
products must be consistent as appropriate to their intended use. This may be
ensured by good manufacturing practice (GMP) and further checked by
quality control (QC).
 Enzymes released onto the market should conform to a number of quality
procedures including regulatory requirements, which are legal and
mandatory.
 This is provided by the quality assurance (QA) within the company. Enzyme
products must be consistent as appropriate to their intended use. This may be
ensured by good manufacturing practice (GMP) and further checked by
quality control (QC).
Thank You