PRESENTATION

NAME : RIMSHA SALEEM, IQRA KANWAL

ROLL : NUMBER 31,24
Presentation Topic

(Microbial Enzymes)
Amylase
Amylase

Outlines
 1) What are amylases.
 2) Types of amylases.
 3) Production of amylases.
 4) Determination of enzyme activity.
 5) Purification.
 6) Industrial Applications.
1) What are amylases

➢ Amylases are important hydrolase enzymes which have been widely

used since many decades.
➢ These enzymes randomly cleave internalglycosidic linkages in starch
molecules. To hydrolyze them and yield

(dextrins )
(oligosaccharides)
2) Types of amylases

 2.1. α-Amylase:
➢ α-Amylase is a hydrolase enzyme that catalyses the hydrolysis of
internal α-1, 4 glycosidiclinkages in starch
➢ to yield products like glucose and maltose.
➢ It is a calcium metalloenzyme i.e. it depends on the presence of a
metal co factor for its activity.
➢ The optimum pH for activity is found to be 7.0
2.1. α-Amylase:

The substrate that α-amylase acts upon is starch.

➢ Starch is a polysaccharide composed of two types of polymers

amylose and amylopectin.
➢ Amylose constitutes 20-25% of the starchmolecule.
➢ It is a linear chain consisting of repetitiveglucose units linked by x-1,4-
glycosidiclinkage.
2.1. α -Amylase:

➢ Amylopectin constitutes 75-80% of starch and is characterized by

branched chains of glucose units.
➢ The linear successive glucose units are linked by x-1, 4-glycosidic
linkage.
➢ while branching occurs every 15-45 glucose units where x-1, 6
glycosidic bonds are present.
2.2. β-Amylase

➢ β-Amylase is an exo-hydrolase enzyme that acts from the

nonreducing end of a polysaccharide chain
➢ by hydrolysis of x-1, 4-glucan linkages to yield successive maltose
units.
➢ Since it is unable to cleave branched linkages in branched
polysaccharides such asglycogen or amylopectin,
➢ the hydrolysis is incomplete and dextrin units remain.
2.2. B-Amylase

➢ Primary sources of B-Amylase are the seeds of higher plants and

sweet potatoes.
➢ The optimal pH of the enzyme rangesfrom 4.0 to 5.5.
➢ B-Amylase can be used for different applications on the research as
well as industrial front.
➢ It can be used for structural studies ofstarch and glycogen
molecules produced byvarious methods.
2.3. y-Amylase

➢ y-Amylase cleaves a(1-6)glycosidic linkages, in addition to cleaving

the last α(1 4)glycosidic linkages
➢ At the nonreducing end of amylose and amylopectin,
➢ Un-like the other forms of amylase, yielding glucose.
➢ y-amylase is most efficient in acidic environments and has an
optimum pH of 3.
3. Production of a-Amylase

3.1. Sources

➢ α-Amylase can be isolated from plants, animals or microorganisms.

➢ The enzyme has been isolated from barley and rice plants.
➢ It has been found that cassava mash waste water is a source of α -
Amylase.
➢ In the recent past, there has been extensive research on microbial
production of α-Amylase.
3.1. Sources

There are 2 major reasons for the increasing interest in microbial

sources:
1) The growth of microorganisms is rapid and this will in turn speed up
the production of enzyme.

➢ Microorganisms are easy to handle whencompared to animals and

plants.
➢ They require lesser space and serve as more cost effective sources.
3.1. Sources

2) Microorganisms can be easily manipulated using genetic

engineering or other means.

➢ They can be subjected to strain improvement, mutations and other

such changes by which the production of a-Amylase can be
optimized.
➢ α-Amylase is produced by several bacteria,fungi and genetically
modified species ofmicrobes.
Bacterial And Fungal Sources
7. β-Amylase is an exo-hydrolase enzyme that acts from the
nonreducing end of a polysaccharide chain by hydrolysis of α-1, 4-
glucan linkages to yield successive maltose units. Since it is unable to
cleave branched linkages in branched polysaccharides such
asglycogen or amylopectin, the hydrolysis is incomplete and dextrin
units remain.8. Primary sources of β-Amylase are the seeds of higher
plants and sweet potatoes. The optimal pH of the enzyme ranges from
4.0 to 5.5. β-Amylase can be used for different applications on the
research as well as industrial front. It can be used for structural studies
of starch and glycogen molecules produced by various methods
3.2. Production Methods

There are mainly two methods which are used for production of x-Amylase
on a commercial scale.
These are:
1) Submerged fermentation (SMF)
2) 2) Solid State fermentation (SSF)

3) Submerged fermentation (SMF):

➢ Submerged fermentation (SmF) employs free flowing liquid substrates,

such as molasses and broths.
➢ The products yielded in fermentation are secreted into the
fermentation broth.
1) Submerged fermentation (SMF):

➢ This fermentation technique is suitable for microorganisms such as

bacteria that require high moisture content for their growth.
➢ SmF is primarily used for the extraction of secondary metabolites
that need to be used in liquid form.
➢ This method has several advantages. SmF allows the utilization of
genetically modified organisms to a greater extent than SSF.
➢ The sterilization of the medium and purification process of the end
products can be done easily.
➢ Also the control of process parameters like temperature, pH,
aeration, oxygen transfer and moisture can be done conveniently
2) Solid State fermentation (SSF)

➢ Solid state fermentation is a method used for microbes which

require less moisture content fortheir growth.
➢ The solid substrates commonly used in this method are,bran,
bagasse, and paper pulp.
➢ The main advantage is that nutrient-rich waste materials can be
easily recycled and used assubstrates in this method
➢ Other advantages that SSF offers over SmF are simpler equipments,
higher concentration of products and lesser effluent generation.
➢ For several such reasons SSF is considered as a promising method
for commercial production ofenzymes.
4. Determination of enzyme activity

▸3 methods
4.1. Dinitrosalicylic Acid Method (DNS):
➢ In the dinitrosalicylic acid method, aliquots of the substrate stock
solution are mixed with the enzyme solution.
➢ Followed by 10 min of incubation at 50°C,
➢ DNS reagent is added to the test tube and the mixture is incubated
in a boiling water bath for 5 min.
➢ After cooling to room temperature, the absorbance of the
supernatant at 540 nm is measured.
4.2. Nelson - Somogyi (NS) Method

➢ In the NS method, an aliquot of stock solution.of substrate is heated

at 50°C for 5min.
➢ Preheated (50°C for 5 min) enzyme solution is added to the
substrate.
➢ This reaction mixture is incubated at 50°C and thereaction is carried
out for 10min.
➢ After incubation Somogyi copper reagent is added toterminate the
reaction.
➢ This is then incubated in boiling water bath for 40 min & cooled to
room temperature.
➢ Finally water is added and the mixture is centrifuged at 13,000 rpm
for 1 min and absorbance ofsupernatant is read at 610 nm
4.3. Determination of Activity Using
lodine
➢ The hydrolytic activity of α-Amylase can be determined based on
the principle that starch and iodine react to form a blue colored
complex.
➢ On hydrolysis of starch this complex changes toa reddish brown
colored one.
➢ The absorbance can be read after the enzyme substrate reaction
has been terminated.
➢ This gives a measure of the extent of hydrolysis of starch by α-
Amylase.
5. Purification of α-Amylase

➢ Purification methods commonly employed are precipitation,

chromatography and liquidliquid extraction depending on the
properties of the enzyme desired.
➢ A combination of the above methods is used in a series of steps to
achieve high purity.
➢ The crude extracellular enzyme sample can be obtained from the
fermented mass by filtration and centrifugation.
➢ The crude amylase enzyme can be precipitated and concentrated
using ammonium sulphate precipitationor organic solvents.
➢ The precipitated sample can be subjected to dialysis against water
or a buffer for further concentration.
5. Purification of α-Amylase

➢ This can be followed by any of the chromatographic techniques like

ion exchange,
➢ Gel filtration and affinity chromatography for further separation and
purification of the enzyme.
➢ In a method of purification of the enzyme produced by Aspergillus
sps
➢ the enzyme was precipitated was followed by dialysis and then
column chromatography
Industrial Applications of α-Amylase
and the Microbial Source used
THANK
YOU
Presentation of industrial microbiology
Acidic protease
Acidic protease

• A protein-digesting enzyme that exhibits maximum activity and stability in acid

conditions (pH 2.0–5.0)
• It is inactivated at pH values above 6.0.
• Acid proteases have a low isoelectric point and are low in basic amino acids.
• Source of Protease Enzyme
• These enzymes are also called proteolytic and proteinases.
• Mainly in the human body, these enzymes are produced in the stomach and the
pancreas.
• Protease enzymes are most commonly known for their role in the digestion of
proteins.
• These enzymes can also be naturally found in certain foods and it is also available
in the form of a supplement.
Protease Enzyme Function

• Some of the important protease functions are given below.

• These functions are very important to regulate various metabolic and cellular
processes.
• Protease enzyme breaks the protein, which helps in digestion and catabolism of
protein.
• This enzyme is very important for the blood coagulation process
• .It is involved in the process like cell division, growth, apoptosis and migration.
• It helps to transport the protein and recycle it between the membranes.
• It helps in the activation of precursor proteins and zymogens.
• It regulates the process of tumor growth, metastasis, inflammation and also
provides immune support to the body.
• enzyme can also be used to heal the wound and muscle soreness.
Acidic proteases in industrial microbiology

• Acid proteases represent an important group of enzymes, extensively used in food

and beverage industries.
• There is an increased demand for acid proteases adapting to the industrial extreme
environment, especially lower pH.
• Thus, this necessitates the search for a better acid protease from fungi that best
performs in industrial conditions.
• Acidic proteases are found in animal cells, yeast and molds but never
in bacteria.
• These microbial renin-like enzymes are derived from Mucor michei,
Mucor pusillus, Mucor racemaeus, Mucor bacilliformis.
• Pepsin like acid proteases are derived from Aspergillus species and
Rhizopus spp.
• Renin like proteases are commonly used in cheese production in
optimum pH 2-4.
Applications of protease enzyme:

• Used in cheese production

• Used in medicine (similar to mammalian pepsin)
• Used in digestion of soya-protein for soy-sauce production.
• Break down wheat gluten in baking industries.
Production process of protease enzyme:

step I: Isolation of proteolytic microbes

step II: Media formulation:
step III: Fermentation:
step IV: Purification:
Application of acid protease in the industrial production of corn
ethanol

• Bioethanol is a renewable, clean energy and a very important basic

chemical raw material, improving production efficiency and
reducing production cost are the goals pursued by ethanol
enterprises.
• Adding acid protease in the process of producing ethanol from
grain will have many effects on fermentation, one of the most
concerned effects is to increase liquor yield.
• Acid protease can hydrolyze the protein in cereals to produce free
amino acids that can be used directly by yeast, thus reducing the
amount of sugar consumed for amino acid metabolism, and
allowing more sugar to be converted to ethanol.
• the results showed that the addition of acid protease at the dosage
of 10 U/g (raw material) during yeast seeding could replace urea,
promote yeast proliferation, reduce the infection rate,
• shorten the fermentation period by 7.5 h and increase the alcohol
yield of raw materials by 0.33 percentage point.
• When acid protease was used in ethanol production line with an
annual output of 180,000 tons, the net profit increased by at least US
$4900/day compared with the control process.
Diagrammatic explanation
 ALKALINE PROTEASE:
ASIFA RIAZ ROLL NO 32
PRESENTED TO: DR . IMRAN SAJID
ALKALINE PROTEASE (FERMENTED LEATHER ENZYME):

 Alkaline Protease
 One of the class of protease enzyme.
 An extracellular enzyme.
 Performs proteolysis, that is, protein catabolism by hydrolysis of the
peptide bonds.
 Active at alkaline pH 8 to 12 and at temperature 30⁰-80⁰C.
GENETIC ENGINEERING AND MICOBES:

Bacteria Fungi
 More than 50% of the industrially
Bacillus subtilis Aspergillus niger important enzymes are now
produced from genetically
Bacillus pumilus Aspergillus melieus engineered microorganisms.

Bacillus proteolyticus Aspergillus fumigatus

 Methods Used:
 Conventional mutagenesis (UV
Bacillus firmus Aspergillus flavus or chemical exposure) or
 Recombinant DNA technology.
 PRODUCTION PROCESS OF ALKALINE PROTEASE ENZYME:
 Isolation of Microorganisms.
 Development and Preparation of
Inoculum.
 Preparation of Fermentation Media.
 Optimization of Media .
 Fermentation Process .
 Enzyme Extraction and Assay.
 Protein Assay .
PRODUCTION PROCESS OF ALKALINE PROTEASE ENZYME:

 Alkaline Protease Purification.

 Ammonium sulphate precipitation .
 Ultracentrifugation.
 Flocculation .
 Chromatography.

 Electrophoresis.

 Characterization of Purified
Alkaline Protease .
 Packaging.
Stages Enzymes involved Function of enzymes

Curing Enzymes are not directly To preserve hides and skins

involved
Soaking Alkaline Protease To remove non fibrillar protein

Dehairing Neutral and alkaline Protease To remove hair

Bating Trypsin and alkaline Protease To make soft, supple and pilable

ALKALINE
Degreasing Lipase and acid protease To remove fats
PROTEASE
Tanning IN Enzymes are not directly To influence the quality of
involved tanning
LEATHER
Waste processing Trypsin and alkaline Protease Tanned wasted processing
INDUSTRY :
ADVANTAGES OF USING ENZYMES IN LEATHER INDUSTRY:

 Significant reduction of using chemicals.

 Simplification of processes.
 Creates of an ecologically conducive atmosphere for the workers.
 Leathers have shown better strength and quality.
 Saves time.
 Environment friendly.
 Leather wastes can be hydrolyzed by enzymes.
GLOBAL MARKET OF ALKALINE PROTEASE ENZYME :

 Of the industrial enzymes, Alkaline protease

covers 25% of total enzyme market. •
 The global market is increasing because
replacement of chemicals and additives, more
demand for environment friendly ingredients,
less production cost, high yield, less time taken
for production and the growing industrial need
in developing countries.