Isolation and screening technique to

produce primary metabolite from

microorganism
Nur Ida Panca N
2017
 Primary metabolites are considered essential to
microorganisms for proper growth.
Primary metabolites are involved in growth,
development, and reproduction of the organism.
The primary metabolite is typically a key
component in maintaining normal physiological
processes
 Primary
metabolites are
typically formed
during the
growth phase as
a result of
energy
metabolism
 Examples of primary metabolites include :
Ethanol
Lactic Acid

Amino Acid

Citric Acid

Enzymes
 Aspergillus niger used in industrial
microbiology for mass production of citric acid
 ENZYME
The majority of industrial
enzymes used nowadays has a
microbial origin replacing
conventional chemical catalysts.
Microbial enzymes are relatively
more stable (temp, pH,
pressure), biodegradable, and
have properties more diverse
than other enzymes derived
from plants and animals.
 ENZYME

1. Screening and Isolation of Protease Producing

Bacteria from Soil
Proteases, one among the three largest groups of
industrial enzymes, account for about 60% of the
worldwide sale of enzymes.
The major use of free proteases occur in dry
cleaning detergents, meat processing, cheese
making, silver recovery from photographic film,
production of digestive and certain medical
treatments of inflammation and virulent wounds.
 ENZYME

Bacterial Proteases are preferred as they :

Grow rapidly

Needless space

Can be easily maintained

Accessible for genetic manipulations

 ENZYME

Currently, a large Proportion of commercially

available proteases are derived from Bacillus
strain
 Matherials & Methods
Source of soil sample :

Farm Soil Oil Spilled Soil

 ENZYME

Below 5-6 cm depth COLLECTED

stored in sterile plastic bags at 4:C

 ENZYME

Isolation of protease producing bacteria :

Serial dilution

1 g of soil sample

0.1 ml of each aliquot was

spread on SKIM MILK AGAR
Note the zone of hydrolysis
(1%) (37:C for 48 hr)
 ENZYME

Grown on nutrient agar plate

Preserved on nutrient agar slant at 4:C

The colony
showing highest
zone of
inhibition
 ENZYME
Identification and characterization of bacteria :
Gram staining
Motility test

Acid Fast test

Endospore staining

Cultural characterization

Biochemical test

16S rRNA gene sequencing

 ENZYME

Preparation of casein solution :

Alkali soluble casein Dissolved in 10 ml distilled water

The pH was adjusted to 8.0 with

0.1 M NaOH

Casein Broth Medium

 ENZYME

Crude enzyme preparation :

The protease Inoculated in casein broth
producing bacterial colony medium (37:C for 48 hrs)

centrifuge Using Whatmann No.1 filter

at Filtrate
paper cultured medium was
10,000 rpm filtered
for 10
minutes
Supernatant

Supernatant enzyme activity assay

 ENZYME

Enzyme activity assay

Protease activity was measured by using
casein as a substrate following the method of
Shimogaki et al. (1991). One unit of protease
activity is defined as the amount of enzyme
liberating 1 g of tyrosine per minute under
assay conditions.
 ENZYME

Treatment factors that can be used :

Effect of pH on enzyme activity

Effect of temperature on enzyme activity

Effect of fermentation period on enzyme activity

 ENZYME

Another type of potential enzymes that can

be produced from soil bacteria :

Amylase

Lipase
 ENZYME

Amylases hydrolyze starch molecules to give

diverse products, including dextrins and
progressively smaller polymers composed of
glucose units.
Potential applications of amylase : food,
textile, and paper sectors.
 ENZYME

Isolation of amylase producing bacteria :

Serial dilution

1 g of soil sample

0.1 ml of each aliquot was

spread on STARCH enriched
Note the zone of hydrolysis
medium
 ENZYME

Isolation of lipase producing bacteria :

Serial dilution

1 g of soil sample

0.1 ml of each aliquot was

spread on OLIVE OIL
Note the zone of hydrolysis
enriched medium
 ENZYME

Crude enzyme preparation :

The amylase/lipase Inoculated in starch/olive oil
producing bacterial colony broth medium

centrifuge Supern Using Whatmann No.1 filter

at atant paper cultured medium was
10,000 rpm filtered
for 10
minutes

Supernatant enzyme activity assay

 ENZYME

Enzyme activity assay

Amylase activity was assayed following the
method of Bernfeld (1955) using starch as a
substrate. One unit of amylase activity was
defined as the amount of enzyme releasing 1
mol of maltose equivalent per minute from
soluble starch under assay conditions.
 ENZYME

Enzyme activity assay

Lipase activity was determined using p-
nitrophenol palmitate (pNPP) as substrate
following the method described by Kilcawley
et al. (2002). One unit of lipase activity was
defined as the amount of enzyme liberating 1
nmol of pNP (p-nitrophenol) per minute under
standard assay conditions.
 ENZYME

2. Screening And Isolation Of Halophilic

Bacteria Producing Industrially Important
Enzymes
Halophiles are excellent sources of enzymes
that are not only salt stable but also can
withstand and carry out reactions efficiently
under extreme conditions.
 ENZYME
Extreme conditions :
High temperature
pH

Presence of salts

Solvents

toxicants
 Matherials & Methods

Source of sample :

Saltern crystallizer Dead Sea

ponds

Salt mine
 ENZYME

Isolation of bacteria :
Serial dilution

1 g of soil/water
sample

Screening of 0.1 ml of each aliquot was

hydrolase spread on salt enrichment
medium + substrat (starch/ olive
activities oil/ gelatin ) (30:C for 96 hr)
 ENZYME

Screening of hydrolase activities :

Amylolytic activity

Proteolytic activity

Lipase activity
 ENZYME

Grown on complete media agar

Stored at 4:C
The colony
showing highest
zone of Identification and
inhibition characterization of potential
halophilic isolates
 ENZYME

Enzyme production :
The colony showing Inoculated in medium containing
highest zone of (gL-1): gelatin/starch/olive oil
inhibition 10.0; peptone 5.0; yeast extract
5.0; NaCl 50.0-200.0 and pH 7.0
centrifuge at (30:C for 96 hrs 150 rpm)
10,000 rpm for 10
minutes at 4 :C

Supernatant enzyme activity assay

 ENZYME

Treatment factors that can be used :

Effect of pH on enzyme
activity

Organic solvent stability

 ENZYME

3. Another hydrolitic enzymes that can be

produced from microorganism :
Carboxymethyl cellulases

Cellobiohydrolases

-glucosidases

Esterase

dll
 Sources :
Boundless. Primary and Secondary
Metabolites. Boundless Microbiology
Boundless, 26 May. 2016. Retrieved 22 Feb.
2017 from
https://www.boundless.com/microbiology/tex
tbooks/boundless-microbiology-
textbook/industrial-microbiology-
17/industrial-microbiology-198/primary-and-
secondary-metabolites-999-5345/
 Sources :

Waites et al. 2001. Industrial Microbiology; An

Introduction. London : Blackwell Science
Rupali, Dalal. 2015. Screening and Isolation of
Protease Producing Bacteria from Soil
collected from Different Areas of Burhanpur
Region (MP) India. Int.J.Curr.Microbiol.App.Sci
(2015) 4(8): 597-606
 Sources :
Shimogaki, H.; Takeuchi, K.; Nishino, T.;
Ohdera, M.; Kudo, T.; Ohba, K.; Iwama, M.;
Irie, M. (1991). Purification and properties of a
novel surface active agent and alkaline-
resistant protease from Bacillus sp., Y. Agric.
Biol. Chem. 55, 22512258
Bernfeld, P. (1955). Amylase, and . Meth.
Enzymol. 1, 149-158
 Sources :
Kilcawley, K.N.; Wilkinson, M.G.; Fox, P.F.
(2002). Determination of key enzyme activities
in commercial peptidase and lipase
preparations from microbial or animal
sources. Enzym. Microb. Tech. 31, 310320
Kumar et al. (2012). Screening And Isolation
Of Halophilic Bacteria Producing Industrially
Important Enzymes. Brazilian Journal of
Microbiology (2012): 1595-1603