MEDIUM OPTIMIZATION FOR THE

PRODUCTION OF AMYLASE BY Bacillus subtilis

RM16 IN SHAKE-FLASK FERMENTATION

Afifah Nur Aini (176090100011006)

Fermentation Technology Class
Master Program in Biology
Brawijaya University April 13, 2018
B A C K G R O U N D
4000 enzymes are known.
Only 200 are used commercially.

Feedstuff Food industry

57

64 158

Technical
ENZYME application
• Natural catalyst 75% of all industrial enzymes are
• Produced by all living organisms hydrolytic enzymes (carbohydrases,
• Involved in all processes essential for life proteases & lipases).
• Perform specific chemical transformations
 Global market for industrial
B enzymes was estimated as
A $4.2 billion in 2014 and
C amylase constributes 25% of it.
K
G Amylase degrades starch
R to produce smaller
O
carbohydrates like
glucose and maltose.
U
N
D Amylase a-(1,6)-glycosidic bond

a-(1,4)-glycosidic bond
 To optimize the culture conditions for Bacillus subtilis RM16 and
AIM
examined the effect of various parameters on the production of amylase

Inoculum
METHODS
preparation

24-hrs-old 37oC 18 hrs

microbial 5 ml
culture used as
inoculum
100 ml Laura
Bertani broth Shaker incubator
 To optimize the culture conditions for Bacillus subtilis RM16 and
AIM
examined the effect of various parameters on the production of amylase

Enzyme
Inoculum
METHODS production
preparation
medium
Tryptone..........10 g
Yeast extract......5 g 40oC 150 rpm 24 hrs 5000 rpm
NaCl.................10 g 48 15 min
Soluble starch..10 g 72
pH  7.0
+ 5 ml
starter Centrifuge
culture Enzyme assay
100 ml Shaker incubator
 To optimize the culture conditions for Bacillus subtilis RM16 and
AIM
examined the effect of various parameters on the production of amylase

Enzyme
Inoculum Estimation
METHODS production
preparation of amylase
medium

1% starch solution................100 ml
CFS containing the enzyme....20 ml 200 ml
20 mM Tris-HCl pH 8.0...........80 ml DNS 5 min 492 nm
solution
37oC 10 min

Boiling Spectro-
Incubator water bath photometer
 To optimize the culture conditions for Bacillus subtilis RM16 and
AIM
examined the effect of various parameters on the production of amylase

Enzyme
Inoculum Estimation Optimization of
METHODS production
preparation of amylase the parameters
medium

Incubation time (hours) Incubation temp. (oC)

24 48 72 40 50 60 70

Inoculum size (%) Medium pH

2.5 5.0 7.5 10.0 5.0 6.0 7.0 8.0 9.0
 To optimize the culture conditions for Bacillus subtilis RM16 and
AIM
examined the effect of various parameters on the production of amylase

Enzyme
Inoculum Estimation Optimization of
METHODS production
preparation of amylase the parameters
medium
Carbon source (1%)

Starch MgSO4

Metal ion (0.1%)

Glucose Nitrogen source
Organic CuSO4
Inorganic (1%)
Sucrose (0.5, 1.0, 1.5, 2.0%)
Yeast CaCl2
Maltose NaNO2 (NH)4SO4 NH4NO3 Tryptone
extract
 Enzyme activity (U/ml)

Enzyme activity (U/ml)

R
E
S
U
L
T
40 50 60 70 5 6 7 8 9
S Temperature incubation (oC) pH

activity (U/ml)
Enzyme activity (U/ml)

Enzyme activity
Enzyme
2.5 5.0 7.5 10.0 Starch Glucose Sucrose Maltose
Inoculum size (%) Carbon source (1%)
 Enzyme activity (U/ml)

Enzyme activity (U/ml)

R
E
S
U
L
T NaNO2 (NH)4SO4 NH4NO3 0.5 1.0 1.5 2.0
S Inorganic nitrogen source (1%) Organic nitrogen source (% yeast extract)

Enzyme activity (U/ml)

(U/ml)
Enzyme activity (U/ml)

MgSO
2.5 4 5.0CuSO4 7.5 CaCl210.0 0 1 2 3
Metal ion (0.1%) Organic nitrogen source (% tryptone)
C
O Bacillus subtilis RM16 was found to produce maximum
N unit of amylase activity:
C
• at temperature of 40°C
L
U • with pH ranges from 7.0–8.0
S • after 24 hours of the incubation time period.
I
O Regarding carbon and nitrogen sources, 1% starch as well
N
as 1.5% yeast extract showed maximum enzyme activity.

Magnesium sulphate (0.1%) and inoculum size (5%) has

shown to increase amylase production in cell-free broth.