第一組 : 黃潔儀 葉冠諝 黃建國
指導教授 : 褚俊傑老師
日期 : 2014.04.24
Introduction
 Lipase are an important group of
biotechnologically relevant enzymes and use in
many ways:
1.
The processing of fats and oils, detergents
2.
Degreasing formulations(脫脂配方)
3.
Food processing
4.
The synthesis of fine chemicals and pharmaceuticals, paper
manufacture
5.
Production of cosmetics and pharmaceuticals
6.
Accelerating the degradation of fatty waste and polyurethane(聚氨酯)
 Production of lipase was available from
many sources:
The most suitable sources for lipase production are microbes
including
1. bacteria
2. fungi
3. yeast
 Lipase production is dependent upon a
number of factors including
1. carbon
2. nitrogen sources,
3. pH,
4. temperature,
5. aeration
6. inoculums size
MATERIALS AND METHODS
Material and method of determination of
Lipases (triacylglycerol hydrolase)
6.Partial Purification of Lipase
1.Collection of
Samples
5.Optimization
4.Fermentation
3.Lipolytic Activity
2.Isolation and Identification
of Bacteria
7.Enzyme Characterization
7.1>Determination of
Optima and Stability of
Temperature and pH
7.2>Application Study
Collection of Samples
1.
Samples such as soil, coconut, milk were subjected to
serial dilution.
2. Serially diluted samples were plated to nutrient agar
plates
3. kept for incubation at 37°C for 24-48 hours and the
plates were observed for growth.
Isolation and Identification of
Bacteria
 Isolated bacterial cultures were examined for various
morphological and biochemical characteristics
 Tested by:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Gram staining
motility
Indole test
MR test
VP test
Citrate test
Gelatin test
Urease test
Catalase test
Lipolytic Activity
 All the isolated cultures were inoculated into
tributyrin agar (三丁酸甘油酯瓊脂平板)plates and
kept for incubation at 37°C for 24 hours and observed
for zone formation
 A clear zone around the colonies indicates the
production of lipase.
Fermentation
1.
Fermentation was carried out in shake flask using a
complex medium
2. The flask containing 40mL fermentation medium
were inoculated by 18 h old vegetative inoculums.
3. The inoculums development and the fermentations
were carried out at 30°C for 7 days at 100 rpm
Optimization
1.
Lipase production was optimized at
different pH (7 - 10),
b. different temperature (30 - 60°C)
c. incubation period (1-5 days)
d. constant shaking at 120 rpm.
a.
2. Bacteria were cultured in nutrient broth with 1%
olive oil.
3. lipase activity will be measured every 12 h to
determine the maximum lipase producing period.
Partial Purification of Lipase
 1.in order to precipitate fatty acids:
the culture supernatant was treated with 0.4M CaCl2
followed by centrifugation at 4°C and 12,857 g for 30
min.
 2.allowing protein precipitation:
The supernatant was collected in a glass beaker and to
it chilled acetone was added slowly, with continuous
stirring, up to 70% (v/v) concentration and kept at
20°C for 4 h
Partial Purification of Lipase
 3.Harvested the precipitates:
centrifugation at 4°C and 12,857g for 30 min.
 4.Allowing the solubilization of proteins:
The pellet thus obtained was resuspended in 34 mL of
20 mM Tris-HCl buffer (pH 8.0)
Supernatant was then subjected to ultrafiltration
and dialyzed overnight against same buffer at 4°C.
Determination of Optima and Stability of
Temperature and pH
 Optimum temperature for activity of the lipase was
determined by carrying out at selected temperatures
from 30 to 70°C.
 The optimum pH was determined by monitoring
lipase activity at pH values between 6- 10.
Application Study
1.
5g of meat was weighed
2. it was autoclaved at 121°C for 15 lbs for 30 minutes.
3. The enzyme filtrate was added to meat
4.
incubated at 37°C for 72 hours for the observation of
the lipolytic activity on the meat.
RESULT AND DISCUSSION
Result and discussion of determination of
Lipases (triacylglycerol hydrolase)
6. Optimization Studies
1. Samples
Collected
5. Total Protein Estimation by Lowry’s
Method
4. Enzyme Assay
7.Partial Purification of
Lipase Enzyme:
3. Fermentation
8.Characterization Study:
2. Microbiological
Examination
9.Application Study
Samples Collected
 47 isolates were obtained which was then inoculated to
tributyrin media and observed for zone formation.
 Among the 47 isolates 3 isolates (KMCC CB301, 302,
303) were found to produce maximum zone formation
of 15, 20 and 35 mm, respectively
 Pseudomonas sp (KMCCCB301)
 Bacillus sp (KMCCCB302)
 Lactobacillus sp (KMCCCB303)
Microbiological Examination
Bacillus sp (KMCCCB302)
Pseudomonas sp
(KMCCCB301),
Lactobacillus sp
(KMCCCB303).
Microbiological Examination
1.Indole test:
determine the ability of the organism to convert tryptophan into the indole.
All the tested organisms give negative response.
It mean it cannot convert tryptophan into the indole
Also, it mean the used bacteria do not produce other material to affect the
production of lipase.
Microbiological Examination
 2.The methyl red test is used to identify enteric bacteria (腸道細菌)based on their
pattern of glucose metabolism.
 All enteric initially produce pyruvic acid from glucose metabolism. Some enteric subsequently
use the mixed acid pathway to metabolize pyruvic acid to other acids. These bacteria are called
methyl-red positive
 Other enteric subsequently use the butylene glycol pathway to metabolize pyruvic acid to
neutral end-products. These bacteria are called methyl-red-negative.
All the tested organisms give negative response.
Also, it mean the used bacteria do not produce other material to affect the
production of lipase.
Microbiological Examination
3.Voges–Proskauer test used to detect acetoin in a bacterial broth
culture.
A cherry red color indicates a positive result, while a yellow-brown color indicates a
negative result.
+VE
-VE
KMCC CB301 and KMCC CB302 give positive response
KMCC CB303 organisms give negative response.
It mean KMCC CB301 and KMCC CB302 can detect
Acetoin and KMCC CB303 cannot
Microbiological Examination
4.The citrate test detects the ability of an organism to use citrate as the sole
source of carbon and energy.
Production of sodium bicarbonate (NaHCO3) as well as ammonia (NH3) from the use of
sodium citrate and ammonium salts results in alkaline pH. This results in a change of the
medium’s color from green to blue.
KMCC CB301 and KMCC CB302 give positive response
KMCC CB303 organisms give negative response
Microbiological Examination
5.Gelatin hydrolysis test is used to detect the ability of an organism to produce gelatinase
Positive: Partial or total liquefaction of the inoculated tube even after exposure to cold
temperature of ice bath or refrigerator (4oC)
Negative: Complete solidification of the inoculated tube even after exposure to cold
temperature of ice bath or refrigerator (4oC)
 All the tested organisms give positive response.
 It mean they can produce gelatinase
Microbiological Examination
6.Urease test is used to detect the ability of an organism to produce urease. Urease
is an enzyme that breaks the carbon-nitrogen bond of amides to form carbon
dioxide, ammonia, and water.
 Positive result purple/pink colour
 Negative result colour of medium remains unchanged
KMCC CB301 give positive response
KMCC CB302 and KMCC CB303 organisms give negative response
Microbiological Examination
7.The presence of catalase in the test isolate is detected using hydrogen peroxide.
 Positive result: bubbling indicates the presence of catalase
 Negative result: No bubbling
KMCC CB301 and KMCC CB302 give positive response
KMCC CB303organisms give negative response
Fermentation
 After fermentation, the turbidity was detected by
using spectrophotometer:
 Lactobacillus sp showed absorbance of 1.34
 Bacillus sp showed absorbance of 1.15
 Pseudomonas sp showed absorbance of 1.20
 Lactobacillus showed highest turbidity
Enzyme Assay
 Pseudomonas sp showed the lipase activity was 10U/mL
 Bacillus sp showed the lipase activity was 18U/mL
 Lactobacillus sp showed the lipase activity was 35U/mL
 The highest the lipase activity was Lactobacillus sp
Total Protein Estimation by
Lowry’s Method
 The Lowry protein assay is for determining the total
level of protein in a solution.
 The total protein concentration is exhibited by a color
change of the sample solution in proportion to protein
concentration, which can then be measured
using colorimetric techniques.
Total Protein Estimation by
Lowry’s Method:
 the total protein content :
 Lactobacillus sp = 1.8mg/ml
 Bacillus sp = 1.63mg/ml
 Pseudomonas sp =1.2mg/ml
 Lactobacillus sp showed highest total protein content
Optimization Studies
 Lipase production increased
with increase in temperature
from 30 to 40°C.
 Maximum production of
lipase was 39 U/ml was
obtained at 40°C
 Lipase production begin to
decline at 50°C.
Optimization Studies
 lipase production increased
with increase in pH from 6
to 9 constantly.
 Maximum lipase production
of 39.6 U/ml was observed at
pH 9.
 Lipase production declined
at pH 10
Partial Purification of Lipase
Enzyme
 The purification of lipase resulted in 2 fold
purification with 75% recovery by ammonium sulfate
precipitation. The purification of crude enzyme
through dialysis gave purification fold of 36.5 with
66.6% recovery of lipase from Lactobacillus sp.
Characterization Study
 The maximum stability
of the enzyme was
observed in the
temperature range of 30
to 40°C for
Lactobacillus sp.
 The lipolytic activity
declined at 50 °C
Characterization Study
 The pH stability curve
showed that the lipase
was stable in the range
of pH 8 -9.
Application Study
 After the incubation period, the beginning of
degradation of meat was observed along with a very
strong smell.
 This may be because of the lipid present in the meat
get degraded by lipase enzyme
conclusion
conclusion
 When compared with production of lipolytic enzymes
between Bacillus sp and Lactobacillus sp and
Pseudomonas sp by fermentation
 It was concluded that Lactobacillus sp could be used as
a new potent microbial source of lipase.
 It is because The maximum lipase activity of
Lactobacillus sp reached was 32 U/ml with a
purification fold of 36.5 and a yield of 66.6 %.
Thanks for your attention !