Novozymes_report

2013
Project done at Novozymes South Asia
Pvt. Ltd.
Development of reducing end sugar assay
for amylase activity using PAHBAH reagent
in the presence of metal ions at low
temperature and Estimation of protein
concentration by ELISA.
By
C.H. YESESRI
Supervisor:Dr. PadmavathiBalumuri&
Mr. Roshan Shetty.
Project done at Novozymes South Asia
Development of reducing end sugar assay
for amylase activity using PAHBAH reagent
in the presence of metal ions at low
temperature and Estimation of protein
Supervisor:Dr. PadmavathiBalumuri&

Development of reducing end sugar assay and protein concentration estimation by ELISA
Dept. of Biotechnology, ANITS Page 2
ACKNOWLEDGEMENTS
A summer internship is a golden opportunity for learning and self-development. I
consider myself very lucky and honoured to have so many wonderful people to
lead me in completion of this project.
I would like to extend my sincere thanks to Dr.V.Sridevi (H.O.D dept. of
biotechnology, ANITS) and Dr.V.S.R.K.Prasad (Principal, ANITS) for
permitting to do this internship.
My sincere Thanks to Dr. Steffen Danielsen, R&D Head, Novozymes India for
giving me an opportunity to do the project in Novozymes.
I choose this moment to acknowledge Dr.AdityaBasu, (Manager, Protein and
Assay Technology, Novozymes) for his grateful contributions in arranging all the
facilities.
My grateful thanks to Dr. Padma (Scientist),who in spite of being busy with her
duties took time out to guide me in the correct path.
I would like to express my special thanks and gratitude to
Mr.RoshanShetty(Senior Research Associate), whose patience I have probably
tested to the limit.He was so involved in the entire process, shared his knowledge
&encouraged me to think. Thank You sir.
My thanks toMr.ChamanMehta (Asst.prof.ANITS) and Mr.PVSRK.Chaitanya
(Research Associate, Novozymes) for their efforts & help provided to me to get
such excellent opportunity.
Last but not the least there were so many who had extended their constant support
and help,I will be always be grateful to them.

CONTENTS:
PART –A
(DEVELOPMENT OF REDUCING END SUGAR ASSAY FOR AMYLASE ACTIVITY USING PAHBAH
REAGENT IN PRESENCE OF METAL IONS AT LOW TEMPERATURE)
1. INTRODUCTION
2. PRINCIPLE
3. MATERIALS REQUIRED
4. PROCEDURES
5. RESULTS
6. DISCUSSION
PART-B
(ESTIMATION OF PROTEIN CONCENTRATION BY ELISA)
1. INTRODUCTION
2. PRINCIPLE
3. MATERIALS REQUIRED
4. PROCEDURES
5. RESULTS
6. DISCUSSION
REFERENCES

Development of reducing end sugar assay for amylase activity using PAHBAH
reagent in the presence of metal ions at low temperature and Estimation of
protein concentration by ELISA.
Part- A
(Development of reducing end sugar assay for amylase activity using PAHBAH reagent in
presence of metal ions at low temperature)
INTRODUCTION:
Enzymes are natural catalysts that are produced by living organisms to increase the rate of an
immense and diverse set of chemical reactions required for life. The ability of an enzyme to
perform very specific chemical transformations has made them increasingly useful in industrial
processes and now they have a diverse array of applications in industries and scientific research,
ranging from the degradation of various natural substances in the starch processing, detergent
and textile industries, to the manipulation of DNA/RNA in biotechnology research.
At present, almost 4000 enzymes are known, and of these, approximately 200 microbial
original types are used commercially. However, only about 20 enzymes are produced on truly
industrial scale, these enzymes are used in various industries such as dairy, food, detergents,
textile, pharmaceutical, cosmetic and biodiesel industries, and in synthesis of fine chemicals,
agrochemicals and new polymeric materials (Saxena et al., 1999; Jaeger and Eggert, 2002) in
order to produce the enzymes of desired characteristics there is a need for greater control over
microorganisms which has led to a greater focus on Genetic Engineering and Recombinant DNA
technology. This technology, allows genetic modification of microorganisms to produce the
desired enzyme under specific conditions. This helps either to produce a particular type of
enzyme or enhance the quantity of enzyme produced from the single recombinant
microorganism. Understanding the structure of enzymes and modifying them to extract benefits
is categorized as Protein Engineering. Studies are being conducted on ways to improve or
modify protein structure and its function thus finally the enzyme. Biotechnology is therefore,
being increasingly viewed as a possible solution against traditional chemical processes. The
production of enzymes from natural sources and their environment friendly characteristics has
led the industry to believe that enzymes are indeed a sustainable alternative to chemicals in
industrial processes.
Enzyme applications in detergents began in the early 1930s with the use of pancreatic
enzymes in pre-soak solutions. It was the German scientist Otto Rohm who first patented the
use of pancreatic enzymes in 1913. Today, enzymes are continuously growing in importance for
detergent formulators. The most widely used detergent enzymes are hydrolyses, which remove
soils formed from proteins, lipids, and polysaccharides. Cellulase is a type of hydrolase that
provides fabric care through selective reactions not previously possible when washing clothes.
Looking to the future, research is currently being carried out into the possibility of extending the

types of enzymes used in detergents. Each of the major classes of detergent enzymes – proteases,
lipases, amylases, mannanases, and cellulases – provides specific benefits for laundering and
proteases and amylases for automatic dishwashing. Historically, proteases were the first to be
used extensively in laundering. Today, they have been joined by lipases, amylases and
mannanases in increasing the effectiveness of detergents, especially for household laundering at
lower temperatures and, in industrial cleaning operations, at lower pH.
Amylase is an enzyme that catalyses the breakdown of starch into sugars. It is classified into two
types based on cleavage of glicosidic linkages they are β-amylase and γ-Amylase.β-amylase
(works from the non-reducing end) catalyzes the hydrolysis of the second α-1, 4 glycosidic bond,
cleaving off two glucose units (maltose) at a time. whereas,γ-Amylase will cleave α(1-6)
glycosidic linkages, as well as the last α(1-4)glycosidic linkages at the nonreducing end of
amylose and amylopectin, yielding glucose.
Some other Industrial applications of AMYLASE:
Application field Enzyme Technical benefit
1. Pulp and paper
industry
2. Textile industry
3. Laundryindustry
4. Baking industry
5. Juice industry
Amylases
Amylases
Amylases
α-amylases
Amylases, glucoamylases
Cleaving starch molecules to
reduce the viscosity for
surface sizing in coatings, but
not used for dry strength
agent additive.
Desizing efficiently without
harmful effects on the fabric
Removing
resistantstarchresidues.
Degrading starch in flours
and controlling the volume
and crumb structure of bread
Breaking down starch into
glucose.
Clarifying cloudy juice,
especially for apple juice

6. Starch processing
7. Brewing industry
β-amylases
α-amylases
Cleaving α-1, 4-linkages
from non-reducing ends of
amylopectin and glycogen
molecules.
Producing low-molecular
weight carbohydrates, such
as maltose and “β-limit
dextrin”.
Hydrolyzing starch to reduce
viscosity.
Increasing maltose and
glucose content
According to a research report from Austrian Federal Environment Agency, Nearly 75% of the
total enzymes are produced by three top enzyme companies, i.e. Denmark-based Novozymes,
US-based DuPont (through the May 2011 acquisition of Denmark-based Danisco) and
Switzerland-based Roche.
PRINCIPLE:
The amylase catalyzes the hydrolysis of starch, usually at the 1,4-glycosidic bond cleaving of
two glucose units i.e. maltose .This reducing sugar reacts with PAHBAH in dilute alkaline media
upon heating at high temperatureto yield glyoxalbis (benzoylhydrazone) and
methylglyoxalbis(benzoylhydrazone) which develop a yellow colour. Addition of metal ions
increases the rate of the reaction and sensitivity of colorimetric method, thereby enabling to use
lowertemperatures and still develop a color of measurable sensitivity.
MATERIAL REQUIRED:
Solutions:
Stock solution of glucose and maltose of concentration 3mg/ml,8mM stock solutions of ions like
copper,calcium,nickel,zinc,cobalt,manganese,magnesium and iron.
Stock solution of PAHBAH(4-hydroxybenzoic acid hydrazide) reagent of concentration
15mg/ml (all the ion and reagent solutions are to be made with sodium tartrate and sodium
hydroxide solution (5g&2g in 100ml water)).

Buffers like HEPES of 50mM concentration pH-8,MOPS of concentration 50mM pH -7.8,BR (
BRITTON-ROBINSON) of concentration40mM pH-8,TRIS of concentration 50mM pH-8,
Amylase enzyme stock (1.6mg/ml),corn starch substrate of concentration 3mg/ml.
Instruments:
Spectramax M5 spectrophotometer, Pipettes, 96 well Micro titre plates, eppendorf master
cycler,PCR plates.
Introduction:The amylase catalyses the hydrolysis of starch, usually at the 1, 4-glycosidic bond
cleaving of two glucose units i.e. maltose. This glucose will form a complex with PAHBAH
reagent (colour intensity) which can be measured by calorimetric analysis and this takes place at
90ºc. Optimization experiments were conducted to improve the complex formation at 40ºc using
ions which enhances the activity.
Procedures:
10µl of sugar solution is added to 190µl of buffer to which 50µl of ion and 50 µl of reagentwere
added(prepared in alkaline media).The mixture was incubated for 20 minutes at 40ºC and the
plate was read in spectramax M5 at 405nm.This procedure is followed if reducing sugar is used
directly.
If enzyme is used then 10µl of enzyme is added to 40µl buffer and 50µl of substrateand
incubated for 20minutes at 40ºC. As a result we will have disaccharide units to which 50µl of ion
and 50µl of reagent is added and incubated for 20 minutes at 40ºC and sample is read at specific
wavelength i.e. 405nm.
In these experiments the reaction mixture was initially loaded into PCR plate and then
transferred to 96 well microtitre plate inorder to read the plate in spectramax M5.
RESULTS:
1. Identification of optimum wavelength for analysis:
Table 1:
300nm 320nm 340nm 360nm 380nm 400nm 420nm 440nm 460nm 480nm 500nm
PAHBAH 3.22 1.59 0.76 0.53 0.44 0.34 0.18 0.08 0.06 0.05 0.05
Glucose +
PAHBAH 3.35 2.67 1.83 1.61 1.50 1.10 0.52 0.17 0.09 0.07 0.06
Ca 3.63 3.13 2.34 2.07 1.97 1.92 1.65 1.14 0.65 0.38 0.32
Ni 3.46 3.36 2.56 2.03 1.49 0.98 0.60 0.36 0.30 0.26 0.23
Fe 3.44 3.89 3.43 3.13 2.49 1.83 0.91 0.32 0.18 0.15 0.13
Cu 3.52 3.93 3.62 3.37 3.08 2.40 1.38 0.63 0.41 0.32 0.28
Mn 3.44 3.39 2.58 2.28 2.04 1.63 1.03 0.50 0.29 0.18 0.13
Zn 3.38 3.13 2.35 2.20 2.10 1.58 0.76 0.23 0.11 0.07 0.06
Mg 3.39 3.33 2.48 2.40 2.38 1.80 0.83 0.22 0.09 0.07 0.06
Co 3.49 3.87 3.61 3.31 3.12 2.43 1.25 0.46 0.28 0.22 0.18

Dept. of Biotechnology, ANITS
Graph 1:
X-axis: wavelength, Y-axis: OPTICAL
Total reaction volume
Glucose concentration
Ion concentration in reaction mixtu
Reaction time& temperature
PAHBAH added in the presence of alkaline media (NaOH + Sod. Tartrate15mg/ml of alkaline
media).
Conclusion: The measurable wavelength identified for the spectr
range.
2. Effect of ion on complex formation (when glucose is used)
Note: one set is kept with normal reaction mixture and in other set equivalent
added instead of glucose.
Table 2:
Ion
with
glucose
Ca 0.546
Mn 0.566
Zn 0.603
Mg 0.891
Co 1.375
PAHBAH 0.367
OPTICAL DENSITY (at 405nm)
volume : 200µl
concentration in reaction mixture : 50µg/ml
in reaction mixture : 1mM
temperature : 20minutes at 40ºc
HBAH added in the presence of alkaline media (NaOH + Sod. Tartrate15mg/ml of alkaline
The measurable wavelength identified for the spectrumis 400nm to 420nm
2. Effect of ion on complex formation (when glucose is used)
one set is kept with normal reaction mixture and in other set equivalent volume
glucose w/o glucose
0.546 0.368
0.566 0.421
0.603 0.399
0.891 0.539
1.375 0.884
0.367 0.235
Page 8
at 40ºc
HBAH added in the presence of alkaline media (NaOH + Sod. Tartrate15mg/ml of alkaline
is 400nm to 420nm
volume of buffer is

Graph 2:
X-axis:ions, Y-axis:OPTICAL DENSITY (At 405nm)
Total reaction volume : 200µl
Glucose concentration in reaction mixture : 50µg/ml
Ion concentration in reaction mixture : 1mM
Reaction time& temperature : 20 minutes at 40ºc
PAHBAH added in the presence of alkaline media (NaOH + Sod. Tartrate15mg/ml of alkaline
media)
Conclusion: Comparison of signals is done when reaction is carried out in presence or
absence of glucose, and was found that signal is high in presence of glucose.
3. Effect of ions during reaction:
Note:The incubation was carried out with and without ions in two sets. For the later set
ions were added after incubation.
Table-3.1:
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Ca Mn Zn Mg Co PAHBAH
with glucose
w/o glucose

Table -3.2:
Graph 3:
X- axis: samples, Y-axis: OPTICAL
Ion concentration in reaction mixtur
Reaction time and temperature
Samples
Glucose+ PAHBAH
Ca
Cu
PAHBAH (reagent blank )
Samples
Glucose+ PAHBAH
Ca
Cu
PAHBAH
OPTICAL DENSITY (at 405nm)
volume : 200µl
and temperature : 20minutes
Before incubation
Glucose+ PAHBAH 0.848
1.770
3.161
(reagent blank ) 0.616
After incubation
PAHBAH 0.782
0.774
1.314
0.574
Page 10
: 200µl
50µg/ml
1mM
minutes at 40ºc

PAHBAH added in the presence of alkaline media (NaOH + Sod. Tartrate15mg/ml
of alkaline media)
Conclusion:Incubation in the presence of ion shows higher signal compared to without ion.
4. Experiments with glucose:
4.1. Determination of ideal glucose concentration:
Note:Experiment was performed with different glucose concentration range from 1µg/ml
to 200µg/ml with Zn and Mn at 1mM.
Table 4.1:
Graph 4.1:
X-axis: Glucose concentration, Y-axis: OPTICAL DENSITY (at 405nm)
0
0.5
1
1.5
2
2.5
3
0 50 100 150 200 250
Zn
Mn
Linear (Zn)
Linear (Mn)
Glucose
concentration Zn Mn
1µg/ml 0.7065 1.2258
5µg/ml 0.6134 1.1973
10µg/ml 0.5687 1.5199
50µg/ml 0.9284 1.8294
100µg/ml 1.3309 2.0163
200µg/ml 1.8322 2.3477
blank 0.3408 0.3584
ion(no buffer) 0.1129 0.2618

Ion concentration in re
Conclusion: The data observed a linear regression line with increase in glucose
concentration.
4.2. Determination of optimal ion concentration:
Table 4.2:
ion
concentration
50µM
100µM
200µM
500µM
1mM
2mM
blank
Graph 4.2:
X-axis: glucose concentration, y
volume : 200µl
: 20minutes at 40ºc
The data observed a linear regression line with increase in glucose
n of optimal ion concentration:
Zn Mn
w glucose w/o glucose w glucose w/o glucose
0.832 0.463 0.698
0.7527 0.3975 0.8488
0.7299 0.4398 0.9594
0.7481 0.4714 1.0544
0.7167 0.4351 1.2185
0.7471 0.4573 1.4895
0.372 0.345 0.3652
axis: glucose concentration, y-axis: OPTICAL DENSITY (at 405nm)
Page 12
at 40ºc
The data observed a linear regression line with increase in glucose
w/o glucose
0.5746
0.3904
0.8037
0.9194
1.156
1.2638
0.2548
(at 405nm)

Glucose concentration in reaction mixture : 50µg/ml
Ion concentration in reaction mixture : 1mM
Reaction time and temperature : 20minutes at 40ºc
Conclusion: Mn ion shows a linear regression, and observed a higher S/N ratio. Hence the
experiment was performed with different buffer to identify the ideal buffer with lower S/N.
4.3. Buffer selection:
Table4.3:
Effect of buffer on reducing sugar Assay:
Buffer
Glucose +ion+
reagent+ buffer
ion+ reagent+
buffer
ion+ reagent+
water
Glucose
+reagent+ buffer
TRIS pH 7 0.6635 0.487 0.215 0.223
MOPS pH 7.8 0.7556 0.4941 0.1973 0.2256
BR pH 8 0.4584 0.197 0.1951 0.061
HEPES pH 8 1.1588 1.045 0.2053 0.3304
TRIS - 50mM
MOPS - 50mM
BR - 40mM
HEPES - 50mM
Blank-1: to test the ion activity in absence of glucose in different buffers
Blank-2: to know the reaction response in absence of buffer required (helps in identify
the effect of buffer)
Blank -3: to compare the difference in amount of complex formation while in presence
and in absence of ion.

Graph-4.3:
X-axis: various buffers, Y
Glucose concentration in reaction mixture :
Ion concentration in reaction mixture :
Conclusion: It was found thatb
with reducing sugar.
5) Selection of ion:
Table 5:
Glucose
Mops-50mM
Mn 0.480
Zn 0.155
Cu 0.803
Ca 0.148
Co 1.305
Mg 0.189
Y-axis:OPTICAL DENSITY (at 405nm)
: 200µl
in reaction mixture : 50µg/ml
both BR buffer and MOPSbuffer are suitable for experiment
Glucose Maltose
BR-40mM Mops-50mM BR-40mM
0.480 0.310 0.619
0.155 0.088 0.507
0.803 0.742 1.089
0.148 0.108 0.398
1.305 0.667 2.003
0.189 0.123 0.515
Page 14
at 40ºc
suitable for experiment
40mM
0.478
0.365
1.017
0.227
1.363
0.305

Graph 5:
X-axis: sugar concentration, Y-axis:
Glucose concentration in reaction mixture
Maltose concentration in reaction mixture
Ion concentration in reaction mixture :
Conclusion: From the above experim
intensity with lower S/N ratio in BR buffer compare
precipitation at higher concentration
concentration.
6. Experiment with maltose:
Mops-50mM
axis: OPTICAL DENSITY (at 405nm)
: 200µl
From the above experiment it is observed that both Zn,Co and Cu
intensity with lower S/N ratio in BR buffer compared to MOPS but all them showed
at higher concentration. Further experimentswere carried out
w/o sugar
BR – 40mM
0.350 0.176
0.103 0.075
0.955 0.588
0.106 0.089
1.134 0.544
0.112 0.103
Page 15
and Cu shows high
all them showed
swere carried out at lower ion

6.1. Determination of maltose c
Table 6.1:
Maltose concentration with Co-
2mg
1.5mg
1mg
500µg
250µg
160µg
80µg
40µg
buffer blank
Graph 6.1:
:
X-axis: maltose concentration, Y
Conclusion: The ideal maltose concentration was found to be
6.2. Determination of ion concentration
Table 6.2:
ation of maltose concentration:
-100µM w/o CO
2.9952 0.4043
2.0874 0.2041
1.7838 0.1784
1.159 0.1089
0.894 0.0873
0.5189 0.0739
0.4482 0.0782
0.4734 0.0733
0.3412 0.3412
axis: maltose concentration, Y-axis: OPTICAL DENSITY (at 405nm)
: 200µl
The ideal maltose concentration was found to be in range of 160
of ion concentration:
Page 16
in range of 160-250 µg.

Co
Ion
concentration maltose 200µg
2mM 2.2581
1mM 1.6368
500µM 1.1777
200µM 1.0434
100µM 0.9371
50µM 0.7442
Graph 6.2:
X-axis: maltose concentration,
Maltose concentration in reaction mixture : 200µg/ml
Conclusion: 100µM of Co can be used to
7. Experiment with enzyme (purified):
Co Zn
w/o maltose maltose 200µg w/o maltose
2.2581 1.4257 0.3567 0.1027
1.6368 1.0322 0.3502 0.1018
1.1777 0.8947 0.3826 0.1056
1.0434 0.5256 0.3258 0.0983
0.9371 0.5472 0.3201 0.0951
0.7442 0.3828 0.3381 0.1068
concentration, Y-axis: O.D (at 405nm)
: 200µl
of Co can be used to get better signal with maltose.
7. Experiment with enzyme (purified):
Page 17
w/o maltose
0.1027
0.1018
0.1056
0.0983
0.0951
0.1068

Note: while tested with maltose both the buffers showing better performance but when tested
with enzyme MOPS is not giving significant result hence BR b
7.1. Determination of enzyme concentration:
Table-7.1:
Graph 7.1:
X-axis – amylase concentration, Y
concentration
80
40
20
10
5µg
2.5
1.25
0.625
0.3125
Substrate
(sub.) blank
Enzyme
(enz.)blank
while tested with maltose both the buffers showing better performance but when tested
with enzyme MOPS is not giving significant result hence BR buffer is finalized with Co ion.
7.1. Determination of enzyme concentration:
amylase concentration, Y-axis –OPTICAL DENSITY (at 405nm)
: 200µl
Amylase
concentration
with Co-100µM w/o CO
80µg 1.0378 0.132
40µg 0.827 0.1114
20µg 0.7855 0.1046
10µg 0.8198 0.0835
µg 0.7775 0.1025
2.5µg 0.7805 0.0895
1.25µg 0.7611 0.0829
0.625µg 0.7432 0.0903
0.3125µg 0.573 0.0794
Substrate
(sub.) blank 0.0595 0.0595
Enzyme
(enz.)blank 0.0638 0.0638
Page 18
while tested with maltose both the buffers showing better performance but when tested
alized with Co ion.
at 405nm)

Conclusion: 40µg/ml was found to be ideal enzyme concentration to get better signal.
Note: 40µg/ml of enzyme concentration used in the assay from
Table7.2:
BR buffer-Co ion concentration gradient
1mM
500µM
200µM
100µM
50µM
25µM
12.5µM
Graph7.2:
X-axis – amylase concentration, Y
40µg/ml was found to be ideal enzyme concentration to get better signal.
centration used in the assay froman enzyme stock of 1.6mg/ml
Co ion concentration gradient
enzyme+ substrate+
reagent
Substrate
+reagent
Enzyme +reagent
2.0332 0.811 1.2348
1.2941 0.5304 0.5763
1.0259 0.3376 0.5069
0.975 0.3233 0.3356
0.5956 0.239 0.2792
0.6055 0.2256 0.2677
0.5618 0.1453 0.1458
amylase concentration, Y-axis –OPTICAL DENSITY (at 405nm)
: 200µl
Page 19
40µg/ml was found to be ideal enzyme concentration to get better signal.
stock of 1.6mg/ml
Enzyme +reagent
at 405nm)

Conclusion: 100µM of cobalt (co
experiment.
8. Optimization of Incubation time
8.1. with Maltose:
Table-8.1:
Graph 8.1:
Co ion concentration in reaction mixture
maltose concentration
2mg
1.5mg
1mg
500µg
250µg
160µg
80µg
40µg
blank(ion+ buffer +reagent)
cobalt (co) ion concentration is optimized from the above
8. Optimization of Incubation time
axis: maltose concentration, Y-axis – OPTICAL DENSITY (at 405nm).
: 200µl
reaction mixture : 100µm
maltose concentration with ion without
ion
1.5104 0.1922
1.4309 0.1256
1.0286 0.1128
0.7587 0.0833
0.5211 0.0703
0.3655 0.0631
0.2591 0.0593
0.2461 0.0611
blank(ion+ buffer +reagent) 0.3082 0.3082
Page 20
ized from the above
at 405nm).
at 40ºc

Conclusion: Experiments were done
intervals i.e. 20, 40, 60 minutes but after comparing the results 20 minutes incubation time
was found to be ideal.
8.2. With enzyme:
Table-8.2:
Graph-8.2:
enzyme conc.
1.6mg/ml
0.8
0.4
0.2
0.1
0.05
0.025
0.0125
0.00625
blank(w/o enz
Blank (w/o subst
Experiments were done by incubating the reaction mixture at various time
20 minutes incubation (2nd step)
conc. with ion without ion
0.9138 0.126
0.8315 0.106
0.8022 0.0857
0.562 0.0925
0.7498 0.0976
0.7498 0.0954
0.6804 0.08
0.7805 0.0787
0.5489 0.0659
enzyme) 0.3979
o substrate.) 0.4195
Page 21
by incubating the reaction mixture at various time

X-axis: enzyme concentration, Y-axis: OPTICAL DENSITY (at 405 nm)
Co ion concentrationin reaction mixture : 100µm
Reaction time and temperature : 20minutes at 40ºc
Conclusion: Experiments were done by incubating the reaction mixture at various time
was found to be ideal.
9 Experiment in presence of detergent:
9.1. with detergent (1% detergent)
Table 9.1:
Graph9.1:
Maltose(mg/ml) with detergent w/o detergent
2 2.489 3.217
1.5 2.697 2.646
1 1.842 2.088
0.5 0.668 1.399
0.25 0.580 0.678
0.08 0.619 0.714
0.04 0.599 0.734

1. detergent blank 0.2157
2. buffer blank 0.4577
3. no ion+ detergent +reagent 0.1318
4. buffer + reagent 0.0873
5. detergent alone 0.0598
Conclusion:It was found that enzyme is showing better activity even in
detergent.
9.2. Triplicate assay with detergent
Table 9.2:
sample Absorbance at
1% detergent 0.329
no detergent 0.523
Enzyme
blank 0.209
Substrate
blank 0.255
Graph 9.2:
axis: maltose concentration, Y-axis: OPTICAL DENSITY (at 405 nm)
detergent blank 0.2157
buffer blank 0.4577
no ion+ detergent +reagent 0.1318
buffer + reagent 0.0873
detergent alone 0.0598
It was found that enzyme is showing better activity even in
with detergent:
orbance at 405 Average SD
0.387 0.309 0.342 0.040
0.626 0.518 0.555 0.061
0.227 0.242 0.226 0.017
0.249 0.260 0.255 0.005
Page 23
axis: OPTICAL DENSITY (at 405 nm)
It was found that enzyme is showing better activity even in presence of
% CV
0.040 12
0.061 11
0.017 7
0.005 2

X-axis: % of detergent,Y-axis: average OPTICAL DENSITY at 405nm
Co 100µM
BR buffer 40mM pH 8
Starch 3mg/ml
Amylase 40µg/ml
Detergent 1%
PAHBAH prepared in sod. Tartrate + NaOH
Conclusion: In order to avoid the experimental error final triplicate assay was performed
with the prescribed conditions (from previous experiment).
10. With PURPALD reagent:
Note: the experiment was performed in the presence of Purpald reagent instead of PAHBAH.
 Few experiments were conducted as done for PAHBAH and we came to a
conclusion that it requires an incubation for a long time at room temperature( as it
is a oxidation reaction, without heating)
 As compare with Purpald and PAHBAH the former one needs a longer incubation
time and slow rate of reaction it has been discontinued in the further assay.
Discussion:
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
1% detergent no detergent enz blank sub blank

After conducting each experiment certain analysis was made and finally a protocol for the assay
was derived. Here are the conclusions made after each experiment, when the first experiment
was done it was found that maximum intensity with low S/N ratio observed in between 400-
420nm so, 405nm wavelength is selected as optimumwavelength and after conducting few more
experiments, we came to a conclusion that better signal intensity was observed with ion during
incubation rather than adding after incubation. This proves the effect of ion in the reaction. Few
more experiments were conducted with glucose and was found that Zn,Ca and Cu ions were
giving better signal in comparison to other ions (some ions are not considered as they precipitate
in solution) and 50µg/ml glucose(final con.) and 100µM ion was found to be the ideal
concentrations for the assay.Optimization for different buffers were carried out and observed
thatB.R. and MOPS are found to be the suitable buffers for experiment with reducing sugar, and
in this case Cobalt ion was found to give better signal than Zn and Ca.
Amylase acts on 1, 4-glucosidic bond of starch and cleaves it into disaccharides units i.e.
maltose.So, in order to mimic the actual product the experiments which were done with glucose
earlier were repeated with maltose and the following conclusions were made i.e. if we use
100µM of co ion and 200µg of maltose in presence of B.R. buffer and PAHBAH we got better
signal when incubated for 20 minutes at 40ºc.
To mimic the actual reaction an experiment was done using purified enzyme and corn starch as
substrate and following conclusions were made. i.e.100 µM concentration of cobalt ion and
40µg/ml of enzyme in presence of B.R. buffer and PAHBAH reagent will give better signal
when incubated at 40ºc for 20minutes.
Finally, experiments were conducted in presence of detergent in order to check the enzyme
activity in the presence of detergent and it was found that enzyme shows a good activity even in
presence of 1% detergent.
Part -B
(Determination of Amylase concentration by Sandwich ELISA)
INTRODUCTION:
Enzyme-linked immuno sorbent assay (ELISA) is a test that uses antibodies and colour change to
identify a substance. The main purpose of an ELISA is to determine if a particular protein is
present in a sample and if so, how much. There are two main variations on this method: you can
determine how much antibody is in a sample, or you can determine how much protein is bound
by an antibody. The distinction is whether you are trying to quantify an antibody or some other
protein.

On basis of the procedure carried out ELISA are mainly classified into three types they are
Indirect ELISA, Sandwich ELISA, Competitive ELISA.
The sandwich ELISA quantifies antigens between two layers of antibodies (i.e. capture and
detection antibody). The sample which is to be measured must contain epitope capable of
binding to antibody, The advantage of Sandwich ELISA is that the sample does not have to be
purified before analysis, and the assay can be very sensitive (up to 2 to 5 times more sensitive
than direct or indirect ELISA) but, Sandwich ELISA procedures are difficult to optimize and
tested match pair antibodies should be used. This ensures the antibodies are detecting different
epitopes on the target protein so they do not interfere with the other antibody binding.
MATERIAL REQUIRED:
384 well Nunc Maxisorp plates
BUFFERS required for ELISA:
1. TBS-T buffer :
 1X TBS
 0.01%triton pH -7.5
 0.15MnaCl
 0.02M TRIS
2. 1X PBS buffer :
(pH-7.2)
 0.137M NaCl
 2.68mM KCL
 2.33mM KH2PO4
(Prepare required volume of buffers). Instrument: TECAN reader
Procedure:
Conjugate antibodies preparation: (using kit)
 Antibody solution of concentration 53mg/ml
 Prepare a stock of concentration 2mg/ml
 Add 10µl of LL-modifier to 100µl of 2mg/ml antibody stock sol.
 Add that 110 ml sol. to HRP vial & mix gently
 Cover with aluminium foil and keep it in dark place overnight
 Add 10µl of LL-quencher
 Leave it for 30 minutes at R.T.
 Add pierce peroxidise conjugate stabilizer (add 1µl for every 10µl of antibody used)
 Now the conjugate antibodies can be used for ELISA
 The antibodies are aliquoted into eppendorf tubes and stored at -20ºc until further use.

Steps involved in sandwich ELISA:
 Coating :
 Prepare a 10µg/ml concentration of Ab from anAb stock solution of
53mg/ml.
 Add 25µl of Ab solution into each well of the plate.
 Incubate the plate at -4ºc, overnight.
 Washing :
 Wash the plates with TBS-T buffer (90µl)
(You can give even a water wash)
 Incubate the plates at room temperature for about an hour.
 Blocking :
 Add small amount of TBS-T buffer and store the plate at -4ºc for
further use.
 Washing:
 Now, again wash the plates TBS-T buffer (100µl) and leave the empty
plates aside.
 Enzyme addition :
 Now, prepare enzyme dilutions (as required) and add 25µl of each
enzyme dilution into that empty plate and incubate it for about
30minutes.
 Washing :
 Wash the plates with TBS-T buffer for about 3 times.
 Addition of Conjugate Antibody
 Add 25µl of HRP conjugated antibodies
 Incubate for 1hour at R.T.
 Washing:
 Wash the plates with TBS-T buffer for about 3 times
 Add 25µl of TMB (substrate), incubate for 20 minutes at R.T.
 Colorimetric Reading :
 Read at 620 nm
(This, protocol is for 384 well, for 96 well the volumes should be doubled)

Results:
1. Protein linearity with various Ab concentrations :
Table 1:
Protein linearity with various Ab concentrations
LASB0000(ppm) 1:1000 1:2000
100 2.011 1.936
20 2.105 1.500
4 1.848 1.621
0.8 1.900 1.636
0.16 1.999 1.559
0.032 1.308 0.744
0.0064 0.676 0.259
0.00128 0.548 0.123
0.000256 0.424 0.095
0.0000512 0.421 0.080
0.00001024 0.398 0.084
0.000002048 0.431 0.098
Graph 1:
X-axis : enzyme dilutions , Y
Protein linearity with various Ab concentrations :
Protein linearity with various Ab concentrations
1:2000 1:3000 1:4000 1:5000 1:6000 1:8000 1:10000
1.920 1.208 0.713 0.375 0.131 0.060
1.786 1.165 0.680 0.386 0.129 0.059
1.667 1.060 0.635 0.342 0.116 0.059
1.691 1.030 0.587 0.349 0.120 0.055
1.249 0.726 0.441 0.245 0.090 0.050
0.432 0.246 0.160 0.095 0.053 0.038
0.136 0.096 0.078 0.051 0.046 0.040
0.083 0.062 0.050 0.038 0.039 0.039
0.067 0.055 0.046 0.036 0.040 0.036
0.062 0.053 0.049 0.038 0.037 0.035
0.065 0.054 0.050 0.038 0.040 0.038
0.088 0.068 0.064 0.046 0.044 0.042
dilutions , Y-axis :Absorbance – 620nm
Page 28
1:10000
0.060
0.059
0.059
0.055
0.050
0.038
0.040
0.039
0.036
0.035
0.038
0.042

2. ELISA optimization 1:3000 HRP-Ab :
Note:
 Enzyme dilutions were done from stock of 1600ppm and dilutions were made from
0.8ppm to 0.00039ppm (2 fold i.e. 0.8, 0.4, 0.2 …..)
 Ab of concentration , 1:3000 dilution is used (from stock of 2mg/ml
TABLE- 2:
ELISA optimization 1:3000 HRP-Ab (standard curve)
Enzyme(ppm)
OD at
620nm Avg SD
0.8 1.404 1.297 1.311 1.34 0.058
0.4 1.222 1.231 1.214 1.22 0.009
0.2 1.040 1.092 1.020 1.05 0.037
0.1 0.722 0.676 0.704 0.70 0.023
0.05 0.436 0.438 0.427 0.43 0.006
0.025 0.277 0.270 0.279 0.28 0.005
0.0125 0.180 0.174 0.176 0.18 0.003
0.00625 0.118 0.113 0.125 0.12 0.006
0.003125 0.093 0.093 0.089 0.09 0.002
0.0015625 0.076 0.083 0.090 0.08 0.007
0.00078125 0.068 0.062 0.064 0.06 0.003
0.000390625 0.060 0.060 0.059 0.06 0.000
Blank 0.075 0.050 0.053 0.06 0.014
GRAPH-2:

X-axis : enzyme dilutions , Y
3.ELISA optimization 1:2000 HRP
Table-3:(Determination of unknown sample
ELISA optimization 1:2000 HRP-Ab
concentration(ppm) Amylase(pur
Blank 0.065
1.95313E-05 0.065
3.90625E-05 0.070
0.000078125 0.072
0.00015625 0.083
0.0003125 0.087
0.000625 0.100
0.00125 0.141
0.0025 0.227
0.005 0.338
0.01 0.530
0.02 0.805
0.04 1.121
0.08 1.426
0.16 1.824
axis : enzyme dilutions , Y-axis : Absorbance – 620nm
ELISA optimization 1:2000 HRP-Ab
(Determination of unknown sample using standard curve)
Ab
Amylase(purified) Amylase(supernatant)
0.070
0.063
0.075
0.073
0.079
0.097
0.135
0.168
0.239
0.405
0.584
0.922
1.270
1.564
1.804
Page 30

Graph-3:
X-axis : enzyme dilutions , Y-axis :Absorbance – 620nm
0.922 O.D. - 0.0359ppm (0.922/ slope of standard curve i.e. 25.741)
Total protein - 146.57ppm (0.0359 * dilution factor i.e. 2^12
)
Discussion:Sandwich ELISA experiment was done in order to estimate the protein
concentration by using the antibody specific to the enzyme, after conducting few experiments the
unknown protein concentration was found to be 147.56 ppm (app.) at 2^12
dilution (stock
assumed to be 100ppm concentration).
y = 25.74x + 0.174
R² = 0.949
y = 29.02x + 0.202
R² = 0.951
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
0 0.01 0.02 0.03 0.04 0.05
Amylase(pur) Amylase(sup)

REFERENCES:
 Gordon E .Anthon, Diane M.Barrett (2002),determination of reducing sugar with 3-
methyl-2-benzothiazolinonehydrazone,dept. of food sci. and tech. ,carlifonia,published
online ,may 9
 Shuangli, Xiaofeng Yang et al...(2012), Technology prospecting on enzymes,
computational and structural biotechnology journal, vol.2, issue 3.
 M.Lever (1972), calorimetric and flurometric carbohydrate determination with p-
hydroxybenzoic acid hydrazine, review paper.
 Matti leisola, jouni jokela et al.(2010),Industrial use of enzymes, physiology
&maintenance ,vol-2
 Fariha Hassan, Aamer Ali shah et al., (2010), Enzymes used in detergent, African journal
of biotechnology, vol.9 (31).
 Arvind Duggat, Kakli Dey et al., (2013), Industrial enzymes-present status and
perspectives for India, journal of scientific and industrial research, vol. 72.
 Enzymes at work (pdf. file), Novozymes.
 Wikipedia.

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