Project on Enzyme

I take this opportunity to express my deep sense of gratitude to my project guide Dr. Padul M.V. under whose guidance the present dissertation was completed. I shall ever remain grateful to him for his scholastic guidance, helpful and constrictive criticism during the entire period of there investigation.
I fee highly indebted to Dr. A.D. Chaugale, Dr. Baladhye and miss. Shaikh R.R. for her Valuable suggestion and instruction.
I must place on record my sincere and grateful thanks to Proff. M.T.Patil and Prof. Sable for their valueable contribution in naming and identification of plants.
I wish to express my profound gratitude to Prof. H.M. Humbe H.O.D. Chemistry for providing necessary facility during the entire course of this project.
I also express my sinsiour thanks to the teaching and non teaching staff of bio chemistry, micro biology, botony for their valuable suggestion and mindful help. Extended to me for getting the work done.
I would remiss if do not acknowledge my debit to my friend and class mate for their wonderful contribution and rendered to me during the experimental work.
No words and thanks to enough to express my heartfelt filling of love and indebtness to parent and brother for there sincere efforts in successfully keeping the best selling through most stable envoirment for their sacrifice in moulding me to a learn citizen




Mr. G.R. Sambare








ABSTRACT


Seed leaves and flower tissue were extracted in P.V.P. the extract were screen qualitatively dot-blot agar plate technique for amylase inhibitor were further analyzed for quantitative estimation of Amylase inhibitor activity by DNSA method. Endogenous amylase and amylase activator also present within the tissue. these are detected by developing specific protocol for each.
The inhibitors were separated in polyacrylamide gel containing 0.5% soluble starch by electrophoresis and visualized by incubation of the gel in salivary amylase solution and staining with iodine. Starch in the gel is hydrolise by amylase during incubation but starch in vicinity of amylase inhibitor is protected from hydrolysis and appear as the blue band after staining.

Keywords: - Amylase inhibitors, Enzyme, Endogenous amylase, Amylase activator, Electrophoresis,















INTRODUCTION

α-Amylase inhibitors:-
Microorganisms, higher plants, and animals produce a large number of different protein inhibitors of α-amylases in order to regulate the activity of these enzymes. These inhibitors can be grouped into six classes based on their tertiary structures. Lectin-like, knottin-like, cereal-type, kunitz-like, γ-purothionin like and thaumatin like inhibitors, some of these inhibitors act by directly blocking the active centre of the enzyme at various local sites.
In animals, α-amylase inhibitors reduce the glucose peaks that can occur after a meal, slowing the speed with which α-amylase can convert starch to simple sugars until the body can deal with it. This is of particular importance in people with diabetes, where low insulin levels prevent extra cellular glucose from being cleared quickly from the blood. Therefore, diabetics tend to have low α-amylase levels in order to keep glucose levels under control, except after taking insulin, which causes a rise in pancreatic α-amylase.
Plants also use α-amylase inhibitors as a defense strategy. These inhibitors impede the digestive action of α-amylases and protinases in the insect gut, thereby acting as insect anti-feedants. As a result, α-amylase inhibitors have potential in various fields, including crop protection and the treatment of diabetes.( Marshal JJ, Lauda)
What do they do?
Amylase inhibitors are also known as starch blockers because they contain substances that prevent dietary starches from being absorbed by the body. Starches are complex carbohydrates that cannot be absorbed unless they are first broken down by the digestive enzyme amylase and other, secondary, enzymes. They are claimed to be useful for weight loss. But when they were first developed years ago, research did not find them very effective for limiting carbohydrate absorption. Later, however, highly concentrated versions of amylase inhibitors did show potential for reducing carbohydrate absorption in humans.
Purified starch blocker extracts. When given with a starchy meal, have also been shown to reduce the subsequent rise in blood sugar levels of both healthy people and diabetics. The is effect could be helpful in the treatment of blood sugar disorders.( Choudhary A, Maeda)
Where are they found?
Amylase inhibitors can be extracted from several types of plants, especially those in the legume family. Currently available amylase inhibitors are extracted from either white kidney bean or wheat.
The relative inefficacy of alpha-amylase inhibitors in affecting human digestion of starch has been highlighted by reacent scientific and public controversy over the commercial sales of so-called starch-blockers or slimming pillis alpha-amylase and its inhibitors is drug-design targets for the development of compounds for treatment of diabetes obesity and hyperlipaemia studies of the structures of the numerous enzyme inhibitors found in cereal grains have led to the recognition of a super family of homologous proteins which includes inhibitors of alha-amylase, proteinase and bifunctional inhibitors of alpha-amylase, proteinase and bifunctional inhibitors active against two or more classes of enzymes.(Alam and Gourinath, 2001, Octavio and Rigden 2002 Richardson, 1991)
The first alpha-amylase inhibitor determined was that of the monomeric 13 kD known as 0.31 form, from wheat (kashlan and Richardson, 1981) other dimeric 0.19, 0.23, 0.28, 0.53 form of weat inhibitors of exogenous alpha-amylase were later shown (oneda et al 2004, kondo and ida1995, Roy and Gupta 2000, Richardson, 1991, Octavio and Rigden 2002)
The favored hyupotheses about physiological roles fo the enzyme inhibitors in seeds is that they act as storage or reserve proteins as regulators of endogenous enzyme or as defensive agents against the attacks of animal predators and insect or microbial pests. It seems likely that in certain species these proteins may fulfill a combination of these proteins may fulfill a combination of these function(Octavia and Rigden 2002, Octivio and Rigden 2000, Richardson 1991) Also plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plant against pests (Octavio and Rigden2002)
Nutritional and metabolic effects of enzyme inhibitors certainly some of inhibitors certainly some of inhibitors found in cereal and legume seeds can inactive the salivary and pancereatic enzymes of human (pick and wober) and their sucseotibility to be rather variable (singhand bbkundel, 2001) many are destroyed by cooking but some retain inhibitory activity even after baking (Richardson 1991)
Amylase inhibitors present in seeds currently used as food present few nutritional problem for healthy people but may have some toxicological significance in the diets if infants who have a lower production of pancreatic alpha-amylase than adults and for patients with impaired peptic or gastric function (brietender and rauduer ,2004. richrdson ,1991 shewry etal2001) also one inhibitor of insect alpha-amylase isolated from barley flour is the major allergen associated with bakers asthma disease (barber et al 1989) the inhibition is strictly competitive and in the1:1 complexes aloof the activities of the enzyme are completely abolished .
Crystallographic nuclear, magnetic resonance (NMR) and mechanistic studies all indicates that the inhibitors act as highly specific substrate for the enzyme they inhibit at a unique peptide bond called the reactive site peptide bond .
The reaction mechanics involved in the inhibition of alpha-amylase by plant protein inhibitors are not clearly understood (silan 1986) but there are suggestion that reducing sugars which are covalently bound to the inhibitor polypeptide chain may play a major role in the mechanism or that the inhibitor may induce confermational changes in the enzyme molecule.















Materials:-
Different part of the plants including leaves, flower, seeds were collected from local region. Some sample were collected from Nanded ,Rahuri,Pathardi total 146 sample were collected.
Start was from qualgen fine chemical company Bombay India
Polyvinylpolypurrolidone (P.V.P) was from Sigma
Human saliva was diluted and used as salivary amylase all other chemicals were of the highest purity available
LEAVES:
Sr. no Common name Botonical name
1 Sitaphal Annona sequmusa
2 Subhabhul Leueaene lapisiliqua
3 Errand Risinus comnuis
4 Naginiche pan Piper bettne
5 Bhabhul
6 Ghaneri Lantana camera
7 Gawti chaha Cymbopogan flexuosus
8 Aalu Calocasia esculanta
9 Rui Calotropis procera
10 Kadhipatta Murraya kotnigii
11 Gahu Triticum aestivum
12 Papai Papaya indica
13 Amba Mangifera indica
14 Mohair Brassia juncea
15 Naral Coccus nucifera
16 Jambul Syzium cumini
17 Aalu Leaves Calocasia esculanta
18 Harbara Cicer aeriantum
19 Dhol Amba
20 Nimbu Citrus cemani
21 Palak Spinach
22 Kadulimb Azadirecta indica
23 Tobacco Nicotiane tobacum
24 Tulas Ocimum Americanum
25 Tarwad Cassia auriculata
26 Kardali Canna indica
27 Wel
28 Nishigandha Polianthus tuberosa
29 Manjiri Ocimum Americanum
30 Tomato Lycopersicon escunentum
31 Beshram Ipomea
32 Sunflower Helianthus annus
33 Chandan Santalum album
34 Maka Zea mays
35 Halad Curcuma aromatica
36 Sadaba Runca chalepensis
37 Ratali Ipomeabatatas
38 Bartodi Morinda, citrifolia
39 Apte Bahunia
40 Palas Bulea monosperma
41 Biba Semecarpus anacardium
42 Rohini
43 Acasia Chandra
44 Agav Americana
45 Glyrisidium capium
46 Ipomea
47 Carissa carandus
48 Sag Tectona granvis
49 Maniplant
50 Cactus
51 Tur Cajanus cajan
52 Pimple

FLOWER:

1 Dhotra Datura alba
2 Jaswand Hibicus rosasinensis
3
4 Tarwad Cassia auriculata
5
6
7 Kaneri Nerium indicum
8 Zendu Tagehun erecta
9 Rose Red
10
11 Rui Calatropis procera
12 Chafa Michella champaka
13
14
15 Jai Jasminium auricylamn
16 Allamnda
17 Shewga Moringa olifera
18 Gandhar
19 Boganwel Boganvillia spectibilis
20 Kunda
21 Nishigandh Polyanthus tuberose
22
23 Gulbakshi Mirabilis jalapa
24 kardali Canna indica
25
26 Ghosali Lutta aegeyptia
27 Danger
28 Gerbera
29 Aboli Crossendra undilifolia
30 Sadafully Catharathus roses
31 Biliat
32 Tur Cajanus cajan
33 Shevari Sesbania sesban
34 Ghevda Psophocarpus tetiagomolobes
35
36
37 Palas Butea monosporne
38 Sunflower Tridax procambers
39
40 Khajawe
41 Sweet potato Ipomebatata
42 Kandhar
43 Lakh Acacia
44 Sag Tectona Gran
45 Chirisidium caprsium

SEED:
1 Mug Phaseomus aureus
2 Mahsoor Lens esculenta
3 Macca Zea maize
4 Chawali Vigna sinensis
5 Udit Phaseolus mango
6 Tur Cajanus cajan
7 watana Pisum Sativum
8 Hulga
9 Jawari Sorghum valgare
10 Tag
11 Methi Trigonella toenum
12 Ubal bee
13 Math
14 Soyabeen Glycine max
15 Karale
16 Kabuli Harbala Cicer grientum
17 Chinchuka Tamarindus indica
18 Airnda Ricimus communis
19 Tandul Orizha Sathiva
20 Bhagar
21 Dudhi Bhopla
22 Kharbuz Cacumis melo
23 Gokarn Clitonia kleio
24 Rale
25 Wange Solalum righhi
26 Khas khas
27 Shengadane Arachis hypogea
28 Coffee Caffia Arabica
29 Danger
30 Mohari Brassica kmphesis
31 Rajgira Amaranthus
32 Mire Piper nigrum
33 Rajma
34 Elayachi Elettoria cordomomum
35 Tulas Ocium santum
36 Hawari
37 Jira Caminum cyminum
38 Dhane Corriander sativa
39 Mirchi Capsium annum
40 Apte Bahunia racemose
41 Badishop Foenicumum valgari
42 Jawas Alhagi pseudalhagi
43 Bajari Pennisenem typhoids
44 Ghosale Laffer cylindrical
45 Sabja Ocimum bascilium
46 God babhul Accacia
47 Gahu Triticum aestivam






















METHOD
Extraction of amylase inhibitors:-
extraction if amylase inhibitors leaves, flower, seed are done by same procedure.
1. Decorticated seeds of all sample were dried and ground in blender to obtain fine flour.
2. Leaves are crused making the fine as possible as in small amount of acetone.
3. Same procedure was used for the flower sample.
4. After removing pigments all the sample are kept in hexane for overnight to remove the fatty material.
5. This procedure was repeated 2 to 3 times.
6. Then samples are carefully dried and packed.
7. Proper method for numbering and identification was used
8. The defatted seed powder was stirred with 1% PVP in 1:5 ratio for 48 hour.
9. Inclusion of PVP helpful in removal of phenolics from extract.
10. The suspension was centrifuged on cooling centrifuged at 10000 rpm for 20 min at 4oC
11. addition of PVP and centrifugation was repeated twice for same sample.
12. The clear supernatant contained amylase inhibitors was stored in freeze and used for analysis.
(Here after mentioned as partially purified extract.)












DOT BLOT INHIBITOR ASSAY:-
1. Extracted sample was tested on agar-starch plat in different concentration for screnning.
2. Using the enzyme (Amylase) Buffer (6.8PH) inhibitor at different proportion dot-blot assay was done.
3. For that 2% agar and 1% starch was used.
4. These are screening method due to this reaction sample which show inhibition are repeated once again and confirm that they show inhibition for amylase.
5. Among the 146 sample 58 sample shown inhibitions.
6. In that these are further categorized in strong inhibitor, medium inhibitor, weak inhibitor.
7. In 146 samples 27 samples are strongly inhibitor, 22 samples are inhibitor, and 9 samples are weak inhibitor.
8. After the screening of 146 samples the extracts which show strong inhibition and medium inhibition was taken for their amylase inhibitor assay by DNSA.















AMYLASE INHIBITOR ASSY :
Amylase activity was assayed by measuring liberated maltose. amylase inhibitory ativity was assyed by measuring reduction in maltose liberated by salivary amylase using dinitrosalisylic acid reagent.
One amylase activity unit is defined as activity resulting in to liberation of 1 mg of maltose from starch at ph 6.9 at 37o C in 3 min.
one amylase inhibitor unit is one amylase unit inhibited under the given assay condition.
In this assay same extract showing the exact inhibition but here again same extract does not show inhibition.
The samples which show inhibition are again tested for the endogenous amylase and amylase activator.


















ENDOGENOUS AMYLASE:-
1. Presence of amylases in biological sample particularly from plant case a major problem in detection of amylase inhibitor. The detection of endogenous amylase is possible with DNSA method.
2. In that tow test are taken in one reaction was arrested by addition of DNSA and in anther the reaction are carried out complete and then DNSA was added.


AMYLASE ACTIVATOR:-
Amylase activator also present within the extract there can be detected by DNSA.
In one test tube enzyme, buffer and substrate are added and in another activator was added with enzyme, buffer substrate. Incubate the both test tube and add DNSA activeter was add in first test tube after incubation the difference in OD indicated the amylase activator.
















ELECTROPHORETIC SEPRATION OF AMYLASE INHIBITOR :-
Amylase inhibitors in the partially purified extract were analyzed on a vertical slab gel electrophoresis system in 7% polyacrylamide gels containing 0.5% soluble starch without stacking gels containing 0.5% soluble starch without stacking gel. For that tris-glycine (PH 8.9) both in gel and electrode tank was used.


VISUALIZATION OF AMYLASE INHIBITORS:-
After electrophoresis gels are placed in 20mm phosphate buffer (PH 6.9) containing 6.7mm NaCl for 5-10 mm for equilibration and incubated in salivary amylase in phosphate buffer (PH 6.9) for 30 min at 37 C. after incubation the gels are wash with D/W and placed in iodine solution. (10 MM iodine in 14mm KI) for 4 to 5 min.
Exact band of amylase inhibiter way not found.



















RESULT AND DISCUSSION:-
Amylase inhibitors are detected by using three different concentrations the strong inhibitor show the inhibitor at all three concentration. Where the weak inhibitor show inhibition only at low concentration of amylase by using this technique determination of amylase activator is also possible. Same extract were help to the activation. amylase hydrolyzes the starch present in the agar. After staining with iodine. It show white area indicate that it is not inhibitor. When these area are broad than the control, then it is activator

DOT-BLOT ASSAY :-





TEST ENZYME BUFFER INHIBITOR
CONTROL 40 40 -
STRONG 40 28 12
MEDIUM 28 24 28
WEAK 12 28 40





SAMPLE NO TYPE OF INHIBITION
Seed- 1,4,7,8,11,9,23,24,26,27,42,43,15,19 * * *

25,30,31,28,43,3,5, * *

6,12,13,15,16,19,20 *

Flower-
1,2,8,10,20,23,27,30 * * *

5,6,7,11,43 * *

9 *

Leaves-
25,29,32,35,49, * * *

4,18,31,33,36,41,46,47,48,49 * *

26 *

*** = Strong Inhibitor
** = Medium Inhibitor
* = Weak Inhibitor







The extracts which show inhibitions for amylase are tested by DNSA assay for each sample two test are done. In first test extract were added after incubation and another, extract added before incubation. amylase inhibitory activity was assayed by measuring reeducation in maltose liberated by salivary amylase using DNSA reagent

AMYLASE INHIBITOR (DNSA)
TEST ENZ EXTRACT BUFF SUB EXTRACT DNSA
(ML) DILU
(ML) O.D
Control - - 1.0 0.5 Incubation for twenty min - 1 Bwb for 10 min 2 -
Test-1 0.4 - 0.5 0.5 0.1 1 2 -
Test-2 0.4 0.1 0.5 0.5 - 1 2 -

SAMPLE T1 T2 SAMPLE T1 T2
1 S2 0.53 0.62 L31 0.77 0.87
2 S3 0.49 0.39 L32 0.79 0.85
3 S5 0.44 0.50 L33 0.78 0.78
4 S7 0.84 0.81 L35 0.80 0.77
5 S8 0.97 1.02 L36 0.76 0.83
6 S9 0.85 0.85 L41 0.89 0.90
7 S10 0.83 0.81 L46 0.93 0.86
8 S12 1.15 1.19 L47 0.97 1.01
9 S13 0.86 0.84 L49 0.88 0.85
10 S16 0.84 0.84 F1 1.38 1.42
11 S19 0.85 0.85 F2 1.40 1.30
12 S24 0.62 0.55 F6 1.38 1.35
13 S26 0.75 0.58 F8 1.41 1.40
14 S27 0.56 0.43 F9 1.31 1.38
15 S28 0.96 0.78 F10 1.40 1.38
16 S30 1.01 0.97 F23 1.11 0.98
17 S31 0.65 0.59 F26 1.32 1.38
18 S4 0.76 0.73 F29 1.07 1.14
19 S42 0.63 0.53 F30 1.19 0.93
20 S43 0.58 0.67 F39 1.28 1.36
21 L4 0.80 0.74 F42 0.98 1.03
22 L18 0.78 0.79 F43 1.17 0.97
23 L26 0.77 0.74
24 L29 0.78 0.75

Amylase Inhibitor:-
S3, S24, S26, S27, S28, S31, S4, S42, L4, L26, L46, L49, F2, F23, F30, F43.


Presence of amylases in biological sample particularly from plant is a major problem in detection of amylase inhibitors. The detection of amylase inhibitors in sample containing endogenous amylases is possible. Endogenous amylase in sample can be detected. In one test tube DNSA are added before incubation and reaction are stopped. In another reaction are carried out. Difference between the O.D. indicate the concentration of endogenous amylase.











ENDOGENOUS AMYLASE :

TEST EXTRACT
(ML) BUFF.
(ML) DNSA
(ML) SUB
(ML)

Incubation
20 min DNSA
(ML)
Boiling
Water
Bath
10 min DILU
(ML) O.D.
Control - 1.0 0.5 1 2 -
T1 0.1 0.5 1 0.5 - 2 -
T2 0.1 0.5 0.5 1 2 -



SAMPLE T1 T2 SAMPLE T1 T2
S3 0.80 0.93 L35 0.63 0.58
S24 0.90 0.94 L49 0.51 0.52
S26 1.26 1.29 L33 0.60 0.63
S27 0.81 1.31 L10 1.06 0.98
S30 1.40 1.49 F2 0.78 0.81
S43 1.40 1.53 F8 0.93 0.95
S5 0.89 1.20 F10 1.10 1.17
S42 0.51 0.84 F23 1.03 1.07
L4 0.41 0.80 F30 1.40 1.51
L26 0.50 0.58 F43 0.84 0.97

Amylase activator is also present within the extract. This can also detect. In one test tube extract were added after addition of DNSA and in another test tube extract were added before incubation. Differences between those indicate the presence or absence of amylase activator.




AMYLASE ACTIVATOR

TEST ENZ
(AMY) BUFF.
(ML) ACT.
(ML) SUB
(ML)
Incubation
20 min DNSA
(ML) ACT.
(ML)
Boiling
Water
Bath
10 min DILU
(ML) O.D.
Control - 1.0 - 0.5 1 - 2 -
T1 0.4 0.5 - 0.5 - 0.05 2 -
T2 0.4 0.5 0.05 0.5 1 - 2 -

SAMPLE T1 T2
L31 0.42 0.50
L32 0.52 0.54
L19 0.56 0.70
L25 0.60 0.58
S43 0.57 0.72
S12 0.98 1.09
S8 1.01 1.10
F9 1.29 1.41

Salivary amylase inhibitors in extract also separated by using starch polyacrylamide gel. But in many attempt the amylase inhibitor bands were not found clearly. The gels are incubate in the amylase solution, amylase hydrolyze the starch as it enters the gel. However, starch in the vicinity of amylase inhibitor band is protected from hydrolysis due to inhibition band is protected from hydrolysis due to inhibition of amylase and appears as blue band after staining with iodine. The size and intensity of blue bands correspond to the extent of inhibition of amylase which depends upon the concentration and activity of amylase inhibitor protein in gel.
This method is sensitive entire procedure takes about one hour after electrophoresis run. In the particle purification of samples by ammonium sulfate fractionation. Increase the possibility of band formation by enriching amylase inhibitory activity. Most of the amylase inhibitor is temp sensitive. So storage at cooling condition is necessary.
Albumins in the seed extract also inhibit starch-iodine complex formation by sequestering iodine and may be confuse for amylase activity bands. In the gel same time starch having the tendency to form clumps and precipitate during polymerization of gels. Slow polymerization and overnight keeping of gels prior to run help in even distribution of starch in the gel.
However, our attempts on visualizing amylase inhibitor band in starch poly acryl amide gas were unsuccessful.



























REFRANCES

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7. Octavio, L. and D. Rigden, 2002. Plant amylase inhibitos and their interaction with amylases. Eur. J. Biochem. 269:397-412
8. Octiva L. And D. Rigden, 2000. Activity of wheat amylase inhibitors towards bruchid. Amylase and structural explanation of observed specificities. Eur. J. Biochem, 267: 2166-2173
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