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DECLERATION

I, Miss. MANSI YADUVANSHI declare that this dissertation work “Isolation, Partial
Purification, Immoblization And Kinetic Study Of L- Arginase From
Tricophytionspp’’.Ms.MansiYaduvabshi being submitted for the degree of B.Sc. in
Industerial Microbiology, is my own project work carried out under the supervision and
guidance of DrAbhinavshrivasatav Department of Biotechnology, College of Life Science
,CHRI Campus Gwalior 474009, M.P. Gwalior.

DATE: MANSI YADUVANSHI


CONTENT

1. INTRODUCTION

2. REVIEW OF LITRATURE

3. AIM OF STUDY

4. MATERIALS AND METHOD

5. RESULT

6. CONCLUSION

7. REFERENCES
CANCER

What is Cancer?
Cancer is a class of diseases characterized by out-of-control cell growth. There are over 100 different

types of cancer, and each is classified by the type of cell that initially affected.

Cancer harms the body when damaged cell divide uncontrollably to form lumps on masses of tissue

called tumors (except in the case of leukemia where cancer prohibits normal blood function by

abnormal cell

division in the blood stream). Tumors can grow and interfere with the digestive, nervous, and
circulatory systems and they can release hormones that alter body function. Tumors that stay in one
spot and demonstrate limited growth are generally considered to be benign.

(More dangerous, or malignant, tumors form when two things occur)


1. A cancerous cell manages to move throughout the body using the blood or lymph systems,

destroying healthy tissue in a process called invasion .

2. That cell manages to divide and grow, making new blood vessels to feed itseif in a process called

angiogenesis.

When e tumor successfullyspreads toother parts of the bodyand grows, invading ant other healthy

tissues, it is said to have metastasized, this process itself is called metastasis, and the result is a

serious condition that is very difficult to treat.

In 2007, cancer claimed the lives of about 7.6 million people in the world. Physicians and

researchers who specialize in the study, diagnosis, treatment, and prevention of cancerare called

oncologists.

ARGINASE

L-Arginase (arginine amidinase, canavanase, L-arginase, arginine transamidinase EC 3.5.3.1) is a

manganese-containing enzyme that catalyzes the deamidation of L-arginine to l-ornithine and urea It

is an essential enzyme in urea cycle. Arginase is widely expressed in various worms, molluscs,

fishes, bac41 LArginase has received significant attention due to its wide range of activity against

cancer cells. It also plays important role in the treatment of neurological disorders, allergic asthma

and rheumatoid arthritis. Unlike normal cells, some of the cancer cells do not demonstrate the -

argininosuccinatesynthetase which is involved in the synthesis of 1-arginine, thus it is dependent on

the exogenous supply of the amino acid for their growth and survival.Arginase (EC 3.5.3.1) is a 105

kDhomotrimeric enzyme which requires an intact binuclear manganese cluster for its catalytic

activity 7 .Arginase is the terminal enzyme of the urea cycle among the six other enzymes. Interest

in the arginases as possible regulatory enzymes is growing because of their potential for regulating the

availability of arginine for the synthesis of NO, polyamines, agmatine, proline and glutamate 8

.Arginase was discovered in mammalian liver by Kossel & Dakin (1904) who showed that the
products of its action are ornithine and urea. Since then, the enzyme has attracted a great deal of

interest from many points of view. The enzyme has been found to exist in two forms that have

evolved with differing tissue distributions, metabolic functions and subcellular locations in mammals

9 . The cytosolic form, Arginase I is found predominantly in the liver or hepatic cells and is

important in ureogenesis. Arginase II is a mitochondrial enzyme that is extrahepatic and more

widely distributed in numerous tissues, for example, kidney, and skeletal muscle 10. It may be found

at lower levels in macrophages, lactating mammary glands, and brain. Important roles of Arginase II

have been reported to be the biosynthesis of polyamines, the amino acid ornithine, proline and

glutamate and in the inflammatory process. Genetic “knockout” experiments suggest that arginase II

functions in L- arginine homeostasis by regulating L-arginine concentrations for cellular

biosynthetic reactions such as nitric oxide biosynthesis. The human type I and type II arginases are

Cancer is a disease characterized by abnormal proliferation of cells. It is one of the leading causesof

death in the world, and it is of serious problem to people living in both the developing anddeveloped

nations 1,2,3. Both benign and malignant tumors manifest as uncontrolled proliferation,but the latter

are distinguished by their capacity to dedifferentiate, their invasiveness and theirability to

metastasise. Cancer can be cured without much problem if it is diagnosed at the earlierstages. The

treatments include chemotherapy, radiation and other forms of therapy. Every therapyhas its own

advantages and disadvantages. Nutritional starvation of cancer cells have been triedby many

researchers and have been found to be successful in a few cases. Cancer cells haveabsolute

requirements for nutrition, a few of its nutrients it can synthesize and for a few others itdepends on

the host cell. Amino acids like methionine and arginine come under these categoriesrelated by 58%

sequence identity and are immunologically distinct.

Arginase is the terminal enzyme of the urea cycle allowing the disposal of nitrogenouswaste from

protein catabolism by catalyzing the hydrolysis of arginine in the final stage of theurea cycle. The

enzyme exists in 2 forms that have evolved with differing tissue distributions,metabolic functions

and subcellular locations in mammals. The cytosolic form, Arginase I, isfound predominantly in the
liver cells and is important in ureogenesis. Arginase II is amitochondrial enzyme that occurs mainly

in extrahepatic tissues, including kidney, brain andskeletal muscle. Apart from being ubiquitously

present in many mammals, there are a widerange of microbial sources of arginase, including bacteria

(many Bacilli, Agrobacterium-

Rhizobium group, cyanobacteria, Proteus spp.)[1, 2], protozoa (Entamoebahistolytica,

andPlasmodium falciparum) [3, 4], yeast (Saccharomyces cerevisiae) [5] and fungi

(Neurosporacrassa, Aspergillusnidulans, Agaricusbisporus) [6-8].Arginase is also found in

plants(Lathyrussativus, Vitisvinifera, Arachis hypogea, Lycopersiconesculatumetc) [9-12]though

plant arginases form a separate group in the Arginase superfamily and are not muchclosely related

(in terms of sequence homology) to non-plant (vertebrate, fungi and bacteria)arginases.

The fact that arginine deprivation has been of considerable therapeutic significance in treating

different forms of cancer has been reported by many researchers [e.g.13-14]. Unlikenormal cells and

tissues, many different tumor cells are auxotrophic for arginine, because theycannot synthesize

arginine endogenously [15-17]. The mechanism of arginine auxotrophy inthese tumors is primarily

related to the downregulation of the ASS gene at the transcriptionallevel by methylation of its

promoter sequence [18]. In vitro arginine depletion, either througharginase or use of arginine-free

medium, leads to rapid tumor death in a wide range of tumorcell lines because of the high

dependency of cells on an adequate supply of this aminoacid[19].

The in vitro anti-tumor activity of arginase has been well documented since the 1960s
[20]
. Arginase released from lipopolyssacharide and zymosan-stimulated macrophages can
beresponsible for the death of V79 Chinese hamster lung cells, L5178Y lymphoma cells, andHSN
hooded rat sarcoma in culture [21]. Murine and bovine liver arginases led the totaldestruction of
lymphosarcoma cells when arginine was reduced to <8 μmol over 24 h [22].Chemically modified
arginase II has been employed for treatment of Taper liver tumor andthe L5178Y murine leukemia
[23]
. Single amino acid (arginine) restriction produced byarginase resulted in death of cultured HeLa
while human diploid fibroblasts moved inquiescence and were largely protected [24].
ARGIINASE AND ITS DEVELOPMENT AS A CHEMOTHERAPEUTIC

AGENT:

The use of Arginaseas a potential chemotherapeutic agent has shown a lot of potential andpromise.

Arginasetreatment of cultured HeLa, human diploid fibroblasts and L1210 cellsproved to be as

efficient as the use of AFM (Arginine Free Medium) by reducing arginine inthe medium to

micromolar levels within 5-30 minutes resulting in cell death in both thecultures 137. Remission of

hepatocellular carcinoma was achieved by arginine depletion throughendogenous human hepatic

arginasereleased from transhepatic arterial embolization 138.Usingstate-of-the-art DNA technology,

researchers of the Hong Kong Polytechnic University(PolyU) produced a human recombinant

arginaseas a novel method in the treatment of livercancer by arginine depletion 139. The

combination of the recombinant arginasewith an antineoplasticagent 5 flourouracil (5FU) for

treatment of human malignancies was tested in nude mice bearing an ADI-resistant HCC xenograft

and this treatment methodology was shown to beeffective for arginine depletion 140. BCT-100

pegylated recombinant human arginasemanufactured by large scale fermentation of a recombinant

B. subtilisstrain LLC101 has beenreported as a novel anti-melanoma agent for treatment of human

melanoma cells as reported byHsuehet al.141. Pegylation of recombinant human arginase(rhArg-peg

5000mw) produced inB. subtilisexpression system was done and the pegylated enzyme has been

shown to have invitro and in vivo anti-proliferative potential and apoptotic activities in human

hepatocellular carcinoma (HCC) by Cheng et al. 142. While studies for conducting clinical trials of

PEG BCT-100 are reported to be in process to assess its safety and efficacy in humansrecently

further studies on pegylation of recombinant arginaseconducted by the Cheng group by conjugation

of rhARGwith methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000) has

beenreported as having comparable anti-tumor efficacy to native rhArg1[143]. That the inhibition

ofhuman hepatocellular carcinoma by recombinant arginase(rhARG1) is by the inducing cellcycle

arrest at the G2/M or S phase, possibly mediated by transcriptional modulation of cyclinsand/or


cyclin dependent kinases (CDKs) has been observed by Lam et al. 144. This group hasfurther

proposed the use of rhArg1 alone or in combination with chemotherapeutic drugs fortreatment of

liver cancer.

In vitro cytotoxicity of human arginase I by replacing the two Mn2+ ions normally present inthe

enzyme with Co2+ significantly lowered the Km value of the enzyme, increased its serumstability

and showed incredible ability to eliminate human hepatocellular carcinoma andmelanoma cell lines

proving it to be a capable new contender for treatment of L-Argauxotrophic tumors 145. A study

conducted by Hernandez et al., published just a few monthsback in the prestigious journal ‘Blood’

reports of the potential therapeutic role of pegylatedArginase I in the treatment of adult patients with

acute lymphoblastic T cell leukemia (T-ALL)through arginine depletion 146Arginase: The

multilateral medical therapeuticThe enzyme has been found to possess profound therapeutic benefits

in treatment of variousphysiological disorders in the body. Measurement of circulating Arginase I

i.e., serum arginaselevels have been used experimentally as rapid marker for liver injury 167.

Arginasehas beenfound to be essential for the treatment of acute neurological disorders168.

Ornithine, producedArginase: The multilateral medical therapeuticThe enzyme has been found to

possess profound therapeutic benefits in treatment of variousphysiological disorders in the body.

Measurement of circulating Arginase I i.e., serum arginaselevels have been used experimentally as

rapid marker for liver injury 167. Arginasehas beenfound to be essential for the treatment of acute

neurological disorders168. Ornithine, produced.

Mechanism of Action:
The arginasescatalyse the divalent cation-dependent hydrolysis of Larginine to form the non-protein

amino acid L-ornithine and urea 11,12.

(The arginase reaction)

In the organisms containing urease, urea is further converted to ammonia and carbondioxide.

OCCURRENCE AND DISTRIBUTION:


Sources of L-arginase: Arginase apart from being ubiquitously present in mammalian tissues has
also been characterized from various worms, molluscs, fishes, bacteria, fungi, yeast, actinomycetes,
algae and plants.
Table No. 1

Mycobacteria 13
Proteus spp 14
T.aquaticus 15
Agrobacterium-Rhizobium group 16
CyanobacteriumAphanocapsa 17
Cyanobacteria 18
Bacillus licheniformis 18
Bacillus subtilis 19
Bacteria
Streptomyces spp 20
Streptomyces calvuligerus 21
Streptomyces calvuligerus 22
Cyanobacterium Anabaena cycadeae 23
Thermophile Bacillus caldovelox 24
Rhodobactercapsulatus E1F1 25
Bacillus anthracis 26
Chlamydia pneumoniae 27
Leishmania, Crithidia and Blastocridhidia 28
Protozoa: Entamoebahistolytica 29
Plasmodium falciparum 30
Fungi: Neurosporacrassa 31
Fungi
Aspergillusnidulans 32
Trichoderma sp. 33
Agaricusbisporus 34
Everniaprunastri 35
Everniaprunastri and Xanthoriaparietinathalli 36
Lichens
Peltigeracanina 37
Leptogiumcorniculatum 38
Saccharomyces cerevisiae 39
Yeast
Schizosaccharomyces 40
Lathyrussativus 41
pumpkin seeds 42
grapevine- Vitisvinifera 43
Arachis hypogea 44
Canavalialineata 45
Soybean 46
Jack bean (Canavaliaensiformis) 47
Plant
kiwifruit vines- Actinidiadeliciosa 48
sources
ginseng (Panax ginseng 49
loblolly pine (Pinustaeda L.) 50
Saccharumofficinarum cv. Mayari 51
tobacco 52
Lycopersiconesculatum (tomato) 53
Quercus ilex 54
Vignacatjang 55
Hyalommadromedarii 56
Insects Drosophila 57
Bombyxmori- the silk worm 58
organisms Larva of Phoronispallida 59
African snail-Achatinafulica 60
Protopterusaethiopicus and Protopterusannectens 61
South American fish pacu
(Piaractusmesopotamicus) 61
Sea South American fish pacu
(Piaractusmesopotamicus) 62
Sparusaurata 63
Antarctic fish Nototheniarossii and
Notothenianeglecta 64
Pacific spiney dogfish shark Squalusacanthias 65
Salamandrasalamandra 66
Genypterusmaculatus 67
Musbooduga 68
Mammals
Raja erinacea 69
(Feliscatus) 70
Ranatemporaria 71
CLASSIFICATION OF FUNGI

Typical isolates of T. rubrum are white and cottony on thesurface. The colony underside is usually

red, although some isolates appear more yellowish and others more brownish.[6]Trichophytonrubrum

grows slowly in culture with sparse production of teardrop or peg- shaped microconidia laterally on

fertile hyphae: Macroconidia when present, an smooth-walled and narrowly club-shaped although

most isolates lack macroconidia Growth is inhibited in the presenceof certain sulfur nitrogen- and

phosphorus-containing compounds isolates of T.rubrum are known to produce pentellit in vitro and

in vivo.[7]

Scientific Classification

Kingdom: Fungi

Phylum: Ascomycota

Subphylum: Pezizomycotina

Class: Eurotiomycetes

Order: Onygenales

Family: Arthrodermataceae

Genus: Trichophyton

Species: T. rubrum
(Showing cottony thread culture of fungi)(Showing fungi pigmentation)

REVIEW OF LITERATURE

In 1928, Krebs and Henseleit conducted a series of experiments using liver slices and manometric

assays to show that in the presence of arginase, ornithine produced urea (Jenkinsonet al. 1996).

Crude preparations of arginase were reported as early as 1931 (Salaskin and Solowjew 1931, and

Waldschmidt-Leitzet al. 1931).Hellerman and Perkins first showed activation by bivalent metal ions

including cobalt, nickel, manganese, and iron (Hellerman and Perkins 1935). In 1940 an improved

purification developed by Richards and Hellerman showed that the activity could be restored to pH-

inactivated arginase by Mn2+ and (to a lesser extent) Fe2+ (Richards and Hellerman 1940).

In 1956, the "partial" purification was further improved by Robbins and Shields. They demonstrated

that the activity of arginase was dependent upon manganese and found the optimal pH to be 9.2

(which has since been adjusted to 9.4) (Robbins and Shields 1956, and Xie et al 2004)

Much work was done investigating inhibitors in the late 1970s and early 1980s (Rosenfeld et al

1975 Bedino 1977, and Pace and Landers 1981). Bedino studied the effect of the product/inhibitor

ornithine, and proposed an allosteric model for the regulation of the enzymes activity (Bedino 1977).
Recent research has investigated the roles of arginases in vascular disease, pulmonary disease,

infectious disease, and cancer (Zimmerman and Rothenberg 2006, Maarsinghet al. 2008,

Santhanamet al 2008, and Morris 2009). Varying levels of arginase have been found in the

reproductive system of cattle (Razmietai. 2005), as well as in the immune system of mice and

humans (Munder 2009).


MATERIALS AND METHODS

 Material

 EQUIPMENTS USED

S.No Instrument Group Product


1 Autoclave Remi
2 Hot plate stirrer Supriya
3 Oven Tempo Instrument & Equation Pvt. Ltd. Mumbai
4 Centrifuge Remi
5 Electronic Balance Roy Electronics
6 Vortex Mixture Remi
7 pH meter Ei
8 Colorimeter Remi

 GLASSWARES

S.No Glassware Group product


1 Beaker Borosil India

2 Conical Flask Borosil India

3 Glass Pipette Borosil India

4 Measuring Cylinder Borosil India

5 Test Tubes Borosil India


 Chemicals

S.No Chemicals

1 L-ornithine

2 Ammonium sulphate

3 Cobaltous chloride

4 Ninhydrine

5 Tris buffer

6 PBS buffer

7 Sodium chloride

8 Copper sulphate

9 TCA (Tri chloro acetic acid)

10 Silica gel

11 SDA (Sabourard dextrose agar)

12 BSA (Bovine serum albumin)

13 PDA (Potato dextrose agar)

14 Lectophenal cotton blue stain (dye)

15 Folien lowery reagent

16 EDTA

17 Nitrocellulose membrane
 BUFFER PREPRATION

1. Sodium phosphate buffer (PBS) [0.05m]


a) Na H2Po4 – 1.170 gm
b) Ba2 HPo4 - 1.334 gm
Weingh the solute A and solute B then individually makeup then volume in different breaker then
mix A and B to make final volume of then buffer.
2.Tris buffer (15mM)
Add .018gm Tris in 10ml distilled water.

REAGENT PREPRATION:-
1) 10mM L-arginin for 5ml-
.010 gmarginin mix with 5ml distilled water

2) 10% TCA-
.010gm TCA add into 1ml distilled water

3) 10mM Cocl2 for 5ml


.01gm Cocl2 add into 5ml distilled water

4) Ninhydrim 0.02gm add into 5ml distilled water

MEDIA PREPRATION

 Preparation of PDA (100ml)

 Cut the small pieces of potato weighing according to making medium.


 Boil the potatos for 30 min then filter (Separate the particular and liquid starch material).
 Add 2gm dextose and 1.5gm aqar then makeup the volume 100ml.
 Then autoclave for proper mixing.

 Preparation of SDA (100ml)


Weighing 6.5gm SDA mix with distilled water and autoclave for proper mixing.
 Sample collection:-

Sample was collected from different places along the cancer research & hospital area in

sterile conical flask and stored the sample at 4oc and sub culturing for further use.

 Inoculum preparation:-
7 loop form 7 days old culture were take in 10ml sterile distilled water & mix it by vortex.

 Enrichment and screening-


Inoculum suspension is equally transfer into 5 test tube, one is control and remaining fungal

suspension were exposal to UV radiation for various time period (5,10,15,20 min).

Then after UV exposal fungal suspension were use as for enzyme production by transferring

it into broth medium (dox’s medium).

Suspension in .5ml each test tube and incubate at 280 c for 5-7 days.

After incubation filtered then suspension and collect myclia present in surface of broth and

fake on take on dry filter paper for dry mass estimation.


 PREPRATION OF CRUDE EXTRACT (ENZYME)

From the UV exposed prepared 5ml sample was transferred to 25ml of production medium which

prepared by the SDA media. The sample medium was kept on a rotary shaker at 100r pm for 48hrs.

then 10% inoculum is used for purification.

A. Enzyme estimation by enzyme activity test:-


Principal –
Arginase activity based on the rate of hydrolysis of L-arginase to L-ornithine and urea by measuring

the amount of urea. Released in the reaction the urea produced given dark pink coloum when heat it

is the presence of ninhydrine.

Method–
Add 100ml enzyme and 100ml Cocl2 in a test tube at 50-55o c for 30 min.
Then add 200ml buffer and 100ml arginase then incubate for 10min at room temperature (35-40o c).

Add 100ml TCA put the sample at 90oc for 1 hrs in water bath for boiling then add ninhydrin

(100ml).

Fig:-Enzyme Activity showing by diffrnt colour formation

B. Folin lowery estimation of protein concentration :-


Principal-
Protein reacts with follin reagents to give a colored complex. The color so formed is due to the
reaction of the alkalin copper with protein. The intensity of color depends on the amount of these
aromatic amino acids present and will thus varey for different protion Protein Reagents:-(Freshly
Prepared)

Ingredients –
Solution - 1 % Cus04
SolutionB - 2 % Na - K Tarterat
SolutionC 0.2N NaOH
SolutionD 4 % Na2CO3

For 100ml Protien Reagent:-


 Solution - 1 % CuSO4-0.0 10gm
 SolutionB - 2 % Na - K Tartarate - 0.02gm
 SolutionC 0.2 N NaoH 0.4
 Solution A dissolves in 49 ml of distilled water
 Solution B dissolves in 49 mi of distilled water
 Solution C dissolves in 1 ml of distilled water
 Solution A dissolves water

Method:-

Solution A and solution B transfer in a measuring flask and prepar the volume up to 98 ml then add
1 ml Solution C in D in it After this add 1 ml solution C drop in it protien reagent is prepared.

PREPERATION OF BSA:-

BSA (Bovine Serum Albumin) used as standard solution in follin Lowery; Experiment 5 mg BSA
dissolve in 5 ml of distilled water.
Fig:3FollinLowrY
C. AMMONIUM SULPHATE PRECIPITAION :-

Principal-
Proteins are of many types and hence they have different solubility factors . The main factor that
which affects is the concentration of salt in the solution. This method is done to remove excessive
amount of salt from the sample mixture. This phenomena consist of generally two stages that is
salting in and salting out. The basic principle of purification by ammonium sulphate is that when it
dissolved in aqueous medium, it dissociates into ammonium (NH and sulfate (SO42-). It is highly
soluble in water and is favourable with most of the physical factors like pH, protein structure, low
density etc. This method is largely used in mass production and purification of proteins in
laboratories.

Salting in:-
The solubility of protein depends upon the concentration of salt in solution. If ion concentrations
(0.5 M) the solubility of proteins increases with increase in salt concentration. This termed as salting
in.

Salting out -

if the concentration of salt is further increased, the solubility of proteins get decreased and at this

high strength the protein get precipitate out from the solution, this termed as salting out.

Method:-
 2.555gm of ammonium sulphate was dissolved slowly in crude extract and overnight incubated

at 4°C.

 Next day ammonium sulphate containing crude extract was centrifuged for 10minutes at 10,000

rpm then collect the pellet for further experiment.

D. DIALYSIS:-

Principal-
Dialysis is method of removing and separating unwanted ions, compounds from the sample by using
semipermeable membrane of a particular pore size. This method is basically based upon the fact that
that the sample molecules of larger size gets trapped within the membrane and the other unwanted
molecules gets removed from sample by the process of diffusion. Here the main focus is to pay on
the selective membrane so that the desired outcome will be achieved easily This dialysis is done
according to the difference in their rate of diffusion.

PROCEDURE:
1) Firstly collect the pallet or precipitated.
2) Take a precipitated in conical flask and add the PBS.
3) These mixture transfer in to the polybags (semi-permeable membrane).
4) Polybags transfer in to tha beaker containing PBS buffur.
5) Then keep for 24hrs on magnatic stirrer.
6) Observed the volume of the sample (during the dialysis only protein is remained and desalting)

E. COLUME CHROMATOGRAPHY:-
Colume chromatography is method used to purify individual chemical compound from mixture of
compound. lt it often ueed or preperative application on scales micro-oraganismup to kilorams.
The stationary phase or adsorbent in colume chromatography is a solid. The most common the
stationary phase for colume chromatography is a silica gel, followed bye alumina. We use sephadax
powder as adsorbentin column.
The flow rate of such a colume can be increased by extending the fress eluent filled colume above
the top of the stationary phase of decreased by the tap controls. Better flow rates can be achieved by
using a pump.
Colume chromatography is set up with peristaltic pumps flowing buffer and the solution sample
through the column the solution and buffers pass through the column where a function collector at
the and of the column setup collect the eluted samples from it. Prior to the fraction collection, the
sample that are eluted form the column pass through the detector such as a spectrophotometer of
mass spectrometer so that the concentration of the separated sample in the sample solution mixture
can be determined.

PREPERATION OF COLUMN:-

Requirement-Glass wool, sephadax powder, phosphate buffer saline, and column.

Method:-
 Weigh1 gm G25 sephadax for 6ml PBS.
 Weigh 0.45 gm G-100 sephadax for 10 ml PBS.
 Appropraiate volume of sephadax was taken in a beaker.
 To this phosphate buffer saline (PBS) was added and kept over night at room temperature.
 Glass wool was taken saturated with PBS kept over night at room temperature.
 The exit of the column was blocked by glass wool then slowly the slurry of sephadax was added
in the column carefully and formation of air bubble was avoided.
 The column was saturated with PBS.

Sample Loading:-

This can be achieved manually via syringe of micropipette.

Protocol:-

 Set column flow rate.


 Check column for cracks of leaks.
 Prepare the autoclave appendroff to collect the apropriate number of fraction.
 Remove the particulate matter by centrifugation immediately prior to the loading.
 Apply the sample.
 Collect fraction for further resolution or assay. Larger fraction will cause higher dilution of the
sample and will decreased the resolution.
 Determine which fraction contains protein by protein estimation.

Resolution of sample:-

 The column was cleaned with PBS by passing the buffer through the column, about 1 ml of
sample was added to the column and elute were collected in fresh eppendrofs. Then estimate the
enzyme by nesselerization.
 Plit a graph by taking fractionson x-axis absorbance in the y-axis.

Spehadex G-25 (desalting):-


 Desalting gel is used to rapidly remove low molecular weight material saphadex G-25
(pharmecia) is used for desalting.
 Sample to be desalted is allowed to pass through desalting columne.

Sephadex G-100:-

 Fraction collected by desalting are allowed the pass through G-100 column for enzyme
purification.
 The rate of swelling is temperature dependent.
 The gel should never be allowed to dry out.

Significance:-

In this techniques protein are separated under physiological condition.


AIM & OBJECTIVE

In this study our aim the isolation of fungi for screening partially purification immobilization and

kinetic study of the therapeutic enzyme L-arginase at different parameter.

For this purpose our objective was:-

 Isolation of fungi.

 Screening of fungi at different temperature tolrence.

 To quantitative estimation enzyme.

 To quantitative and qualitative estimation of protein.

 Immobilization of enzyme and its kinetic study.


ACKNOWLEDGMENT

Firstly I pay my best regards in the name of god and my father and mother the
most beneficent and merciful. Indeed no work is possible without the "Grace of
God "guidance of teacher blessings of elders, love and encouragement of family
members and support from the friends. I am grateful and have no words to
express my hearty thanks to Late Shri SHEETLA SAHAY JI founder trustee.
Dr. B.R. Shrivastav, C.H.R.I. Director, Dr. ArchanaShrivastav, Director
College of Life Sciences, C.H.R.I. Gwalior.Dr. MeenuRai Principal College of
Life Sciences, C.H.R.I. Gwalior.

It is my privilege and it gives me great pleasure to pay may sincere thanks to


my guide Dr. ABHINAV SHRIVASTAVA, Assistant Professor, Department
of biotechnology, College of Life Sciences, Cancer Hospital and Research
Institute, Gwalior, under whose kind guidance I had great honor in carrying out
the present work without his support, vast knowledge, thought provoking
suggestion and confidence incapability, would not have been able to complete
my dissertation work.

I express my special thanks to Mr. MuneshRajak Computer Faculty, Mr.


UmeshKushwah Sir Librarian., and department of B.ScBiotechnology Mathura,
Dean of collage.

I heartily thank and respect my parents and blessings to carry out my work
successfully and my loving friend Monika goswami

MANSI YADUVANSHI
KINETIC STUDIES

STUDY OF DIFFERENT FACTORS ON IMMOBILIZED L-ARGIANSE:-

Effect of various concentration of substrate on the immobilized enzyme:-


substrate of various concentration ranging from 5,10,20 and 40 micro molar concentration were
taken and the effect of various concentration of substrate was observed by enzyme activity assay.

Effect of various temperature:-


various temperature ranging from 0,10,20,30,40,50,60 degrees centigrade. were taken to study the
effect of temperature on L-Arginase The effect was observed by enzyme activity assay.

Effect of various time of incubation:-


Incubation period of enzyme was altered with the same concentration of substrate ranging from
5,10,15,20,25,minute studied by enzyme activity assay.

Effect of various pH:-


Effect of various pH was observed with same concentration of the substrate ranging from 6,7,8,and
10 under similar physiological condition. The effect was studied by enzyme activity assay..

KINETIC OF ENZYME IMMOBILIZATION:-

Enzyme immobilization-
Enzyme immobilization may be defined confining enzyme molecules to a district phase from the one
in which the substrates and the product are present, this may be achieved by fixing the enzyme
molecules or within some suitable material.

The materials used for immobilization of enzyme, called carrier materials, are usually inert. (v1na,
karasakevich and M Beker).

Requirements:-

 0.300gm sodium alginate

 7gm calcium chloride

 Distilled water

 Glutraldihyde

 Glass ware

 Activate charcoal

Preparation for 0.75gm sodium alginate:-

 0.75gm sodium alginate dissolved in 25ml distilled water and autoclave for proper mixing.

Preparation for 3.5gm calcium chloride:-


 3.5gm calcium chloride dissolve in 25ml distill water.

 Chilled calcium chloride use for enzyme immobilization.

Method for enzyme immobilization:-

 Take 0.75gm sodium alginate dissolve in 25ml distill water and transfer the conical flask and
autoclave it.
 Hot solution allowed to cool at room temperature with continuous stirring with magnetic bead.
 Transfer 1.8ml enzyme and mix with continuous stirring for 15min .
 Then 1ml mix glutraldehyde and stirring for 15min.
 Now bead should left in calcium chloride solution for 30 min to harden.
 Decent supernatant & beads washed with distill water.
 Store in refrigerator.

Advantages of immobilized enzyme:-

 Immobilization permits repeated use of enzyme.


 It saved cost of downstream processing of the product.
 Immobilized enzymes can also be used in aqueous system.
 Thermo stability of enzyme is increased.
 Immobilization can increase the localized concentration.

Fig:-Immoblization of Enzyme Sample

RESULT
5.1 ENZYME ESTIMATION BY ENZYME ACTIVITY ASSAY
S.No. Onithine (µl) Distilled water (µl) Ninhydrine (µl) O.D. (515nm)

Blank 0.00 3000 100 0.000

S1 100 2800 100 0.049

S2 200 2700 100 0.068

S3 300 2600 100 0.087

S4 400 2500 100 0.110

Table:4 (a) Standard (4Mm)

Y-Values
0.14

0.12 y = 0.0003x + 0.0112


R² = 0.9543
0.1
O.D(515nm)

0.08

0.06 Y-Values
Linear (Y-Values)
0.04

0.02

0
0 100 200 300 400 500
concentration

S.NO. Enzy Cobltous Incubat Arginin Incubat TCA(µl Ninhy Incub D.W(M O.D.(5
me chloride ion e (µl) ion ) drine( ation( L) 15nm)
extra (µl) Time( Buffer Time µl) Min)
ct Min) (µl)
(µl) (Min)

Blank 100 100 30 200 100 10 100 100 1hrs 2.3

Crude 100 100 30 200 100 10 100 100 1hrs 2.3


Ammoniu 100 100 30 200 100 10 100 100 1hrs 2.3
m sulfate

Dialysis 100 100 30 200 100 10 100 100 1hrs 2.3

Column 100 100 30 200 100 10 100 100 1hrs 2.3

Table:4 (b) Enzyme sample

5.2 PROTEIN ESTIMATION BY FOLIN LOWRY METHOD

S.No. B.S.A (µl) D.W. Protein Incubation FC Reagent Incubation O.D.


(µl) reagent Time (Min.) (µl) Time (660nm)
(ml) (Min.)

Blank 00 500 2.5 30 250 30

S1 20 480 2.5 30 250 30

S2 40 460 2.5 30 250 30

S3 60 440 2.5 30 250 30

S4 80 420 2.5 30 250 30

Table: 5 Standard (B.S.A 1mg/m

Y-Values
100

80

60
y = 0.8313x - 16.388
O D(660nm)

R² = 0.5055
40
Y-Values
20 Linear (Y-Values)

0
0 20 40 60 80 100
-20

-40
CONCENTRATION(BSA)
S.No. Enzym D.W. Protein Incubati FC reagent Incubati O.D.
e (µl) Reagent on Time (µl) on
(660nm)
sample (ml) (Min.) Time
(µl) (Min.)

Blank 00 500 2.5 30 250 30

Crude 50 450 2.5 30 250 30

Ammonium sulfate 50 450 2.5 30 250 30

Dialysis 50 450 2.5 30 250 30

Column 50 450 2.5 30 250 30

Table 4. ENZYME SAMPLE

Sample name Enzyme Protein Specific Purification Recovery


activity fold (%)
Activity Concentration
(IU/ml)
(IU/ml) (mg/ml)

Crude

Ammonium
sulphate

Dialysis

Column

Table:-PURIFICTION TABLE
5.3 KINETIC STUDY (Enzyme Activity)

5.3.1 Effect of various concentration of substrate on enzyme.

TABLE:-6. EFFECT OF CONCENTRATION


S.NO CONC.(Mm) Enzym activity of Enzyme activity of free
Immoblized sample Sample(IU/ml)
(IU/ml)
Blank 0 00 00
T1
T2
T3
T4
T5

12

10

8
CONCENTRATION

6 Y-Values
Column1
4 Linear (Y-Values)

0
0 2 4 6 8 10 12
ENZYME ACTIVITY
5.3.2 Effect of various temperature on enzyme.

S.NO Temprature Enzym activity of Enzym activity of free


immobilized (IU/ml) enzyme (IU/ml)
Blank 0 00 00
S1
S2
S3
S4
S5
S6
S7
S8

12

10
ENZYME ACTIVITY

6
Y-Values
Column1
4

0
0 20 40 60 80 100
TEMPRATURE
5.3.3 Effect of various pH on enzymes

S.No. pH Enzyme Activity of Enzyme Activity of free


ImmoblizedSampl(IU/ml) Sample(IU/ml)

Blank

S2

S3

S4

S5

90

80

70

60
ENZYM ACTIVITY

50

40 Y-Values
Column1
30

20

10

0
0 2 4 6 8 10 12
pH

.
5.3.4 Effect of various time on enzymes.

S.No. Time (Min.) Enzyme Activity of Enzyme Activity of


Immoblized free Sample(IU/ml)
Sample(IU/ml)

Blank 0 0.00 0

S1

S2

S3

S4

S5

12

10
ENZYME ACTIVITY

6
Y-Values
Column1
4

0
0 5 10 15 20 25 30
TIME OF INCUBATION
REFERENCES

1. Journal of Bioactive and Compatible Polymers January 1999; 14(1): 31-53.

2. Laishley, E.J. and Bernlohr, R.W. J Bacterial 1968; 96(2): 322-329.

3. Nakamura, N. Fujita, M. and Kimura, K. AgrBiolChem 1973; 37(12): 2827-2833.

4. Patchett, M.L., Daniel, R.M. and Morgan, H.W. BiochimBiophysActa 1991; 1077(3):295.

5. Igeno, M.I., Moral, C.G., Caballero, F.J. and Castillo, F. FEMS Microbial Lett 6.Lishko VK,
Lishko OV, Hoffman RM. Depletion of serum methionine by methioninase

in mice. Anticancer Res 1993; 13: 1465-14681993;

7. Kuldeep Kumar, TeenaPhathak and ShefaliWalia. L-arginase Based Biosensor for

Detection of L-arginine in Juice Samples. J. Nat. Prod. Plant Resour ., 2012, 2 (4):494-499.

8..MarkusMunder. Arginase: an emerging key player in the mammalian immune system.

British Journal of Pharmacology (2009), 158, 638–651.

9. Kossel, A. and Dakin, H.D.(1904). Physiol. Chemie 41: 321-331

10. Jenkinson CP, Grody WW, Cederbaum SD (1996). Comparative prop- erties of arginases.

Comp BiochemPhysiol B BiochemMolBiol 114: 107–132

11 Kuldeep Kumar, TeenaPhathak and ShefaliWalia. L-arginase Based Biosensor for Detection of
Larginine

in Juice Samples. J. Nat. Prod. Plant Resour ., 2012, 2 (4):494-499.

12. Zellar, A., Van Orden, L.S. and Vogtili, A. (1954) Enzymology of mycobacteria.VII.

13. Prozesky, O.W., Grabron, W.O.K., Merwe, S.V., and Coetzee, J.N. (1973) Arginine cluster in
Proteus
Providence group. J. Gen. Microbiol., 77: 237-240.

14. Degryse, E.N., Glandroff, N. and Pierard, A. (1976) Arginine biosynthesis and degradation

in an extreme thermophile strain Z05. Arch. Int. Physiol. Biochim., 84: 599-601.

15. Dessaux, Y.A., Petit, A., Tempe, J., Demarez, M., Legrain, C. and Wiame, J.M. (1976)

Arginine catabolism in Agrobacterium strains: role of the Ti plasmid. J. Bacteriol., 166: 44-50.

16. Weathers, P.J., Chee, H.L. and Allen, M.M. (1978) Arginine catabolism in Aphanocapsa

6308. Arch Microbiol., 118(1): 1–6.

17. Gupta, M. and Carr, N.G. (1981) Enzymology of arginine metabolism in heterocyst

forming cyanobacteria. FEMS Microb.Lett., 12: 179-181.

18. Simon, J.P. and Stalon, V. (1976) Purification and structure of arginase of Bacillus

19. Baumberg, S. and Harwood, C.R. (1979) Carbon and nitrogen repression of arginine

catabolic enzymes in Bacillus subtilis. J. Bacteriol., 137: 189-196.

catabolic enzymes in Bacillus subtilis. J. Bacteriol., 137: 189-196.

20.Vargha, G., Szabo, G. (1983) A conditional aerial mycelium mutant of Streptomyces

fradiae with deficient ornithine cabamoyltransferase activity. J. Gen. Microbiol., 129: 539-542.

21.Bascaran, V., Haridsson, C. and Bran, A.F. (1989) Regulation of nitrogen catabolic

enzymes in Streptomyces calvuligerus. J. Gen. Microbial., 135(9): 2465-2474.

22. Singh, S. (1994) Arginine metabolism in cyanobacterium Anabaena cycadeae: Regulation

of arginine uptake and arginase by ammonia. Current Microbiology, 29(1): 49-52.

23. Patchett, M.L., Daniel, R.M. and Morgan, H.W. (1991) Characterization of arginase from

the extreme thermophile ‘Bacillus caldovelox’. BiochhimBiophysActa., 1077(3): 291-298

24. Wheatley, D.N., Philip, R. and Campbell, E. (2003) Arginine deprivation and tumor cell

death: Arginase and its inhibition. Molecular and Cellular Biochemistry, 244: 177-

25.Cheng, N.M., Lo, W.H. (2005) Pharmaceutical preparation and method of treatment of
human malignancies with arginine deprivation. US Patent Application No. 20050244398.

26. Cheng, N.M. (2006) Patent application title: Pharmaceutical Composition and Method of

Treating Hepatitis with Arginases.International Application No.:PCT/CN2005/001411.

Conclusion and Future Prospects

L-Arginase is successfully extracted and partially purified from the tricophyton


spp.(fungi)provided from the region of Gwalior, Madhya Pradesh, India. It is done by
mainly two methods i.eEnzym activity and Follin Lowry method. Purified by column
chromatography Sephadex G-100. The study is carried out by the calculation of its
enzymatic activity and purification fold of enzyme.

L-Arginase has a great role in curing and treating tumor cells and it works as
antitumor.

This enzyme can be widely purified and extracted from plants, animals and
microorganism.