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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.
1. INTRODUCTION
2. REVIEW OF LITRATURE
3. AIM OF STUDY
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.
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
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
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 -
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
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
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
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%
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
andPlasmodium falciparum) [3, 4], yeast (Saccharomyces cerevisiae) [5] and fungi
plant arginases form a separate group in the Arginase superfamily and are not muchclosely related
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
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.
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
arginaseas a novel method in the treatment of livercancer by arginine depletion 139. The
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
B. subtilisstrain LLC101 has beenreported as a novel anti-melanoma agent for treatment of human
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
beenreported as having comparable anti-tumor efficacy to native rhArg1[143]. That the inhibition
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
multilateral medical therapeuticThe enzyme has been found to possess profound therapeutic benefits
i.e., serum arginaselevels have been used experimentally as rapid marker for liver injury 167.
Ornithine, producedArginase: The multilateral medical therapeuticThe enzyme has been found to
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
Mechanism of Action:
The arginasescatalyse the divalent cation-dependent hydrolysis of Larginine to form the non-protein
In the organisms containing urease, urea is further converted to ammonia and carbondioxide.
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
Material
EQUIPMENTS USED
GLASSWARES
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
10 Silica gel
16 EDTA
17 Nitrocellulose membrane
BUFFER PREPRATION
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
MEDIA PREPRATION
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.
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
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
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.
the amount of urea. Released in the reaction the urea produced given dark pink coloum when heat it
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).
Ingredients –
Solution - 1 % Cus04
SolutionB - 2 % Na - K Tarterat
SolutionC 0.2N NaOH
SolutionD 4 % Na2CO3
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
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:-
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:-
Protocol:-
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.
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 study our aim the isolation of fungi for screening partially purification immobilization and
Isolation of fungi.
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.
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
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:-
Distilled water
Glutraldihyde
Glass ware
Activate charcoal
0.75gm sodium alginate dissolved in 25ml distilled water and autoclave for proper mixing.
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.
RESULT
5.1 ENZYME ESTIMATION BY ENZYME ACTIVITY ASSAY
S.No. Onithine (µl) Distilled water (µl) Ninhydrine (µl) O.D. (515nm)
Y-Values
0.14
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)
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.)
Crude
Ammonium
sulphate
Dialysis
Column
Table:-PURIFICTION TABLE
5.3 KINETIC STUDY (Enzyme Activity)
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.
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
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.
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
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
Detection of L-arginine in Juice Samples. J. Nat. Prod. Plant Resour ., 2012, 2 (4):494-499.
10. Jenkinson CP, Grody WW, Cederbaum SD (1996). Comparative prop- erties of arginases.
11 Kuldeep Kumar, TeenaPhathak and ShefaliWalia. L-arginase Based Biosensor for Detection of
Larginine
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
17. Gupta, M. and Carr, N.G. (1981) Enzymology of arginine metabolism in heterocyst
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
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
23. Patchett, M.L., Daniel, R.M. and Morgan, H.W. (1991) Characterization of arginase from
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
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.