Application of enzymes/immobilized

enzymes in Pharmaceutical Industries

L-Asparginase
Source of the enzyme:
Bacterial sources- Bacterial sources proved to be an abundant source of L-asparaginase as
they are easy to manipulate. These include E.coli, Erwinia cartovora, Pseudomonas flouroscens
AG , Mycobacterium phlei , Staphylococci , Tetrahymena pyriformis , Thermus aquaticus , a
marine luminous bacteria [23] and Thermus thermophilus which was reported as not
hydrolyzing L-glutamine [24] and a new Erwinia sp.

Yeast sources- Yeast sources include Rhodotorula sp., red imperfect yeast, Rhodosporoidum
toruloides which produces a homodimer enzyme has been reported by Ramakrishnan .

Actinomycetes sources- L-Asparaginase production from Nocardia sp. and Streptomyces sp.
has been reported.

Plant sources-Among plants L-Asparaginase has been reported to be produced from the
Bryophyta Sphagnum fallax , Lupin araboreus & Lupin angustiplius and Lupinus leuteus . L-
asparaginase activity has also been reportedly found in soil of the roots of Pinus pinaster and
Pinus radiata due to ectomycorrhizal fungi in the wheat belt of Western Australia .

Properties of the enzyme:

The extracellular asparaginase from Cyberlindnera jadinii were quite stable at pH 4 for about
10 min at 50⁰C and upto 20h at 37⁰C. However, it remains stable upto a maximum of pH 10. The
purified enzyme from Pseudomonas stutzeri MB 405 having the optimum activity at pH 9 and
37⁰C showed maximum stability in the range of pH 7.5 to 9.5. The enzyme derived from
Helicobaterium pylori J99 remain stable near pH 10 but rapidly loses its activity irreversibly at pH
10 or above . The enzyme from Streptomyces gulbargensis to be more stable at alkaline pH and
retained its 80% of activity in the pH range of 7-10
Immobilization of the enzyme:
1) Covalently conjugated the enzyme on the microparticles of natural silk sericin protein
followed by cross-linking with glutaraldehyde which maintained 62.5% of the original activity of
the enzyme.

2) The multi-subunit covalent immobilization of L-asparaginase from E.coli onto the activated
supports (agarose-glutaraldehyde) had been followed by solid-phase intersubunit cross-linking
with aldehyde-dextran. This resulted in a longer half-life and a nil risk of subunit release into the
blood circulation

3) Bacillus sp. L-asparaginase was efficiently immobilized by covalent binding with activated
carbon. This enhanced the enzyme yield (73.6%) and showed the activity of 33U/g carrier. The
immobilization process greatly enhanced the thermo-stability of the enzyme showing 100%
activity upto 80˚C as compared to 50˚C for that of the native enzyme.

Structure of the enzyme:

Industrial applications:
1) Antitumour drug - L-asparaginase is a therapeutically important protein in combination with
the other drugs in the treatment of various types of blood cancer such as acute lymphocytic
leukemia, Hodgkin disease, acute myelocytic leukemia, acute myelomonocytic leukemia,
chronic lymphocytic leukemia, lymphosarcoma treatment, reticulosarcoma, and
melanosarcoma surrounding tissue. The first studies showed that intraperitoneal injection
of guinea-pig serum-which has a high content of asparaginase produced obvious regression
of the Gardner lymphosarcoma in mice. Later on a potent source of L-Asparaginase was
found in Escherichia coli. Since then L-Aparaginase derived from E.coli has been used for
treating human malignant disease.

2) Role in aminoacid metabolism - L-Asparaginase also play a very critical role in biosynthesis
of the aspartic family of aminoacids. Corynebacteria- producing aminoacids are of great
industrial interest as they excrete large amounts of various aminoacids . Lysine, threonine
 and methionine, commercially important aminoacids produced by C.glutamicum are derived
from the aspartic acid, which under normal physiological conditions, might be limiting for
lysine and/or threonine biosynthesis. Apart from Kreb’s cycle (using glutamic acid as
aminoacid donor) aspartic acid is formed from asparagine by the action of asparaginase. L-
Asparaginase was produced constitutively and its role may be that of an overflow enzyme,
converting excess asparagines into aspartic acid, the direct precursor of lysine and
threonine.

References:
1)L-Asparaginase Immobilized within Semipermeable Microcapsules: in vitro and in vivo
Stability ,Chang T.M.S. Department of Physiology, McGill University, Montreal, Que.
2) Purification and Properties of L-Asparaginase from Serratia marcescens1 JOHN W. BOYD2
AND ARTHUR W. PHILLIPS Department of Biology, Biological Research Laboratories,
Syracuse University, Syracuse, New York
3) MICROBIAL L-ASPARAGINASE: PRESENT AND FUTURE PROSPECTIVE
Rati Sinha1, H R Singh2, S K Jha2
Research Scholar, Department of Biotechnology, Birla Institute of Technology, Mesra,
Ranchi, Jharkhand, India
Assistant Professor, Department of Biotechnology, Birla Institute of Technology, Mesra,
Ranchi, Jharkhand, India

Arginase
Source of the enzyme:
L-Arginase is widely distributed in the plants, animals, human beings and the
microorganisms. Arginase extracted from the livers of the animals exhibits low
recovery, huge cost and contamination with the viruses.
Bacterial sources showed the presence of huge number of inclusion bodies, hence difficult
to purify.
From this case study, Marine strain Idiomarina sediminium gives more efficient expected
action.
Properties of the enzyme:
L-Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme in the urea cycle
that catalyses the formation of urea in the mammalian liver. L-arginase is considered as
an important enzyme for its use in the cancer therapy for arresting the growth of L-arginine
dependent cancer cells. The immobilized enzyme displayed the most activity from pH 7.5
to 11. The optimal temperature was found to be 35˚C and the immobilized enzyme
retained more than 80 % of its activity after incubation for 240 min at 40 °C.

Structure of the enzyme:

Industrial Applications:
The chitosan-HAP immobilized l-arginase can be employed on the large scale for
industrial production of l-ornithin. The free enzyme demonstrated a high level of
enzyme activity, selectivity and specificity but lacked the long-term stability, recovery
and recyclability, which restricted its application whereas the immobilized L-arginase
overcomes the above issue.
It is also being developed as a biosensor for monitoring the levels of the amino acid (l-
arginine) in the blood samples.

References:

1)L-Arginase immobilization on Chitosan Hydroxyapatite Complex: Effects of

Immobilization Conditions Rahamat Unissa1*, Mohd. Abdul Hadi2, Maddineni Sindhu3
1Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Malla Reddy
College Of Pharmacy, Maisammaguda, Dhulapally, Secunderabad, Telangana, India
2Department of Pharmaceutics, Faculty of Pharmacy, Bhaskar Pharmacy College,
Moinabad, R.R. Disct. Hyderabad, Telangana, India 3Department of Pharmacy Practice,
Faculty of Pharmacy, Malla Reddy College of Pharmacy, Maisammaguda, Dhulapally,
Secunderabad, Telangana, India.