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6.
BRIEF RESUME OF THE INTENDED WORK:
ENCLOSURE – I
6.1 INTRODUCTION
Enzymes can be defined as soluble colloidal organic catalysts which are produced by
living cells and are capable of acting independently of the cell. Glucose oxidase belongs to
oxidoreductase and is also called as glucose aerodehydrogenase. Glucose-oxidase (β-D-glucose:
oxygen-1-oxidoreductaseEC 1.1.3.4) is a fungal enzyme which catalyses the oxidation of β-Dglucose to gluconolactone and the concomitant reduction of molecular oxygen to hydrogen
peroxide. The gluconolactone is then hydrolyzed spontaneously to Gluconic acid.17 The sources
of Glucose oxidase are Penicillium variabile,13 Penicillium species,15 Aspergillus niger,
3
Penicillium funicolosm.14
APPLICATION
 Commercial diagnostic application is in the form of paper test strips for diabetic
patients.
 Used in de-sugaring eggs before they are dried. Such removal of glucose greatly
enhances shelf life of dried egg products by preventing the occurrence of "browning"
and other deteriorative processes. 20
 Used as a bio-sensor for detection of glucose in human blood serum.8
 In aerobic fermentation, Glucose oxidase is used to prevent the accumulation of
hydrogen peroxide.5
 Used in the baking industry as a processing aid to strengthen gluten in dough
systems.
 Glucose oxidase is used in fluorescence enzymatic determinations.4
 Glucose oxidase is considered as an antibiotic due to peroxide formation. It is applied
as an antimicrobial agent in oral care and other products.
6.2 NEED FOR STUDY:
Filamentous fungi produce a broad range of enzymes, organic acids, plant regulators,
mycotoxins and antibiotics. Aspergillus and Penicillium are useful model organisms employed
in industries and have much economical value. Glucose oxidase is one of the economical
enzymes which is predominantly produced by fungal species. This enzyme is responsible for the
breakdown of glucose to gluconic acid and hydrogen peroxide and this regulatory mechanism is
applied in the industries and diagnostics.
Considering the vast commercial application of glucose oxidase in diagnostics and
industry and its large scale production from fungal species, there is a need for improved
production of this enzyme commercially. This study will focus on strain improvement for better
production of glucose oxidase from Penicillium species.
6.3 REVIEW OF LITERATURE:
1. Stosz. et.al., (1998) have shown that production of the enzyme glucose oxidase was
involved in the biological control of Verticillium wilt by Talaromyces flavus. In this
study, the location of glucose antibody by immunostaining revealed that glucose oxidase
was both intracellular and extracellular. Old, as well as young, hyphal cells contained
glucose oxidase, but labeling of the cell wall-associated enzyme decreased as the cells
aged. Exocytosis rather than cell lysis was the primary means of release of glucose
oxidase from cells. Enzyme stability studies confirmed that the glucose oxidase of T.
flavus is an extremely stable enzyme, retaining 13% of its original activity after 2 weeks
at 25°C and retaining activity for several days at temperatures up to 50°C.
2. Sherbeny et.al., (2005) have produced the enzyme glucose oxidase by fermentation
technology, using Aspergillus niger as fermentation organism and glucose as substrate.
The highest specific activity of glucose oxidase and enzyme recovery was found to be
35.54 Umg-1 protein and 97.5% respectively, in the fractions precipitated with 80%
ammonium sulphate saturation. Another partial purification by dialysis resulted in raising
the specific activity of glucose oxidase to 37.44 Umg-1 with a purification fold of 16
times. Further purification of the enzyme by gel filtration technique on Sephadex G200
Column at pH 5.6 increased the specific activity of the enzyme to 65.2 Umg-1 proteins
with 3.76 purifications fold.
The maximum activity of the purified glucose oxidase A. niger was reached on using
0.25% of glucose as a substrate in the enzymatic reaction mixture and incubating at 370C
for two hours at pH 5.6. Amino acid composition of pure glucose oxidase was
characterized by containing predominantly 13 amino acids, among which serine, glycine,
glutamic acid, aspartic acid and alanine constitutes more than 60%.
3. Hesham- Enshasy et.al., (2006) have genetically engineered recombinant Aspergillus
niger
to produce glucose oxidase using the constitutive gpdA promoter and the
glucoamylase signal sequence for secretion. It was grown in batch cultures at agitation
speeds of 200 – 800 rpm covering the industrial relevant power input range of 0.1 – 5 W
kg-1. The growth morphology ranged from large pellets with an average diameter of
1500 μm at low power input up to micropellets embedded in a filamentous network at
high power input. A correlation of agitation intensity with growth morphology and
glucose oxidase production revealed an increase of the protein production capability with
the change of the growth morphology from pelleted to filamentous growth forms.
However, the exposure to higher shear stress with increasing power input also resulted in
lower biomass yields as well as increased transient formation of polyol (xylitol) and
higher final concentrations of oxalic acid. The highest specific production rates were
found in young filamentous growth forms at high power input. Although intermediate
agitation intensity leading to small pellets became more favourable during prolonged
cultivation. An acridine orange staining procedure discriminating between RNA rich
(red) and RNA poor regions (green) of the fungal biomass proved that active protein
production is restricted to filamentous growth forms and the outer layer of fungal pellets.
A correlation between the RNA rich fraction of the biomass determined by image
analysis and the productivity is shown.
4. Shazia Sabir et.al., (2007) have worked on production of glucose oxidase (GOD) which
is an important enzyme that finds a wide range of application in food and pharmaceutical
industry. In this investigation the feasibility of using rice polish as a substrate for the
production of GOD by Penicillium notatum in submerged fermentation (SmF) has been
evaluated. The intention was to enhance total GOD activity by the selection of
economical substrate, microorganism and consecutive optimization of various cultural
conditions.
Maximum GOD activity of (112±5) U/mL was achieved under optimum growth
conditions: rice polishes 5 g, incubation period 72 h, and buffering agent 3 % (by mass
per volume), incubation temperature (30±1)°C and pH=6.0. Addition of carbon and
nitrogen sources further enhanced the enzyme yield, indicating an economically
attractive process for GOD production.
5. Jafari. et.al., (2007) has studied dissolved oxygen tension and shear stress which are two
very important factors in fungal fermentation in batch culture of Asperigillus niger. The
intention was to maximise the total activity of GOx produced in a 5-1 bench top bio
reactor .300 rpm found to be optimum for enzyme production however, in higher mixing
rates higher growth was achieved .The maximum activity of GOx was obtained in 1.5
vvm while the best aeration rate for growth was 2 vvm. GOx with the activity of 548
UmL-1 was produced in 1.5 vvm and 300 rpm as the optimum condition.
6. David Lubertozzi (2008) has shown that filamentous fungi can be used for production of
wide range of valuable products. With the advent of molecular biology, it became
apparent that these fungi possess considerable potential as expression hosts for the
production of heterologous proteins and small molecules. Aspergillus is an important
genus, including well known species of economically significant molds, and widely used
for basic genetic research. The development of a genetic engineering “toolkit” for
Aspergillus, such as those existing for the simpler yeasts and bacteria, was delayed due to
the added complexity of the filamentous fungi, and also to the lesser resources devoted to
their study. History of the development of Aspergillus as an expression host, current state
of the art and future directions are reviewed, touching on related research in other fungi
when discussing the areas of greatest potential for future biotechnological applications,
focusing on the large and diverse families of fungal secondary metabolites.
6.4 OBJECTIVES OF THE STUDY
1. Isolation of penicillium from different soil sources and screening for the production of
glucose oxidase.
2. Production and purification of glucose oxidase by SmF.
3. Strain improvement by radiation and chemical mutagens.
4. Characterization of glucose oxidase.
ENCLOSURE: II
7.
7.1 METHODOLOGY :
1) Isolation of penicillium from different soil sources and screening for the production of
glucose oxidase.
a) Isolation by serial dilution.
b) Identification of penicillium.
c) Screening for the production of glucose oxidase.
2) Production and purification of glucose oxidase by SmF.
a) Production by SmF.
b) Downstream processing.
c) Precipitation.
d) Dialysis.
e) Column chromatography.
3) Strain improvement by radiation and chemical mutagens.
a) Mutation by UV
b) Mutation by EtBr
4) Characterization of glucose oxidase.
a) SDS-PAGE
b) Native PAGE
c) Effect of pH
d) Effect of temperature.
e) Effect of activators.
f) Effect of inhibitors.
g) Effect of substrate concentration.
h) Thermal stability of glucose oxidase.
7.2 SOURCE OF DATA:
1. Bharati College of Pharmacy.
2. E-library from Bharati College of Pharmacy.
3. Relevant compact discs regarding information on Enzymology.
4. Relevant compact discs regarding information on Biotechnology
7.3 METHOD OF COLLECTION OF DATA:
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Internet
Scientific abstract
Scientific journals
Relevant books on application of Biotechnology
Relevant books on Enzymology
Relevant compact discs on Enzymology and Biotechnology
7.4 Does the study require any investigation or interventions to be conducted on
patients or other humans or animals?
No
7.4
7.5 Has ethical clearance been obtained from your institution in case of 7.4?
Not applicable
ENCLOSURE: III
8.
REFERENCES:
1. Bentley R. The Enzymes glucose oxidase. 1963;7:567-86.
2. Burchardt M, Wittstock G. Kinetic studies of glucose oxidase in polyelectrolyte
multilayer films by means of scanning electrochemical microscopy. Bioelectrochemistry
2008;72:66-76.
3. Chen S, Chen W, Xue Macromol G, Biosci. Electrogeneration of polypyrrole/alginate
films for immobilization of glucose oxidase. 2008;8:478-83.
4. Del Barrio M, Puertas S, Grazu V, De Marcos JS, De la Fuente M, Galban. GBA
immobilization of labeled glucose oxidase to magnetic nano particales for development
of glucose nano bio sensor. Analytical chemistry department. Nanospain 2009;9-12.
5. Fabian.J. Simple method of anaerobic cultivation with removal of oxygen by a buffered
Glucose Oxidase-catalase system. Department of Technical Microbiology. 1964.
6. Gupta S, Mukhopadhyay L, Moulik SP. Glucose oxidase catalyzed oxidation of beta-Dglucose in aqueous micellar and water-in-oil microemulsion media. Indian J.
Biochem2003;40:340-9.
7. Hesham El-Enshasy, Joachim Kleine, Ursula Rinas. Agitation effects on morphology and
protein productive fractions of filamentous and pelleted growth forms of recombinant
Aspergillus niger Biochemical engineering division.
8.
Jianzhong Zhu, Ziqiang Zhul, Zongsheng Lai1, Rong Wang, Xiaoming Guo, Xiaqin
Wu, Guoxiong Zhang, Zongrang Zhang, Yiting Wang, Zongyou Chen. Planar
amperometric glucose sensor based on glucose oxidase immobilized by chitosan film on
prussian blue layer.2002;4(2):127-36.
9. Kurt Brunner, Susanne Zeilinger, Rosalia Ciliento, Sheridian LWoo, Matteo Lorito,
Christian P, Kubicek,Robert L. Improvement of the fungal biocontrol agent trichoderma
atroviride to enhance both antagonism and induction of plant systemic disease resistance.
Applied and environmental microbiology. 2005:3959–65.
10. Liping Wanga, Darin Ridgwaya, Tingyue Gua, Murray Moo-Youngb Bioprocessing
strategies to improve heterologous protein production in filamentous fungal
fermentations Biotechnology Advances 2005; 23:115–29.
11. Luis Henrique S. Guimarães Simone C. Peixoto-Nogueira Michele Michelin, Ana
Carolina S. Rizzatti1, Valéria C. Sandrim, Fabiana F, Zanoelo1 ,Ana Carla M.M.
Aquino, Altino B. Junior, Maria de Lourdes T M. Polizeli1 Screening of filamentous
fungi for production of enzymes of biotechnological interest Brazilian Journal of
Microbiology 2006;37:474-80.
12. Manivannan S, Kathiresan K, Effect of medium composition on glucose oxidase
production by pennicillum fellutanum isolated from mangrove rhizosphere soil, Research
Journal of Microbiology. 2007;2(3):294-8.
13. Pulci S, Brozzoli, V, Petruccioli M, Federici F, Expression of Penicillium variabile P16
glucose oxidase gene in Pichia pastoris and characterization of the recombinant enzyme
Crognale, Enzyme Microb. Technol. 2006;39:1230-5.
14. Semashko V, Mikhailova R V, Eremin A N, Extracellular glucose oxidase of Penicillium
funiculosum Appl; Biochem. Microbiol. 2003;39:368-74.
15. Simpson C , Jordaan J, Gardiner N S, Whiteley C B. Isolation, purification and
characterization of a novel glucose oxidase from Penicillium sp. CBS 120262 optimally
active at neutral pH Protein Expression and Purification. 2007;51:260–6
16. Shazia Sabir, Haq Nawaz Bhatti, Muhammad Anjum Zia and Munir Ahmad Sheikh
Enhanced Production of Glucose Oxidase Using Penicillium notatum and Rice Polish
Industrial Biotechnology Laboratory. 2007;45(4)443–6.
17. Sherbeny GA, Shindia AA, Sheriff YM. Optimization of various factors affecting
Glucose Oxidase activity produced by Aspergillus niger international journal of
agriculture and biology.2007;1560–8530.
18. Stosz SK, Roy S, Murphy C. Wergin W, Fravel DR. Localization of Glucose Oxidase
with immunocytochemistry in the biocontrol fungus Talaromyces flavus biochemistry
and cell biology. 1998;88(6):581.
19. Sumitra Ramachandran, Pierre Fontanille, Ashok Pandey and Christian Larroche.
Gluconic Acid: Properties, applications and microbial production food technol.
Biotechnology. 2006;44(2):185–95.
20. Underkofler LA, Barton RR, Rennet SS, Production of microbial enzymes and their
applications. Takamine laboratory, Division of Miles Laboratories. 1957.