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A Research Proposal
A thermostable alpha amylase from Geobacillus sp.:
Production, Purification and Biochemical
Characterization.
Submitted to
DEPARTMENT OF BIOTECHNOLOGY
New Delhi
Principal Investigator
DR (MRS) SUSHMA G. SABHARWAL
Associate Professor in Biochemistry
Department of Chemistry,
Division of Biochemistry.
University of Pune,
Pune – 411 007.
November 2010
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Project title: A Thermostable alpha amylase from Geobacillus sp.:
Production, Purification and Biochemical
Characterization
Broad Subject: Biochemistry, Life Sciences.
Basic Research
Duration: 36 months
Total cost: Rs. 20, 26,530
Principal Investigator: Dr. Sushma G. Sabharwal
Designation: Associate Professor in Biochemistry
Department: Department of Chemistry,
Division of Biochemistry.
Institution Name: University of Pune
Address: Department of Chemistry,
University of Pune,
Pune – 411 007
E-mail: [email protected]
Fax: +91(020)25691728
Date of Birth: 5th March, 1958
Gender: Female
Telephone: +91(020)25601230
Telex: 1457719 UNIP IN
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Co- Investigator: Dr. Shobhana Vishnu Bhide
Designation: Retired Professor in Biochemistry
Address: Department of Chemistry,
University of Pune,
Pune – 411 007
E-mail: [email protected]
Date of Birth: 13th July, 1945
Gender: Female
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PROPOSAL FOR MAJOR RESEARCH PROJECT
Project title: A Thermostable alpha amylase from
Geobacillus sp.: Production, Purification
and Biochemical Characterization.
Principal Investigator: Dr. Sushma G. Sabharwal
Institution: Department of Chemistry,
Division of Biochemistry.
University of Pune,
Pune – 411 007
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PART I: GENERAL INFORMATION
1. Name of the Institute/University Department of Chemistry,
submitting the Project Proposal: Division of Biochemistry,
University of Pune, Pune.
2. State: Maharashtra
3. Status of the Institute: UGC recognized.
4. Name and designation of the Mr. M.Jadhav, Registrar,
Executive Authority of the University of Pune.
University: PUNE 411 007.
5. Project Title A thermostable alpha
Amylase from Geobacillus sp.:
Production, Purification and
Biochemical Characterization
6. Category of the Project: R&D (Basic Research)
7. Specific Area: Basic Research Biochemistry
8. Duration: Three Years
9. Total Cost: Rs. 20, 26, 530
10. Is the project Single Institutional or S
Multiple-Institutional (S/M):
11. If the project is multi-institutional, NA
please furnish the following :
12. Project Coordinator/Sole/: Dr. Sushma G. Sabharwal /Principal Investigator: Associate Professor In Biochemistry, 13. Institution/ Address: Department of Chemistry, Division of Biochemistry,
University of Pune,
Pune- 411007
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14. Scope of application:
Enzymes are currently among the well established products in biotechnology; about
85 enzymes are currently in commercial production. The present enzyme market in India is
limited to 20 crores INR, with a predicted annual growth rate of 20 to 30%. However, India
imports 70% of the total enzyme consumed. Thus there is an immediate need for indigenous
production of industrially important enzymes. Amylases, the starch degrading enzymes,
constitute a class of industrial enzymes having the major world market share of enzymes.
Alpha Amylases (E.C.3.2.1.1) have several applications in various industries such as food,
brewing, distilleries, textile industries as well as in effluent treatment, in clinical laboratories,
medicine and analytical chemistry.
Considering the increased demand and requirement of indigenous industrial
amylases, especially thermostable amylase, studies on production, purification and
biochemical characterization of a thermostable alpha amylase is commercially relevant and
industrially important.
12. Project Summary:
Today, production of the enzymes has been identified as one of the most immediate
applications of biotechnology in industry, with nearly 85 enzymes currently in commercial
production. The annual worldwide sale of industrial enzymes is around US $ 1.7 – 2 billion.
The present enzyme market in India is limited to 20 crores INR, with a predicted annual
growth rate of 20 to 30%. However, India imports 70% of the total enzyme consumed. Thus
there is an immediate need for indigenous production of industrially important enzymes.
Amylases, the starch degrading enzymes, have several applications in various
industries, medicine and analytical chemistry. They constitute a class of industrial enzymes
having the major world market share of enzymes. The annual sale of the amylolytic enzymes
accounts for almost US $ 225 million Microbial and plant amylases meet the industrial
demand for amylases. Thermostability is a key feature of amylases sold for bulk industrial
usage, as they can be used for saccharification processes as well as other processes occurring
at high temperatures.
The ever increasing demand of highly active preparation of amylases with appropriate
specificity continues to stimulate the search for new enzyme sources and to identify such
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microorganisms that produce amylases with desired properties. The microbial sources are the
preferred sources due to: cost effectiveness, easy cultivation, rapid growth rate, ability to
respond to various stimuli and stress conditions, secretion of the extra cellular enzymes and
the ease with which they can be genetically modulated to generate enzymes with altered
desired properties. Although a number of microbial source exist for the efficient production
of amylases, only a few selected strain of fungi and the bacteria meet the criteria for
commercial production.
In the present investigation soil samples will be screened for the presence of amylase
producing microorganisms. The primary aim is to identify thermophilic bacteria producing
extracellular amylase with considerable activity and optimize the culture condition for
maximal amylase production. Also, in a developing nation like ours the ideal strategy for cost
effective production of industrially important enzymes is to explore the use of agro industrial
residues as substrates for enzyme production. Thus in the proposed study our aim is to
identify thermophilic bacteria from soil and optimize culture conditions for production of
thermostable amylase, an industrially important enzyme. Identification of microorganism will
be done by 16s rRNA sequencing. Further we propose to optimize the culture conditions for
optimal production of thermostable alpha amylase using various substrates including agro
wastes like oil seed cakes. This will be followed by isolation purification and biochemical
characterization of the enzyme.
This study is commercially relevant and industrially important considering the
increased demand and requirement of indigenous industrial amylases, especially thermostable
amylase.
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PART II: PARTICULARS OF INVESTIGATORS Principal Investigator:
14. Name: Dr. (Mrs.) Sushma Gurucharan Sabharwal
Date of Birth: 5th March 1958,
Sex (M/F): Female
Designation: Associate Professor in Biochemistry
Department: Department of Chemistry,
Division of Biochemistry.
University: University of Pune.
Pune.
Address: Department of Chemistry,
Division of Biochemistry
University of Pune,
Pune- 411 007.
.
Telephone: 020-25601230
Fax: 020-25691728
E-mail: [email protected]
Number of research projects: NIL
being handled at present: (completed ongoing UGC project
On 30th Sept.2010).
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PART III: TECHNICAL DETAILS OF PROJECT
INTRODUCTION 16.1 Origin of the proposal
In present day biotechnology, enzymes the biocatalyst, are helping industry to make
cost effective products, used by the society, of improved quality with less contaminants in a
way that is less harmful to the environment. Today, production of the enzymes has been
identified as one of the most immediate application of biotechnology in the industry, with
nearly 85 enzymes currently in commercial production.
Amylases, the starch degrading enzymes, constitute a class of industrial enzymes
having the major world market share of enzymes. Amylases have several applications in
various industries such as food, brewing, distilleries, textile industries as well as in effluent
treatment, in clinical laboratories, medicine and analytical chemistry. Thermostability is a key
feature of amylases sold for bulk industrial usage. As they can be used for saccharification
processes occurring at high temperatures (Peixoto et al, 2003). The advantages of using
thermostable amylases in industrial processes include the decreased risk of contamination,
cost of external cooling and increased diffusion rate (Lin et al, 1998). Thermophilic
organisms are thus of special interest.
Further , apart from VALUE ADDED PRODUCTS, use of agro industrial residues as
substrates for the production of amylases offers several advantages in productivity, cost
effectiveness in labour, time and medium components in addition to environmental
advantages like less effluent production, waste minimization, etc
16.2 (a) Rationale of the study supported by cited literature (b) Hypothesis (c) Key questions.
The ever increasing demand of highly active preparation of amylases with appropriate
specificity and stability over a wide range of pH and temperature and retention of activity in
the presence of ions, surfactants and organic solvents continues to stimulate the search for
new enzyme sources and to identify such microorganisms that produce amylases with desired
properties. Microbial and plant amylases meet the industrial demand for amylases. But it is
13
the microbial sources which are the preferred sources due to: cost effectiveness, easy
cultivation, rapid growth rate, ability to respond to various stimuli and stress conditions,
secretion of the extra cellular enzymes and the ease with which they can be genetically
modulated to generate new enzymes with altered desired properties. Although a number of
microbial source exist for the efficient production of amylases, only a few selected strain of
fungi and the bacteria meet the criteria for commercial production.
In the present investigation the primary aim is to optimize the culture conditions for
maximal amylase production by the thermophilic Geobacillus isolate (isolated from soil)
producing extracellular amylase with considerable activity and. Also, in a developing nation
like ours the ideal strategy for cost effective production of industrially important enzymes is
to explore the use of agro industrial residues as substrates for enzyme production. There are
several reports describing use of agro industrial residues for the production of amylases, e.g,
wheat bran and Bacillus amyloliquefaciens (Gangadharan et al, 2006), wheat bran and
Bacillus cereus (Anto et al, 2006), wheat bran and Thermomyces lanuginosus (Adinarayana
et al, 2005), wheat bran and rice husk and B. subtilis (Baysal et al, 2003), oil seed cakes and
B. licheniformis CUMC305 (Krishnan and Chandra, 1982), banana waste and Aeromonas
caviae CBTK185 (Krishna and Chandrasekaran, 1995), raw starch and Bacillus sp. TS-23
(Lin et al, 1998 al, 2000)
Apart from getting value added products use of agro waste for production of
amylases offers cost effectiveness and environmental advantages like less effluent
production, waste minimization. (Pandey et al, 2000).
Thus our aim is to identify the thermophilic Geobacillus isolate and optimize
culture conditions for production of thermostable amylase, an industrially important enzyme,
using cheap agro wastes.
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16.5 Current status of research and development in the subject International Status:
The annual worldwide sale of the industrial enzymes is around US $ 1.7 – 2 billion.
The versatile applications of the industrial amylases have touched new horizons thus
increasing its demand. Amylases have the major share in world market. The annual sale of
the amylolytic enzymes accounts for almost US $ 225 million.
National Status
The present enzyme market in India is limited to 20 crores INR, with a predicted
annual growth rate of 20 to 30%. However, India imports 70% of the total enzyme consumed.
Thus there is an immediate need for indigenous production of industrially important
enzymes.
16.6 The relevance and expected outcome of the proposed study
Relevance of the proposed study:
In the proposed project our primary aim is to identify amylase producing thermophile
Geobacillus from soil. Further we propose to optimize the culture conditions for optimal
production of thermostable alpha amylase using various substrates including agro wastes like
wheat bran, rice bran, oil seed cakes etc. This study is commercially relevant and industrially
important considering the increased demand and requirement of indigenous industrial
amylases, especially thermostable amylase.
Expected outcome:
Identification of amylase producing thermophilic Geobacillus and optimization of
culture conditions for production of thermostable amylase
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16.7 Preliminary work done so far
Alpha amylase producing thermophile Geobacillus from soil have been isolated by
enrichment of the soil sample with starch for 24 hours at 60⁰ C. The isolated bacteria can
tolerate high temperature upto 70 ⁰C.
The isolated strain is aerobic and Gram positive growing optimally at pH 7.0.
This bacterium was identified as Geobacillus toebii sp. by biochemical tests; scanning
electron microscopy and 16s rRNA sequencing. Pure culture has been submitted to
National Centre for Biotechnology Information (NCBI) to obtain the strain number.
The growth pattern of the bacteria was studied at various stress conditions. The
maximum alpha amylase activity was detected in the culture supernatant after 10 to 15 hours
of incubation period. The strain also used different carbohydrates as the carbon source, but
maximum enzyme production was seen when the organism was supplied with starch
The maximum production of alpha amylase occurred when 1% (w/v) starch was used. The
use of organic nitrogen source favored the production of alpha amylase in the isolated strain
compared to inorganic nitrogen source.
17a. Specific objectives: Pure culture of Geobacillus toebii ,an aerobic Gram positive thermophile isolated in
our laboratory from soil on Pune University campus (identified. by biochemical tests
scanning electron microscopy and 16s rRNA sequencing) will be used for the studies,
Presently,the pure culture has been submitted to National Centre for Biotechnology
Information (NCBI ) to obtain the strain number. The specific objectives of these studies are
as follows:
Production of extracellular, thermostable alpha amylase by Geobacillus toebii:
• Optimization of culture conditions for maximal enzyme productivity.
• Evaluation of various cheap Agro industrial waste residues such as wheat bran, rice
bran, oil seed cakes as substrates ( as carbon and nitrogen sources) for maximal
production of the Goebacillus toebii alpha amylase ,so as to get value added
products from agro wastes.
• Purification of α-amylase from the culture filtrate in minimum possible steps by using
conventional and advanced techniques of protein purification.
16
Determination of biochemical properties of purified α-amylase
• The molecular mass, pH and temperature optima of the purified enzyme
• Effect of various factors on alpha amylase activity.
• Substrate protection and kinetic studies
Structural Studies of the pure enzyme
• Active site directed chemical modification studies for the determination of amino
acid residues present at the active site.
• Determination of partial amino acid sequence with MALDI-TOF and peptide mass
fingerprinting.
• Fluorescence and Circular dichroism studies.
Immobilization of Geobacillus α-amylase.
Evaluation of applications of α-amylase.
17. b. Novelty of the strain: The alpha amylase producing thermophile, isolated from soil in our laboratory, can
tolerate high temperatures upto 70ο C. Based on 16s rRNA homology studies the isolate
demonstrated more than 95% homology with Geobacillus toebii species. Pure culture has
been submitted to National Centre for Biotechnology Information (NCBI ) to obtain the strain
number.
Enzymes from thermophilic microorganisms are generally thermostable.
Thermostability is a key feature of industrial amylases. Although, presence of extracellular
alpha amylase from Geobacillus toebii .has been reported, there is a need for an indepth
study on its isolation and biochemical characterization Evaluation of various cheap Agro
industrial waste residues such as wheat bran,rice bran,oil seed cakes as substrates ( as
carbon and nitrogen sources) for maximal production of the Goebacillus toebii alpha
amylase , to get value added products from agro wastes.
17
18. Work Plan: 18.1 Work plan: First year: Optimization of culture conditions and Isolation of thermostable α-amylase: The extracellular alpha amylase producing isolate (isolated from soil on Pune
University campus) is a gram positive aerobic thermophile, identified as Geobacillus species
(by Gram staining, surface studies using scanning electron microscopy and by 16s rRNA
gene sequencing). Optimisation of culture conditions for maximal enzyme production will be
carried out by studying the effect of pH, temperature as well as different carbon and nitrogen
sources on enzyme production. Also, the effect of starch concentration, various sugars, and
incubation period on amylase production will be assessed. Further, Use of agro waste
residues viz wheat bran, rice bran, oil seed cakes, soya waste as substrates for enzyme
production will be explored .The antibiotic sensitivity of the isolated bacterial strain will be
studied. After optimization of culture conditions, the enzyme will be isolated from the culture
filtrate and purified. Purification of α-amylase in minimum possible steps will be studied
using the different conventional protein purification techniques viz ammonium sulphate
precipitation, ion exchange chromatography, gel filtration as well as others.
Second year:
Biochemical characterization of the α-amylase:
This will include Polyacrylamide Gel Electrophoresis (PAGE) and SDS-PAGE.
Molecular weight of purified alpha amylase will be determined by MALDI-TOF, SDS-PAGE
and Gel filtration. Kinetic studies will be carried out to determine Km and Vmax of purified
alpha amylase. Influence of pH, temperature, detergents etc on the amylase activity will be
studied. Determination of effect of metals on purified alpha amylase, Isoelectric point of
purified alpha amylase.
Third Year:
In the third year, extensive structural studies of the purified alpha amylase will be carried
out. This will include;
1. Circular Dichroism Studies of purified alpha amylase will be carried out to determine
the secondary structure of the protein
2. Determination of Carbohydrate content of purified alpha amylase.
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3. Determination of total disulfide content, if any, of purified alpha amylase
4. Fluorescence studies of purified alpha amylase.
5. Amino acid analysis of purified alpha amylase
6. Chemical modification studies for the determination of amino acid residues present
at active site.
7. Determination of partial amino acid sequence with MALDI-TOF and peptide mass
fingerprinting.
18.2 Connectivity of the participating institutions and investigators NA.
18.3 Alternate strategies:
Screening of soil from different sources and regions will be done for identification
of thermostable bacteria producing extracellular
19. Timelines:
Period of study Achievable targets
6 Months. Optimization of the culture conditions for maximal enzyme
productivity.
12 Months Use of various agro industrial waste residues as substrates for
maximal production of the enzyme.
18 Months Purification of enzyme from culture filtrates in minimum
possible steps by using conventional and advanced techniques of
protein purification.
24 Months Biochemical Characterization of the Enzyme.
30 Months Extensive structural studies of the enzyme.
36 Months To study the application of the Enzyme.
19
References:
1. Isolation, characterization, and identification of Geobacillus thermodenitrificans HRO10, an amylase and alpha-glucosidase producing thermophile. Thaddeus C. Ezeji, Arite Wolf, and Hubert Bahl Can. J. Microbiol.
2. Industrial enzymes in bioindustrial sector development: An Indian perspective Journal of Commercial Biotechnology (2007) 13, 283–291. doi:10.1057/palgrave.jcb.3050065
3. Fograty W. M. and Kelly C.T., (1980) Economic Microbiology. In: Microbial Enzymes and Bioconversion, Rose AH (eds.) Academic Press, London, 5, 115-170
4. Veille C. and Zeikus G. J., (2001) Microbiology and Molecular biology reviews, 65, 1-43
5. Pandey A., Nigam P. and Socool C.R., (2001) Biotechnol. Appl. Biochem, 31, 135-152
6. Murao S., Ohyama k. and Arai M., (1979) Agric. Boil. Chem., 43, 719 7. Godfrey T. and West S. I., (1996) Introduction to Industrial Enzymology In – The
world market and current future 2nd T. Godfrey (ed.) West Macmillan Press Ltd, UK, 3
8. Aehle W. and Misset O., (1999) Enzymes for industrial application In- Biotechnology, second edition, H. J. Rehm and G. Reed (eds), Wiley- VCH, Germany, 189 -216
9. Van Lenen J. m. and Smith M. B., (1968) US Patent 3, 418, 211 10. Meima R. and Van Dij J. M., (2003) Protein secretion in Gram positive bacteria. In
Protein secretion Pathway in Bacteria, Boudega, (eds) kluwer Academic publishers, Dordrecht, 273.
11. Bolton D. J., Kelly C.T. and Fograty, W.M., (1997) Enzy. Microbio. Technol., 20.304.
20. Name and address of 5 experts in the field
Sr.No. Name Designation Address 1.
Prof. S. Sivakami
Professor of Biochemistry.
Department of Life Sciences, Mumbai University, Mumbai. [email protected]
2.
Dr. Lalita Kumar Scientists E
Division of biochemical sciences, National Chemical Laboratory, Pune. [email protected]
3.
Dr.S.M.Gaikwad Scientists E
Division of biochemical sciences, National Chemical Laboratory, Pune. [email protected]
4.
Dr. N. S. Punekar Professor Biotechnology
Department of Biosciences and Bioengineering IIT-Bombay, Powai Mumbai 400 076 [email protected]
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PART IV: BUDGET PARTICULARS
Budget (In Rupees): Summary: Item 1st Year 2nd Year 3rd Year Total Recurring Staff(Project Assistant) 1,93,200/- 1,93,200/- 2,22,080/- 6,07,200/- Contingency 1,00,000/- 1,00,000/- 1,00,000/- 3,00,000/- Chemicals and Glassware 75,000/- 75,000/- 75,000/- 2,25,000/- Non-Recurring Equipments 6,00,000/- Overhead charges 2,64,330/- Total Budget 20,26,530/-
A. Non-Recurring
S. No.
Item
1 2 3 4
Spectrophotometer Shaking incubator Refrigerator Computer with printer
Rs5,00,000/- Rs. 50,000/- Rs. 20,000/- Rs. 30.000/-
Sub-Total (A) = Rs 6, 00,000/- Justification:
To carry out enzyme assays and other quantitative studies like protein estimation a
spectrophotometer is essential.
Protein solutions and several chemicals have to be preserved under cold conditions, hence
a refrigerator is required.
For optimum microbial growth a shaking incubator is essential.
A computer is most essential for preparing reports, records and other documentation.
B. Recurring
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B.1 Manpower
S. No.
Position No.
Consolidated Emolument
Year 1 Year 2 Year 3 Total
1
Project assistant (one)
- Rs14000/- pm for two year + HRA - Rs16000/- pm for third year + HRA
1,93,200/- 1,93,200/- 2,22,080/- 6,07,200/-
Sub-Total (B.1) =Rs 6, 07,200/-
Justification for staff : A project assistant is necessary for carrying out the optimization, purification and other studies. B.2 Consumables
S. No.
Item
Year 1 Year 2 Year 3 Total
1 Chemicals and glassware
75,000/- 75,000/- 75,000/- 2,25,000/-
Sub-Total (B.2) = Rs 2,25,000/- Justification for Chemicals and glassware; Most of the fine chemicals required for protein purification and characterization are costly
chemicals and some chemicals have to be imported.
B.3 Travel
10,000/- 10,000/- 10,000/- 30,000/-
B.4 Contingency
1,00,000/- 1,00,000/- 1,00,000/- 3,00,000/-
B.5 Overhead@15% (If applicable)
2,64,530/-
Sub-total of B (B.1+B.2+B.3+B.4+B.5)
14,26,530/-
Grand Total (A + B) 20,26,530/-
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Justification for Contingency:
The contingency is required for the maintenance and purchase of spares for existing
equipments, semiconsumables like micropipettes, quartz cuvettes, cartridges for computer,
HPLC columns etc.
Justification for Travel Grant: To attend symposia, conferences.
V: EXISTING FACILITIES Resources and additional information
1. Laboratory:
a. Manpower : NA b. Equipments:
Laminar Air Flow, Microscope, Cold Centrifuge, Incubator, Oven,
Lyophiliser, HPLC, Colorimeters, Balances, Mechanical and Magnetic
stirrers.
2. Other resources such as clinical material, animal house facility, glass house.
Experimental garden, pilot plant facility etc. NA
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PART VII: BIOGRAPHICAL SKETCH OF PRINCIPAL INVESTIG ATORS
Name: Dr. (Mrs.) Sushma G. Sabharwal
Date of Birth: 5th March 1958, Sex (M/F): Female
Designation: Associate Professor in Biochemistry
Department: Department of Chemistry, Division of Biochemistry.
University: University of Pune. Pune
Address: Department of Chemistry, Division of Biochemistry
University of Pune, Pune 411 007.
Education (Post-Graduation onwards & Professional Career)
M.Sc. in Biochemistry University of Pune 1981 Ist Class (67%) Ph.D. in Biochemistry University of Pune 1988
Title of the Thesis: “Studies on the Isolation and Characterization of the Lectins
From Gliricidia sepium”
• Position: Associate Professor in Biochemistry Department of Chemistry, Division of Biochemistry,
University of Pune, 1st February 2002 onwards todate
Permanent Lecturer in Biochemistry, Department of Chemistry, Division of Biochemistry
University of Pune, 1st February 1991 to 31st January 2001.
Temporary Lecturer in Biochemistry Department of Chemistry, Division of Biochemistry
University of Pune, 5th March 1990 to 4th September 1990.
RESEARCH EXPERIENCE :
Junior Research Fellow, Council of Scientific & Industrial Research,
New Delhi, February 1982 to January 1984.
Senior Research Fellow, Council of Scientific & Industrial Research, New Delhi,
February 1984 to January 1987.
Research Associate, Council of Scientific & Industrial Research, New Delhi,
March 1988 to 4th March 1990, and 5th September, 1990 to 31st January 1991.
24
Professional Experience and Training relevant to the Project Research Specialization:
“Biochemical Aspects of lectins , enzyme inhibitors and
Glycosidases”
Students working / have worked under my guidance: (Ph.D. / M.Phil.)
1. Mr. Ashish S. Uzagare (University stipend, SET qualified), has obtained
his M.Phil degree in June 1999.
2. Dr. Sheeja John, Ph.D. (SRF on project, NET qualified), has obtained
her Ph.D degree in July 2005.
3. Mr. Tarun Sharma, Ph.D. (JRF, SRF on project, NET
qualified) thesis submitted in 2010.
4. Miss. Sibi Mani, M.Phil (Degree awarded in May 2006.).
5. Dr. Aditi Devanhalli, Ph.D. (since July 2004, University Stipend, UGC
fellowship), has obtained her Ph.D. degree in May 2009.
6. Mr.Tejas Kulkarni, Ph.D. (Degree awarded in October 2010.
7. Ms. C. Meenakshi, Ph.D. (Thesis submitted in May 2010.
8. Mr. Sanjay Chavan Ph.D. (Since 2007, NET qualified,CSIR SRF)
9. Miss Laxmi Gupta Ph.D. (Since 2007,UGC Project Fellow)
10. Ms. Roopa Nair Ph.D. (since June 2009)
11. Rajesh Gupta Ph.D (since June 2008)
12. Sameer Walunj.Ph.D (since June 2008)
13. Miss Aparna Phirange M.Phil (since November 2010)
14. Mr. Kunal Shukla Ph.D. (since August 2011, GATE qualified)
15. Mr. Chandrakant Tagad Ph.D. (since August 2011, GATE qualified)
25
Research Projects carried out:
1. Studies on Lectin Glycoprotein Interactions using Frontal Affinity Chromatography”,
sanctioned by CSIR, New Delhi, (1989-1993)- Co-Investigator.
2. Studies on the Structure and Carbohydrate binding specificity of the lectin from
Amarenthus paniculatus and its applications, sanctioned by University Grants
Commission, New Delhi (1992-1994) - Principal Investigator.
3. Biochemical Studies on Starches, Carbohydrases and Carbohydrate binding proteins
of seeds of different Amaranthus species cultivated in India, sanctioned by CSIR,
New Delhi, (Oct. 1994 to Oct. 1997) - Coinvestigator.
4. Biochemical Studies on Plant Defense Proteins : alpha amylase inhibitor from
chickpea and grain Amaranth, sanctioned by CSIR Dec. 1999 to November 2002.
Principal Investigator.
5. Polyhydroxylated Indolizindine, Pyrrolizidine and Azepane alkaloids as Glycosidose
Inhibitors, Synthesis and Biological Screening 2002 to 2005. Co- Principal
investigator.
6. Studies on Trypsin inhibitors from plant seeds as Biocontrol Agents sanctioned by
University of Pune 2006-2008 Principal Investigator
7. Studies on hypoglycemic and insecticidal effects of alpha amylase inhibitors from the
seeds of cowpea, horse gram and jack beans sanctioned by University Grants
Commission, New Delhi (2007- Sept 2010) - Principal Investigator
8 “Natural inhibitor of peroxidase and their photo regulation” seed money sanctioned
by University of Pune (2008 to 2010 ) Principal Investigator .
26
Publications in peer-reviewed journals: 30
Papers Presented in Symposia/Conference: 21
Ten selected peer-reviewed publications
1 Immobilised human salivary amylase: An affinity matrix for the isolation of alpha
amylase inhibitor from chickpea seeds Sheeja John and Sushma G. Sabharwal, J.
Food Sci. Technol., 2002, 39, 296-298.
2 Purification and some properties of a D-galactose binding leaf lectin of Erythrina
indica and further characterization of seed lectin. E. E. H. Konzoy, Ranjana Mulay,
Vitor Faca, Richard John Ward, Lewis Joel Greene, Martina cristina Roque –
Barriera, Sushma Sabharwal, and S.V.Bhide , Biohcemie. 84, 1035- 1043 (2002).
3 Studies on the Bio-efficacy of an Alpha Amylase inhibitor from seeds of Cowpea
(Vigna unguiculata L) against cereal pests. Sheeja John, C. S. Chaudhari, A. G.
Chandele, and Sushma Sabharwal, Annals of Plant protection sciences 2005,13
(2)275-277.
4 1-Aza-sugars from D-Glucose, Preparation of 1-Deoxy-5- dehydroxymethyl)
nojirimycin, its analogues and evaluation of Glycosidase Inhibitory Activity.
Nitin T. Patil, Sheeja John, Sushma G. Sabharwal and Dilip D. Dhavale.
Bioorganic and Medicinal Chemistry. 2002,10, 2155-2160.
5 Synthesis and evaluation of glycosidase inhibitory activity of 5-hydroxy substituted
isofagomine analogues. M. M. Matin, Tarun Sharma, Sushma G. Sabharwal, Dilip
D. Dhavale, Organic & Biomolecular Chemistry (Article); 2005(9), 1702-1707.
6 A Versatile Access to Calystegine Analogues as Potential Glycosidases Inhibitors
Krishna P. Kaliappan,* Prasanta Das, Sanjay T. Chavan, and Sushma G. Sabharwal
Journal of organic chemistry 2009,74,6266-6274.
7 Detection of alpha amylase producing bacteria (Bacillus species 5250), from soil:
Isolation and optimization of various conditions of growth. Meenakshi C.Kumar
Narendra, Ambekar Vikrant, K. Suresh, S. Bhide And Sabharwal Sushma. Res. J
Biotech.Vol 4 (1) 2009.50-56.
8 Purification and biochemical characterization of alpha amylase from Bacillus sp.
(NCIM 5250). Meenakshi C. Bhide.S. Suresh K. and Sabharwal Sushma. Res. J
Biotech.Vol 4 (4) 2009.70-75.
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9 Isolation Identification and media Optimization of Thermostable alpha galactosidase
producing Geobacillus sp. Chavan Sanjay and Sabharwal Sushma. Research Journal
of Chemistry and environment vol.14 (3) sept.2010, 22-26.
10 Antidiabetic potential of α amylase inhibitor from the seeds of Macrotyloma
uniflorum in streptozotocin-nicotinamide induced diabetic mice" Laxmi H. Gupta,
Sachin L. Badole, Subhash L. Bodhankar and Sushma G. Sabharwal
Pharmaceutical Biology. . Volume 49, Number 2,February 2011 , pp. 182-189(8)
List of maximum five recent publications relevant to the proposed area of work.
1 Detection of alpha amylase producing bacteria (Bacillus species 5250), from soil:
Isolation and optimization of various conditions of growth. Meenalshi C.Kumar
Narendra, Ambekar Vikrant, K. Suresh, S. Bhide And Sabharwal Sushma. Res. J
Biotech.Vol 4 (1) 2009.50-56.
2 Purification and biochemical characterization of alpha amylase from Bacillus sp.
(NCIM 5250). Meenakshi C. Bhide.S. Suresh K. and Sabharwal Sushma. Res. J
Biotech.Vol 4 (4) 2009.70-75.
3 Isolation Identification and media Optimization of Thermostable alpha galactosidase
producing Geobacillus sp. Chavan Sanjay and Sabharwal Sushma. Research
Journal of Chemistry and environment vol.14 (3) .2010, 22-26.
4. Immobilised human salivary amylase: An affinity matrix for the isolation of alpha
amylase inhibitor from chickpea seeds Sheeja John and Sushma G. Sabharwal, J.
Food Sci. Technol., 2002, 39, 296-298.
5. Immobilized Mucin - An Affinity Matrix for the isolation of Winged Bean Acidic
and basic lectins”, Sushma Y. Sawhney and Shobhana V. Bhide, J. Chromatogr.
(1990), 503, 272-276.
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PART VII: BIOGRAPHICAL SKETCH OF CO -INVESTIGATOR
Name : Dr. Shobhana Vishnu Bhide Designation : Prof in Biochemistry(Retd.)
Department of Chemistry University of Pune
Pune-411007 Date and place of birth : 13th July, 1945, Solapur, India Address : Vigyan Vishwa Apartments
Senapati Bapat Road, Pune-411053 E-Mail Address : [email protected] Marital status : Unmarried Nationality : Indian Educational Qualifications:
Name of the Board/University Year of Passing Marks Obtained Examination S.S.C S.S.C.E 1962 1stClass 71%
Board Maharashtra B.Sc University of Pune 1966 1st Class 60% M.Sc University of Pune 1968 1st Class 66% Ph.D University of Pune 1973
Title of the thesis: Biochemical nature of amylose and its complexes. Diploma in University of Pune 1974 2nd Class 68% German Language
Teaching Experience:
1. Lecturer in Biochemistry, Department of Chemistry. University of Pune, Sept. 1976 to Dec 1988. 2. Temporary Reader May 1982 to April 1983. 3. Reader in Biochemistry, Dec. 1988 to June 1998 4. Prof in Biochemistry July 1998 till retirement on 31st July 2005 5. In charge, MSc Biochemistry,Department of Chemistry,Fergusson College, Pune since July 2007. 6. Contributory Teacher at IBB, University of Pune.
Research Experience:
1. Junior Research Fellow. (CSIR), Oct. 1968 to Dec. 1971. 2. Junior Research Fellow. (DAE), Oct. 1971 to Dec.1971. 3. Senior Research Fellow, (DAE), Jan. 1972 to Jan. 1975. 4. Research Associate, (UGC), Aug. 1975 to Aug 1976. 5. Post Doctoral Fellow, Mombusho Scholarship of Japan, Laboratory of Microbiology,
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Nagoya University, Nagoya, Japan, Jan 1979 to March 1980. 6. Mombusho short-term research program. Laboratory of Microbiology, faculty of Agriculture, Nagoya University, Nagoya, Japan, Oct 1986 to Dec 1986.
Research Specilizations:
Biochemical aspects of polysaccharides, lectins and glycosidases.
Students who/ have worked under Co PI guidance: (Ph.D. / M.Phil.)
1. Usha.N.Nandedkar, Ph.D (UGC teacher fellow) Studies on Concanavalin A- polysaccharide interactions, Ph.D. University of Pune, (1984).
2. Meena.S.Behere, M.Phil (UGC teacher fellow) Studies on Yeast invertase on cross-linked gelatin with gluteraldehyde, M.Phil , University of Pune, (1986).
3. Sushma G. Sabhrawal nee Sawhney, Ph.D. (CSIR JRF, SRF) Studies on the isolation and characterization of the lectins from Gliricidia Sepium, Ph.D , University of Pune, (1987).
4. Vaijayanti.V.Pethe, M.Phil (Pune University Stipend) Some aspects of Concanavalin A– Polysaccharide interactions, M.Phil, University of Pune, (1987).
5. Leela.K.Kunchur, M.Phil (UGC teacher fellow ) Studies on isolation and partial characterization of B-Galactosidase from Erythrina indica, M.Phil , University of Pune, (1990).
6. Varsha.S.Kokil, M.Phil, Studies on isolation and partial characterization of N-acetyl B-D-glucosaminidase from Erythrina indica, MPhil , University of Pune, (1994).
7. Smita.S.Zuting, M.Phil Acid phosphatase from Erythrina indica, purification and partial characterization, M.Phil, University of Pune (1996)
8. Ranjana Mulay, M.Phil Studies on isolation and partial characterization of the lectin from leaves of Erythrina indica, M.Phil, University of Pune, (1996)
9. Emadeldin Hassan EMA Konozy, Ph.D Biochemical studies on Erythrina indica lectin and its endogenous glycosidase receptors, University Of Pune, Dec. 1999
10. P.Anuradha, Ph.D Macromolecular properties of isolectins from Trichosanthes anguina and purification of lectins from other cucurbitaceae plantsUniversity of Pune, August 2001
11. Abdulmonam Razok, Ph.D Biochemical Characterization of Starch from commonly consumed pulses University of Pune, 2005
12. Mohammad Aberomand,Ph.D. Sudies on α-mannosidase,N-acetyl-β-D-glucosaminidase,β-galactosidase acid
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phosphatase from Cicer arietinum seeds and human serumchick University of Pune,2006
13. Rakesh Kestwal,Ph.D. Isolation and characterization of α-D-mannosidase and β-D.galactosidase from Erythrina indica seeds University of Pune, 2006
14. Manju George,Ph.D Induced mutations in Soybean (Glycine max (L)merr)parent MACS-450 Screening and Biochemical characterization of selected mutant lines affecting lectin level University of Pune 2007
15. Ashish Uzgare,Ph.D. Structural and reconstitutional studies of acid phosphatase from Erythrina Indica University of Pune 2008 Publications in peer-reviewed journals: 34
Papers Presented in Symposia/Conference: 30
Ten selected peer-reviewed publications 1. Purification And Partial Characterization Of α-D-mannosidase From Erythrina indica seeds Rakesh Kestwal and Shobhana Bhide,Indian Jour.Biochem.Biophys.42,156-160,(2005) 2. Induced Mutations in Soybean [Glycine max (L.)Merril)Cv.MACS 450 D.D.Ahire,R.J.Thengane,J.G.Manjaya, Manju George and S.V.Bhide Soybean Reserch,3,1-8(2006) 3. Characterization of α-mannosidase from Erythrina indica seeds and ifluence of endogeneous lectin on its activity M.Kestwal,E.H.E/Konozy,C.D.Hsiao,M.C.Roque- Barreira and S.V.Bhide,Biochim.Biophys.Acta, 1770,(2007)24-28 4. Induced Mutations in Soybean [Glycine max(L.)Merril) Cv. MACS 450 Manju
George,D.D.Ahire,S.V.Bhide,J. G. Manjaya and R. J. Thengane Soybean Reserch,3,1-8(2006)
5. Purification and characterization of α-mannosidase from chickpea Mohammad Aberomand and Shobhana V. Bhide, Biochem. Cell. Arch. Vol 7 No.1,pp.33- 40(2007) 6. A novel human serum α-mannosidase:determination of molecular weight,chemical modification and effect of metal ions Mohammad Aberomand and Shobhana V Bhide, Biochemie Vol.7,No1, pp.111-115(2007) 7. Purification and characterization of a novel acid phosphatase from chick pea seeds Mohammad Aberomand and Shobhana V Bhide,Biochem.Cell.Arch.Vol.7 No.2,pp.171-176(2007) 8. Purification of β-galactosidase from Erythrina indica ;Involvement of Rakesh M Kestwal and Shobhana V Bhide, Biochim. Biphys.Acta 1770,1,506-1512(2007) 9. Separation and partial characterization of N-acetyl-β-D-glucosaminidase from chick
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pea Mohammad Aberomand and Shobhana V Bhide,Biochem.Cell.Arch.Vol.7 No.2,pp.225-230(2007) 10. Study on novel β-D-galactosidase from chick pea Mohammad Aberomand and Shobhana Bhide,Biochem.Cell.Arch.Vol.7 No.2,pp.245-251(2007) 11. Characterization of a lectin from Gonatanthus pumilus D.Don having anti
proliferative effect against human cancer cell lines. Vikram Dhuna,Sukhdev Singh Kambhoj,Amandeep Kaur,Ajit Kumar Saxsena,Shobhana V Bhide.Shanmugavel and Jatinder Singh Protein and Peptide Letters,2007,14,71-78
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