A Project Proposal on

Cloning and Production of a Genetically Improved L-Asparaginase from

Escherichia coli

Submitted to

Department of Biotechnology for

financial support

Submitted by

Prof RP Singh Department of Biotechnology

Indian Institute of Technology Roorkee Roorkee 247667

&

Prof Ashok Pandey Biotechnology Division

National Institute for Interdisciplinary Science and Technology, CSIR Trivandrum-695 019

March, 2011

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PROFORMA – I

PROFORMA FOR SUBMISSION OF PROJECT PROPOSALS ON RESEARCH AND DEVELOPMENT, PROGRAMME SUPPORT

PART I: GENERAL INFORMATION

1. Name of the Institute/University/Organisation submitting the Project Proposal: Indian Institute of Technology, Roorkee-247667 National Institute for Interdisciplinary Science and Technology, CSIR, Industrial Estate P.O., Trivandrum-695019 2. State: Uttarakhand/Kerala 3. Status of the Institute: IIT & Institution of CSIR 4. Name and designation of the Executive Authority of the Institute/University forwarding the application: Dean, Sponsored Research & Industrial consultancy, Indian Institute of Technology, Roorkee Dr Suresh Das, Director, NIIST, CSIR, Trivandrum-695019 5. Project Title: Cloning and Production of a Genetically Improved L-Asparaginase from Escherichia coli 6. Category of the Project (Please tick): R&D 7. Specific Area (Please see Annexure - II): Industrial processes 8. Duration: Three Years 9. Total Cost (Rs.): 78,48,000=00 10. Is the project Single Institutional or Multiple-Institutional (S/M)? : M 11. If the project is multi-institutional, please furnish the following:

Name of Project Coordinator: Prof. R. P. Singh

Affiliation: Department of Biotechnology, IIT Roorkee, Roorkee Address: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667 12. Scope of application indicating anticipated product and processes:

L-asparginase is used for therapeutic purpose. It is used to treat acute lymphoblastic leukemia, lymphosarcoma. It may be also used for the treatment of diseases which respond to the

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asparagine depletion such as rheumatoid arthritis. Apart from the therapeutic use it can also be used for reduction of acrylamide in baked food preparations. Thus, it is highly beneficial to develop bioprocess to produce L-asparaginase having desirable properties. 13. Project Summary

L-Asparaginase II is a widely used therapeutic enzyme for the treatment of selected types of haematopoietic diseases such as acute lymphoblastic leukemia and non-Hodgkin lymphomas. The cancerous cells responsible for the disease have an absolute requirement for L-Asparagine due to a decreased or missing asparagine synthetase activity. Asparaginase hydrolyzes L-Asparagine to L-Aspartic acid and ammonia in leukemic cells, resulting in the depletion of asparagine, inhibition of protein synthesis, cell cycle arrest in the G1 phase, and apoptosis in susceptible leukemic cell populations. The enzyme characteristics such as half life in blood and thermostability can be improved by Site Directed Mutagenesis or through chemical modification of the enzyme.

Asparaginase from fungal sources such as Aspergillus species and Saccharomycetes are mainly used for food applications like acrylamide reduction in potato based food products. Acrylamide, a compound present in potato chips, has been classified as probably carcinogenic in humans, so it is important to reduce the contaminant levels in these products. As acrylamide was considered to be formed by Maillard reaction.

The gene encoding L-asparaginase will be isolated from E. coli and will be cloned by PCR based cloning approach and expressed in E. coli. Cloned gene will be used for site directed mutagenesis in pMAL vector and the expression product will be studied for improved half life and thermostability. The stable mutant will be selected for production of the enzyme.

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PART II: PARTICULARS OF INVESTIGATORS Principal Investigator & Coordinator: 14. Name: Prof. R.P. Singh

Date of Birth: 25.3.1956. Sex (M/F): Male

Designation: Professor Department: Biotechnology

Institute/University: Indian Institute of Technology, Roorkee Address: Department of Biotechnology, Indian Institute of Technology, Roorkee-247667

Telephone: (1332) 285792 Fax: (1332) 273560 E-mail: rpsbsfbs@iitr.ernet.in Number of Research projects being handled at present: See CV

Principal Investigator: 14. Name: Prof. Ashok Pandey

Date of Birth: 1.1.1956. Sex (M/F): Male Designation: Deputy Director and Head,

Department: Biotechnology Division Institute/University: National Institute for Interdisciplinary Science and Technology, CSIR

Address: Industrial Estate P.O., Pappanamcode, Trivandrum-695 019. Telephone: (0471) 2515368 Fax: (0471) 2491712. E-mail: ashokpandey56@yahoo.co.in

Number of Research projects being handled at present: See CV

Co-Investigator 15. Name: -NA-

Date of Birth: Sex (M/F): Designation:

Department: Institute/University:

Address: E-mail:

Number of research projects being handled at present:

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PART III: TECHNICAL DETAILS OF PROJECT

16. Introduction 16.1 Origin of the proposal Asparaginase (EC.3.5.1.1; asparagine amidohydrolase) catalyses the deamination of asparagine into aspartate and ammonia. A major potential therapeutic application of enzymes is in the treatment of cancer. Asparaginase has proved to be particularly promising for the treatment of acute lymphocytic leukaemia. Its action depends upon the fact that tumor cells are deficient in Aspartate-ammonia ligase activity, which restricts their ability to synthesize the normally non-essential amino acid L-asparagine. Therefore, they are forced to extract it from body fluids.

The bacterial sources of asparaginase includes E. coli, Erwinia, Serratia, Helicobactor pylori and several Streptomyces species, of these Helicobactor pylori asparaginase is characterized by extremely low glutaminase activity. The fungal sources include Aspergillus species and Saccharomycetes. Asparaginase from E. coli and Erwenia are widely used for treatment of leukemia. L-Asparaginase II was synthesized at constant rates by Escherichia coli under anaerobic conditions. The enzyme was produced optimally by bacteria grown between pH 7 and 8 at 37°C. Although some enzyme was formed aerobically, between 100 and 1,000 times more asparaginase II was produced during anaerobic growth in media enriched with high concentrations of a variety of amino acids. Bacteria grown under these conditions should provide a rich starting material for the large-scale production of the enzyme. Recombinant L- asparaginase was produced by cloning and the recombinant enzyme from E. coli and Erwinia were widely used as therapeutic agents. 16.2 (a) Rationale of the study supported by cited literature An ideal asparaginase for therapeutic use should posses charecterstics such as high activity, a low Km, and a strong preference for asparagine over glutamine. Decrease in glutaminase activity employs site directed mutagenesis of amino acid residues determining substrate specificity of this enzyme. EcA2 and ErA best meet these criteria, whereas other asparaginases preferentially hydrolyze glutamine. In the 1980s, an asparaginase from Wolinella succinogenes (WsA) appeared to be promising, because in vitro studies indicated that its glutaminase activity was negligible (Durden & Distasio, 1981; Distasio et al., 1982). However, the crystal structure of WsA showed that its active site is almost identical to that of EcA2. The thermo stability of the E. coli enzyme was increased by by replacing Asp178 with proline in the hydrogen bonded turn which contributes to the thermo stability of the enzyme (Sun et al, 2007). Effective employment of asparaginases in the antitumor therapy is also limited due to rapid proteolytic degradation of this enzyme in blood. This underlines the necessity of preparation of modified forms of asparaginases, exhibiting resistance to proteolytic degradation in blood and also low immunogenicity and with preservation of high catalytic activity. Enhancing the stability of L-asparaginase by protein engineering improves its body-residence time, and thereby minimizes immunosuppressive effects by lowering the therapeutic dose. Mutations can be introduced by amino acid substitutions at specific sites for improving the half life of enzyme in blood. Also the asparaginase for chemotherapeutic use can be modified chemically using PEG, acetic anhydride, dextran etc. an increase in vitro half life was obtained when asparaginase was chemically modified with 2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-S-triazine (mPEG2) in the presence of L-asparagine (Zhang et al 2004). A noticeable

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increase in half-life of the enzyme was observed by Qian et al. (1996) when the enzyme was chemically modified by N, O-carboxy methyl chitosan in the presence of L-aspartic acid. (b) Hypothesis Administration of L-Asparaginase leads to the degradation of L-asparagine and thus inhibiting the malignant cells from their rapid proliferation. A longer concentration of L-asparaginase is desirable because the plasma can maintain a higher concentration for clinical effectiveness. Amino acid substitution and chemical modification are usually used to improve the half life of the enzyme. (c) Key question Development of an enzyme, which has the better half life.

16.5 Current status of research and development in the subject (both international and national status) International status: The fermentative production of L asparaginase has been studied by various researchers. The effect of culture condition on production of L asparaginase on E. coli was studied by Barnes et al.,1977. Borek and Jaskolski in 2001 analyzed different asparaginase gene sequences and classified the asparaginase type I and type II under bacterial type asparaginases. Immunosuppressive effect of L-asparaginase from E. coli and Erwinia has been compared in 1974 by Leslie et al,. Work on molecular cloning of L-asparaginase started in late 80’s.The complete nucleotide sequence of L-asparaginase gene of Erwinia chrysanthemi was reported by Minton et al., 1986, and it consist of an open reading frame of 1044bp with a 21 amino acid signal peptide. ansB gene from E. coli K12 was cloned and sequenced in 1990 (Bonthron et al,.) and the gene map to position 3114kb on physical map of E. coli. The crystal structure of Escherchia coli Asparaginase II has been determined at 2.3 Å resolution by using data from a single heavy atom derivative in combination with molecular replacement (Jaskolski et. al.). L-asparaginase from Erwinia chrysanthemi 3937 (Erl-ASNase) was cloned and expressed in E. coli BL21(DE3) pLysS was subjected to mutagenesis for increased the thermo stability by Kotzia et. al. Apart from E. coli and Erwinia the L-asparaginase gene of Helicobactor Pylori was cloned and expressed by Krasotkina et al,. in 2009. ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOX1 gene promoter. Similarly to S. erevisiae the heterologous enzyme was expressed to the P. pastoris cell periplasmic space (Ferrara et al,2006.). Approaches have been made to increase the half life, thermostability and substrate specificity of the enzyme by amino acid substitution through site directed mutagenesis. A higher thermo stable mutant L -asparaginase II was created in the present study by replacing Asp178 with proline in a hydrogen-bonded turn (178–180DGR) which had contributed to the thermostability of the enzyme. The results displayed that values of Km and Kcat for the mutant enzyme are not affected although the energy of activation is increased when compared with the wild-type enzyme. These data suggest that such alteration for L-asparaginase II enhances the thermostability of the enzyme without changing the enzyme’s activity and thus the therapeutic use of L-asparaginase II may be benefitted from these results. Replacement of amino acids at

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position 248(asp) was found to reduce the glutaminase activity and thereby had increased the substrate specificity of E. coli L asparaginase II (Roehm et. al.,2000). National Status: Few groups in the country are actively working on fermentative production of L–asparaginase and also on recombinant L-asparaginase. For example, Centre for Biotechnology, Jawaharlal Nehru University has been actively working on recombinant L-asparaginase. Asparaginase II gene was cloned and different host vector combinations were tried by Mukherjee et al (2004). They observed that E. coli BLR (DE3) proved to be the most productive host, in terms of total secretory expression and extracellular production at the shake-flask level. Fusion of pelB signal sequence to the asparaginase gene under the T7 lac promoter had resulted in enhanced asparaginase levels in the culture medium, as compared with the asparaginase native signal sequence. Group of researchers from IIT, Guwahati had worked on production of glutaminase-free L- asparaginase from Pectobacterium carotovorum. Statistically based experimental designs were applied to maximize the production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428. L-asparaginase production was also accomplished from marine actinomycetes by submerged and solid state fermentation by Basha et al, (2009). References: J.F Zhang, LY Shi & D.Z Wei, Chemical modification of L-asparaginase from Escherichia coli with a

modified polyethyleneglycol under substrate protection conditions, Biotechnology Letters 26: 753–756, 2004.

N. Verma, K. Kumar; G. Kaur; S. Anand, L-Asparaginase: A Promising Chemotherapeutic Agent,

Critical Reviews in Biotechnology, 27:45–62, 2007 C. Derst, J. Henseling, and K.H Röhm, Engineering the substrate specificity of Escherichia coli

asparaginase II. Selective reduction of glutaminase activity by amino acid replacements at position 248, Protein Science ~2000, 9:2009–2017.

E. Harms, A. Wehner, H.-P. Aung and K.W. Rohn, A catalytic role for threonine- 12 of E. coli

asparaginase II as established by site-directed mutagenesis, Volume 285, number I, S-58 FEBS 098570 1991.

Leslie A. E. Ashworth and Alastair P. MacLennan, Comparison of the L-Asparaginases from Escherichia

coli and Erwinia carotovora as Immunosuppressants, CANCER RESEARCH 34, 1353-1359, June 1974

Georgia A. Kotzia, Nikolaos E. Labrou, l-Asparaginase from Erwinia Chrysanthemi 3937: Cloning,

expression and characterization, Journal of Biotechnology 127, 657–669 (2007) H. J . Gilbert, R. Blazek, H. M. S. Bullman AND N.L P . Minton, Cloning and Expression of the Erwinia

chrpanthemi Asparaginase Gene in Escherichia coli and Erwinia carotovora, Journal of' General Microbiology, 132, 15 1 – 160, 1986.

N S Basha, R Rekha, M Komala1 and S Ruby, Production of Extracellular Anti-leukaemic Enzyme L-

asparaginase from Marine Actinomycetes by Solid state and Submerged Fermentation: Purification and Characterisation, Tropical Journal of Pharmaceutical Research; 8 (4): 353-360, August 2009

A. Swain, M. Jask6lski, D. Houssett, J. K. M RAO, AND A. Wlodawert, Crystal structure of Escherichia

coli L-asparaginase, an enzyme used in cancer therapy (amidohydrolase/leukemia/active site/aspartate), Proc. Natl. Acad. Sci. USA Vol. 90, pp. 1474-1478, February 1993

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Georgia A. Kotzia and Nikolaos E. Labrou, Engineering thermal stability of L-asparaginase by in vitro

directed evolution, FEBS Journal 276 (2009) 1750–1761 Yu. A. Gladilina, N. N. Sokolov, and J. V. Krasotkina, Cloning, Expression, and Purification Of

Helicobacter Pylori L-Asparaginase, ISSN 1990-7508, Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, Vol. 3, No. 1, pp. 89–91, 2009.

A. V. Kuchumova, Y. V. Krasotkina, P. Z. Khasigov, and N. N. Sokolov, Modification of Recombinant

Asparaginase From Erwinia carotovora with Polyethylene Glycol 5000, ISSN 1990-7508, Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, Vol. 1, No. 3, pp. 230–232 , 2007.

A Khushoo . Y Pal . K. J. Mukherjee, Optimization of extracellular production of recombinant

asparaginase in Escherichia coli in shake-flask and bioreactor, Appl Microbiol Biotechnol 68: 189–197, (2005)

S. Kumar, K. Pakshirajan, V. V Dasu, Development of medium for enhanced production of glutaminase-

free L-asparaginase from Pectobacterium carotovorum MTCC 1428, Appl Microbiol Biotechnol DOI 10.1007/s00253-009-1973-0

A Khushoo, Y Pal, B. N Singh, K.J. Mukherjee, Extracellular expression and single step purification of

recombinant Escherichia coli L-asparaginase II, Protein Expression and PuriWcation 38, 29–36 (2004)

M. A Ferrara, N. M.B. Severino, J J Mansure, A. S Martins , E M.M. Oliveira , A. C Siani, N. Pereira,

Fernando A.G. Torres , E P.S. Bonb, Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene, Enzyme and Microbial Technology 39 1457–1463, (2006)

D. Borek and M. Jaskólski, Sequence analysis of enzymes with asparaginase activity, Acta Biochimica

Polonica, Vol. 48 No. 4/2001 W. R. Barnes, G. L. Dorn, AND G. R. Vela, Effect of Culture Conditions on Synthesis of L-Asparaginase

by Escherichia coli A-1. Applied and Environmental Microbiology, 257-261 ,(1977) Minton NP, Bullman HM, Scawen MD, Atkinson T, Gilbert HJ, Nucleotide sequence of the Erwinia

chrysanthemi NCPPB 1066 L-asparaginase gene. Gene 46:25-35, (1986). Bonthron D.T., L-asparaginase II of Escherichia coli K-12: cloning, mapping and sequencing of the

ansB gene. Gene .91:101-5,(1990) Kotzia G.A, Labrou N.E, L-Asparaginase from Erwinia chrysanthemi 3937: cloning, expression and

characterization. J Biotechnol 127:657–669,(2007) Imada K., Sato M., Tanaka N., Katsube Y., Matsuura Y, Oshima T, Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2A resolution. J. Mol. Biol. 222:725-738. (1991) 16.6 The relevance and expected outcome of the proposed study: An effective drug used for the treatment of cancer should be specific to differentiate between cancer cells and the normal cells. The chemotherapeutic agent must exploit this cellular

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difference in such a way that normal cells are spared and only cancer cells are injured. Tumor cells have unusually higher requirement of asparagine and therefore L-asparagine could of potential significance for targeting this specific process. As the L-asparagine is a major requirement by the cells for protein synthesis, the cells depends on L-asparagine synthatase as well as dietary source for asparagines. In the case of tumor cells, to keep up their rapid malignant growth, they would in need of a huge supply of asparagine and depends mainly on the dietary supply rather than the limited asparagine synthatase. Thus for the growth of tumor cells the L-asparagine is an essential amino acid. An enzyme with increased half life will be stable in blood than the normal enzyme, thus the frequent administration of the drug can be avoided. Based on the information generated from sequencing and X-ray structure mutagenesis has been employed to improve the half life and thus increasing the persistence of the drug in the body. The protein having desired mutation can be produced in large scale through fermentation technology. 16.7 Work done at NIIST, Trivandrum L-asparaginase II gene (ansB) of E. coli DH5 alpha and E. coli MTCC 739 was isolated from the genomic DNA by PCR with specific primers for mature region of L-asparaginase II gene. The ansB gene was cloned in expression vectors pET28a and pET20b system for soluble expression of recombinant protein in E. coli and the gene was sequenced. The cloned gene was successfully expressed in the soluble fraction of E. coli cytoplasm and characterized. 17. Specific objectives Cloning of ansB gene in prokaryotic expression and in Pichia pink expression systems

( NIIST, Trivandrum )

Site-directed mutagenesis of ansB gene for improvement of the half-life and stability of

the recombinant enzyme ( IITR ).

Purification and analysis of the mutagenized recombinant asparaginase B, analysis of the

recombinant ansB from Pichia system for evaluation of its stability ( IITR ).

Selection of improved mutants and scaling up of the production of the developed enzyme

in fermenter (5- & 30-l) ( NIIST, Trivandrum ).

17.I Method(s) to be followed

The ansB gene will be isolated and cloned in a suitable expression vector by PCR based cloning approach. The cloned gene will be sequenced and used for site directed mutagenesis. Mutageneses at positions 248, 11, 57, 88, 178 would be focused for increasing the thermo stability as well as substrate specificity. Mutated proteins would be expressed and analyzed against wild controls and bioprocess will be developed for production of mutant enzyme. 17.II Variable indicators of progress Isolation, cloning and sequencing of asparaginase gene in E. coli system.

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Over-expression of recombinant asparaginase in suitable prokaryotic and eukaryotic

expression systems.

Genetic modification of the enzyme and selection of improved mutants.

Purification, biochemical and molecular analysis of recombinant asparaginase.

To analyze the glycosylation pattern and thermostability of the recombinant asparaginase

from the Pichia system.

Bioprocess development for the production of asparaginase from mutants.

18. Work Plan

18.1 EXPERIMENTAL PROCEDURES

Isolation and cloning of L-asparaginase gene

1044bp ansB gene of E. coli will be amplified by PCR using the specific primers designed from the available gene sequences of E. coli and cloned in suitable expression system. The cloned gene will be sequenced using the commercial services, and compared.

Cloning of ansB gene in yeast expression system (Pichia pastoris) for effective glycosylation of the predicted sequons in ansB gene. The glycosylated L asparaginase II from the yeast cells will be purified and characteristics like stability and half life will be studied and comparison has to be carried out with the non-glycosylated counterpart expressed from E. coli.

Pichia Pink Expression system

Pichia expression system offers several advantages like robust expression, scalable production and ability to perform N – linked glycosylation. Several therapeutic proteins expressed in yeast system limit their application in clinical use because of their high mannose content. Hence researchers made efforts to engineer the glycosylation pathway of Pichia so that the expressed protein resembles mammalian glycosylation pattern. Pichia Pink expression system is the one that could be effectively exploited for expressing proteins particularly for therapeutic applications.

Site-directed mutagenesis

Substitutions already reported in E. coli were at positions 178 for thermo stability (M.Sun et al,.2007) , half life etc and at 248, 11, 57, 88 for substrate specificity (Derst et al,.2000). Accordingly the primers will be designed for site-directed mutagenesis. The site directed mutagenesis would be carried out using Stratagene Quick change site directed mutagenesis kit.

Expression of protein and purification

The desired mutants will be selected and expressed in pET20b vector. The expressed protein with C-terminal His Tag will be collected from the periplasmic fraction by osmotic shock and purified through Ni-NTA column. The ratio of turn over number to Km will be determined to find the half life of the improved mutants.

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Half life and thermo stability studies

The temperature effect on stability of the wild type enzyme and the mutant will be determined by incubating the purified enzymes at a series of increasing temperatures, the residual activities will be measured at 37oC. Half life of the enzyme preparation will be analyzed by incubating the enzyme at 37°C for different time intervals along with the wild type and performing the assay (Imada et al, 1991)

Scaled up production of improved recombinant asparaginase at fermenter level

Large scale production of highly pure therapeutic protein is a prerequisite for pharmaceutical industries. Commercial production of improved L asparaginase can be achieved by a scale up of recombinant protein expression employing fermenter.

18.3 Alternate strategies: We are confident that the project would attain the goals. 19. Timelines:

Period of study

Achievable targets

6 months Cloning of ansB gene and to achieve over-expression in E. coli 12 months Cloning of the ansB gene in Pichia pink expression system 18 months Primer designing and site directed mutagenesis, sequencing,

identification and selection of improved mutants 24 months Expression, purification and characterization of improved

recombinant asparaginase 30 months Analysis of glycosylation pattern and evaluation of thermostability of

recombinant asparaginase 36 months Development of bioprocess for large scale production of mutant

enzyme 20. Name and address of 5 experts in the field

Sr.No. Name Designation Address 1

Prof. S B Chincholkar Professor & Head School of Life Sciences, North Maharashtra University, Jalgaon-425 001; email chincholkar_sb@hotmail.com

2

Prof U C Banerjee Professor & Head Biotechnology Department, National Institute of Pharmaceutical Education and Research, Sector 57, SAS Nagar-160 062- email ucbanerjee@niper.ac.in

3

Prof. D Madamwar Professor & Director

Department of Biosciences, SP University, Vallabh Vidyanagar-388 120, email datta_madamwar@yahoo.com

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4 Prof Arun Goyal Professor & Head Department of Biotechnology, IIT Guwahati, Guwahati, Assam; Email: arungoyl@iitg.ernet.in

5 Prof P Gunasekaran Professor & Head Department of Genetics, Madurai Kamaraj University, Madurai, TN; Email: gunagenomics@gmail.com

PART IV: BUDGET PARTICULARS IIT Roorkee A. Non-Recurring (e.g. equipments, accessories, etc.) IITR Rs

S. No. Item Year 1 Year 2 Year 3 Total 1 2

Thermo cycler Deep Freezer (-20°C to -80°C ) ( 500 lts ) and accessories for table-top refrigerated centrifuge

4,00,000 5,00,000

4,00,000 5,00,000

Sub-Total (A): Rs 9,00,000 only. The instruments are required for carrying out the site directed mutagenesis work and for controlled temperature storage of molecular biological, enzymes and other DNA constructs. B. Recurring B.1 Manpower (See guidelines at Annexure-III)

S. No.

Position No.

Consolidated Emolument

Year 1 Year 2 Year 3 Total

1

JRF/SRF/RA

20,000/- +HRA

2,40,000

2,40,000

2,40,000

7,20,000

Sub-Total (B.1) = Rs7,20,000 only. The project envisages developing low pH active and high temperature tolerant enzyme. An RA/SRF/JRF would be required for taking up the studies independently. B.2 Consumables

S. No.

Item

Year 1 Year 2 Year 3 Total

1 2

Analytical/molecular biology grade chemicals/Kits Glass wares/plastic wares

5,00,000 50,000

5,00,000 50,000

2,00,000 50,000

12,00,000 1,50,000

Sub-Total (B.2) = 13,50,000 only. Required for purchase of media inputs, assay reagents, molecular biology kits for site directed mutagenesis, DNA preparation, purification and molecular analysis of recombinant product. The kits and consumables being costly in nature require sufficient money under consumables.

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B.3 Other items Items Year 1 Year 2 Year 3 Total

Travel

50,000 50,000 50,000 1,50,000

Contingency 50,000 50,000 50,000 1,50,000

Sub-Total (B3) = 3,00,000 only. Travel and contingency grant are required to meet the traveling expenses for attending DBT meetings and seminar /symposia and for the project related minor expenses. The overhead charges @15% are payable to host institution for providing various support and services. B.4 Year wise Total

Total 17,90,000 8,90,000 5,90,000 32,70,000

B.5 Overhead Charges

Overhead charges @ 20% of the total cost of the project

3,58,000 1,78,000 1,18,000 6,54,000

Grand total of (A+B) (B.1+B.2+B.3+B.4+B.5)

21,48,000 10,68,000 7,08,000 39,24,000

GRAND TOTAL Rs. 39,24,000

PART IV: BUDGET PARTICULARS NIIST, Trivandrum Non-Recurring (e.g. equipments, accessories, etc.) Rs

S. No. Item Year 1 Year 2 Year 3 Total 1 2 3

Sonicating water bath Mini Protein Electrophoretic System Fermenter accessories (electrodes for pH, oxygen, tem for 5- and 30-l fermenters and septa, etc

2,00,000 1,00,000 -

- - -

- - 5,00,000

2,00,000 1,00,000 5,00,000

Sub-Total (A): Rs 8,00,000 only. The first two equipment are required for the cloning/expression related experiments. Fermenter accessories required for the 5-l and 30-l fermenters in the third year for bioprocess work. B. Recurring B.1 Manpower (See guidelines at Annexure-III)

S. No.

Position No.

Consolidated Emolument

Year 1 Year 2 Year 3 Total

1.

JRF /SRF

16,000/- +20% HRA pm

2,30,400 2,30,400 2,30,400 6,91,200

2. Lab Asstt 6000/pm - - 96,000 96,000 Sub-Total (B.1) = Rs 7,87,200 only. JRF/SRF to do the project work; Lab Asst in the third year to assist with fermenter studies.

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B.2 Consumables S. No.

Item

Year 1 Year 2 Year 3 Total

1 2

General/molecular biology grade chemicals/Kits Glasswares/plastic wares

5,00,000 50,000

5,00,000 50,000

3,00,000 50,000

13,00,000 1,50,000

Sub-Total (B.2) = 14,50,000 only. Required for purchase of media inputs, assay reagents, molecular biology kits for total RNA, mRNA, site directed mutagenesis, DNA preparation, PCR cloning and DNA purification kits, expression vector, Pichia Pink Expression Kit etc. The kits and consumables being costly in nature require sufficient money under consumables. The mRNA being very sensitive separate sets of glass wares and plastic wares will be required for carrying out the work.

Other items Year 1 Year 2 Year 3 Total

B.3 Travel

75,000 75,000 75,000 2,25,000

B.4 Contingency 50,000 50,000 50,000 1,50,000

B.5 Overhead 1,80,810 1,35,810 1,95,210 5,11,830 Sub-total of B (B.1+B.2+B.3+B.4+B.5)

10,86,210 10,41,210 9,96,610 31,24,630

Grand Total (A + B) 13,86,210 10,41,210 14,96,610 39,24,630

Travel and contingency grant are required to meet the traveling expenses for attending DBT meetings and seminar /symposia and for the project related minor expenses. The overhead charges @15% are payable to host institution for providing various support and services.

PART V : EXISTING FACILITIES

Resources and additional information 1). Laboratory:

a. Manpower: PI and Co-I b. Equipments

SN Instrument Make 1 Gel documentation system Assembled 2 PCR thermal cycler Eppendorf 3 DNA concentrator Eppendorf 4 DNA hybridization chamber BIOgene 5 Gel electrophoresis unit Bangalore Genei 6 Refrigerated centrifuges Benchtop model Eppendorf 7 Refrigerated Hi-speed centrifuge (Floor model ) Hitachi 8 Refrigerated centrifuge (benchtop model) Remi India 9 Fermentor: Biostat B (5-L) and Biostat C (30-L) B. Braun Biotech Int. 10 Millipore water purification unit

Millipore

11 SSF fermentor with related downstream and upstream assemblies

Murhopye Scientific company, India

12 Millipore Ultrafiltration unit

Millipore

13 Gel filtration column Biorad 14 Gilsonfraction collector Gilson

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15 HPLC Varion 16 Gas Chromatograph Chemito 17 Phase contrast microscope Leica 18 Refrigerated incubator shaker New Brunswick 19 Environmental test chamber Sanyo 20 Shaking and nonshaking water bath Julabo 21 UV-VIS spectrophotometers Shimadzu 22 -800 C freezer Sanyo 23 Laminar air flow work station Clean air systems, India

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PART VI: DECLARATION/CERTIFICATION It is certified that

a) The research work proposed in the scheme/project does not in any way duplicate the work already done or being carried out elsewhere on the subject.

b) The same project proposal has not been submitted to any other agency for financial support.

c) The emoluments for the manpower proposed are those admissible to persons of corresponding status employed in the institute/university or as per the Ministry of Science & Technology guidelines (Annexure-III)

d) Necessary provision for the scheme/project will be made in the Institute/University/State budget in anticipation of the sanction of the scheme/project.

e) If the project involves the utilization of genetically engineered organisms, we agree to submit an application through our Institutional Biosafety Committee. We also declare that while conducting experiments, the Biosafety Guidelines of the Department of Biotechnology would be followed in toto.

f) If the project involves field trials/experiments/exchange of specimens, etc. we will ensure that ethical clearances would be taken from concerned ethical Committees/Competent authorities and the same would be conveyed to the Department of Biotechnology before implementing the project.

g) It is agreed that any research outcome or intellectual property right(s) on the invention(s) arising out of the project shall be taken in accordance with the instructions issued with the approval of the Ministry of Finance, Department of Expenditure, as contained in Annexure-V.

h) We agree to accept the terms and conditions as enclosed in Annexure-IV. The same is signed and enclosed.

i) The institute/university agrees that the equipment, other basic facilities and such other administrative facilities as per terms and conditions of the grant will be extended to investigator(s) throughout the duration of the project.

j) The Institute assumes to undertake the financial and other management responsibilities of the project.

Signature of Principal Investigator & Signature of Executive authority Co-Ordinator of Institute/University with seal Date : Date :

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PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS

Name: RAJESH PRATAP SINGH Designation: Professor Address: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667 Date of Birth: March 25th, 1956 Sex: Male SC/ST: No Academic Qualification B. Sc. 1975 Chemistry, Botany, Zoology Avadh University Faizabad M. Sc. 1979 Biochemistry G.B. Pant University of Agriculture & Technology Ph. D. 1983 Microbiology Kanpur University, Kanpur ( Central Drug Research Institute, Lucknow ) Position and Employment 1983 –85 Research Associate, IMTECH, Chandigarh 1985 –86 Visiting Fellow, NIH, USA [July-Oct] 1986-90 Research Fellow, Harvard University, USA [Nov-Apr] 1990-96 Lecturer, University of Roorkee, Roorkee 1996-2003 Assistant Professor, Department of Biotechnology, IIT Roorkee [Apr-Oct] 1997-1998 Visiting Scientist, UAMS, USA [Nov-Dec] 2001-2003 Head, Department of Biotechnology, IIT Roorkee [Jan-Dec] 2003-2006 Associate Professor, Department of Biotechnology, IIT Roorkee [Oct-Mar] 2006-Till date Professor, Department of Biotechnology, IIT Roorkee, [Oct-Mar] Honors/Awards/Fellowship

1. Cancer Research Institute, New York, USA: Fellowship award of US $ 50, 000 for the project entitled “ Regulation of Rpt-1: an intracellular inducer /helper T-cell protein that affects HIV-1 and IL-2r expression “, Harvard Medical School, DFCI, Boston, USA, 1988 – 90

2. The Ph.D. thesis supervised was awarded “Innovation Potential Award 2001” by “Indian National of Academy of Engineering”.

3. Member contributor, Scope Water team, German Organization, Strategic Science Consult Ltd, Granted by German by German Federal Ministry of Education and Research Feb 2004-to date

4. Member, Process Innovation and Intensification Network, Whitley Bay, UK, March 05 to date 5. Convener, Biotechnology, IIT Roorkee, National Programme on Technology Enhanced Learning, MHRD,

Govt. of India. June 2009 – to date 6. Member - Expert , Bioprocess and Bioproduct Development, Department of Biotechnology, Govt. of India,

Ministry of Science & Technology, New Delhi, 2008 - to date 7. Member-Expert, Committee on Genetically modified Organisms and Food, Food Safety and Standards

Authority of India, Govt. of India, New Delhi, May 2009 – to date 8. Member, Expert Committee of AICTE, Govt of India for reviewing the AICTE sponsored projects for

North Western region. 9. Member, Expert Committees of Department of Science & Technology (DST) and Department of

Biotechnology (DBT), Govt. of India, to review the project proposals submitted for funding. 10. Reviewer of various research articles from Elsevier, Springer and Blackwell publishers

11. Best paper award in the technical session at National Symposium ‘Biohorizon 2006, IIT Delhi. 12. University Teaching/Research Assistantship, G B Pant University, Pantnagar, (1976 - 78) 13. National Merit Scholarship (1972 - 75)

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Details of the Ph.D. thesis supervised: Awarded : 14 ; In Progress : 03 M.Sc. Dissertations : Completed – 47 ; In progress – 01 M.Tech. Dissertations : Completed – 03 B. Tech Dissertations : Completed – 0 In progress – 02

Publications

Patents - 01 Reports- 07 Chapters in the Books/Proceedings – 04 Research Papers in National and International Journals - 48

Research Papers in National and International Conf/Symposia - 49 Some recent publications:

1. Penicillium oxalicum SAEM-51: a mutagenised strain for enhanced production of chitin deacetylase for bioconversion to chitosan. Nidhi Pareek, Vivek Vivekanand, Pallavi Dwivedi, and Rajesh P. Singh. New Biotechnology, 28: 118-124 (2010)

2. Bleaching applications and scaled-up production of xylanase-laccase mixture in a intermittent rotating drum bioreactor. Dwivedi P, Vivekanand V, Pareek N, Sharma A and Singh RP. J Biotechnol 150(S1), 75-76 (2010)

3. Bleach enhancement of mixed wood pulp by using xylanase-laccase concoction derived through co-culture strategy. Dwivedi P, Vivekanand V, Pareek N, Sharma A and Singh R.P .Appl Biochem Biotechnol 160(1), 255-268 (2010)

4. Optimization of medium composition for enhanced chitin deacetylase production by mutant Penicillium oxalicum SAEM-51 using response surface methodology under submerged fermentation. Pareek N, Singh R P and Ghosh S. Process Biochemistry. (2010) (Accepted)

5. Parthenium sp. as a plant biomass for the production of alkalitolerant xylanase from mutant Penicillium oxalicum SAUE-3.510 in submerged fermentation. Dwivedi P, Vivekanand V, Ganguly R and Singh RP. Biomass Bioenergy 33, 581-58 (2009)

6. Enhanced delignification of mixed wood pulp by Aspergillus fumigatus laccase mediator system. Vivekanand V, Dwivedi P, Sharma A, Sabharwal N and Singh RP. W J Microbiol Biotechnol 24(12), 2799-2804 (2008)

7. Solid-state fermentation for gluconic acid production from sugarcane molasses by Aspergillus niger ARNU-4 employing tea waste as the novel solid support. Sharma A, Vivekanand V and Singh RP. Biores Technol. 99(9), 3444-3450 (2008).

8. Enhanced delignification of mixed wood pulp by Aspergillus fumigatus laccase Mediator system. Vivekanand, V., Dwivedi, P., Sharma, A., Sabharwal, N., Singh, R.P. World J Microbiol Biotechnol. 24: 2799-2804 (2008).

9. Solid-state fermentation for gluconic acid production from sugarcane molasses by Aspergillus niger

ARNU-4 employing teawaste as the novel solid-support Sharma, A., Vivekanand and Singh, R. P.. Bioresource Technol. 99(9), 3444- 3450 (2007).

10. Effect of xylynases from Aspergillus niger NKUC3-0.2 mutant strain on prebleaching of wheat straw

and mixed hardwood pulps. Kapur N., Dutt,D., Singh, R.P., Tyagi, C.H. and Vivekanand Cellulose. Chem Technol.40 (8), 635-641 ( 2007 ).

11. A multi-kinetic approach to predict gluconic acid production in an airlift bioreactor Mayani, M., Mohanty,

B. and Singh, R.P.Biotechnology J. 2, 1-9 ( 2007 ).

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12. Production of lactic acid with loofa sponge immobilized Rhizopus oryzae RBU2-10Ganguly, R., Dwivedi,

P and Singh, R.P. Bioresource Technol. 98 (6), 1246-1251( 2007 ).

13. Effect of Xylanase from Aspergillus niger NKUCN 3.410 mutant strain on prebleaching of wheat straw and mixed hard wood pulps. Kapur N, Dutt D, Singh RP Tyagi CH and Vivekanand V .Cellu Chem Technol 40(8), 635-641 (2006).

14. Bioconversion of Grape must into modulated gluconic acid production by Aspergillus niger ORS- 4.410. Singh, O.V and Singh, R.P. J Appl Microbiol.100 ( 5 ), 1114-1122. (2006).

Research Support A. Ongoing Research Projects

Sl No. Title of Project Funding Agency Amount, Rs Date of sanction and Duration

1 Evaluation of antimicrobial ingredients of some Indian ethnomedicinal plants mainly from Compositae, Verbanaceae, Apocyanaceae and Liliaceae families from outer Himalayan regions

DRDO, New Delhi 9.48 lakhs November 24, 2010

B. Completed Research Projects (only projects of last 3 years)

Sl No. Title of Project Funding Agency Amount, Rs Date of completion 1 Fund for Improvement for

Infrastructure in Science & Technology

DST, New Delhi 28.0 Lakhs,

2003-2008

Place: Roorkee Signature of PI Date:

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PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS Name Ashok PANDEY Designation: Head of Division, Address: Biotechnology Division, NIIST, CSIR, Trivandrum-695 019 Date of Birth: 1st January 1956........................ Sex: Male................ SC/ST: No Academic Qualification B. Sc. 1974 Biology, Chemistry Kanpur University M. Sc. 1976 Chemistry Kanpur University Ph. D. 1979 Microbiology Allahabad University Position and Employment 1979-82 Post-doctoral Fellow, Allahabad University, Allahabad 1982-85 Scientist, National Sugar Institute, Kanpur 1985-86 Scientist, Biotechnology, Zentral Labotorium, Sudzucker, Germany 1987-till-date* Scientist, National Institute for Interdisciplinary S&T, CSIR, Trivandrum 1992* Visiting Scientist, GBF, Germany [Sept-Oct] 1992-1993* Visiting Scientist, University of Louis Pasteur, France [Dec-Jan]

1995* Visiting Scientist, University of Louis Pasteur, France [July-Dec] 1997* Visiting Scientist, University of Sunderland, UK [May] 1998-2000* Professor Titular, Federal University of Parana, Brazil

2000* UNESCO Professor, Malaysia, Thailand [December] 2003-2010* Visiting Professor, University of Blaise Pascal, France [June/July each year] 2008-2010* Visiting Professor, EPFL, Lausanne, Switzerland [December each year] Honors/Awards/Fellowship:

Editor-in-chief, Bioresource Technology (2011-cont) Fellow, Association of Microbiologists of India (2010) Fellow, International Organization for Biotechnology and Bioengineering (2008) SciTopic Author, Elsevier (2008) Visiting Professor, EPFL, Switzerland, 2008, 2009 Appreciation Award, Federal University of Parana, Brazil (2007) Honorary Doctorate degree, Univesite Blaise Pascal, France (2007) Thomson Scientific Award- India Citation Laurate (2006) Fellow, Biotech Research Society of India (FBRS) (2005) Lupin Visiting Fellow (2005) Editor, Bioresource Technology, Elsevier Science, UK, 2004-cont President, Biotech Research Society of India, 2004-cont Chair (F&H), International Society of Food, Agriculture & Environment (Finland), 2003-2004 Visiting Professor, Universite Blaise-Pascal, Clermont-Ferrand, France, 2003-2009 Best Scientific Achievement Award for the year 2001, Govt of Cuba, jointly with three others, 2002 UNESCO Professor, 2000 Visiting Faculty [Professor Titular], Federal University of Parana, Curitiba, Brazil, 1998-2000 Visiting Scientist, University of Sunderland, Sunderland, UK, 1996 Raman Research Fellowship Award, CSIR, New Delhi, 1995 CNRS, France Fellowship, France, 1993 GBF International Fellowship, Germany, 1992 Young Scientist Award, DSTE, Trivandrum, 1989

Publications 827 Patents - 12 Popular Science Book - 2 Encyclopaedia- 1 Books- 24 Journals as special issue editor – 18 Reports - 25

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Chapters in the Books/Proceedings –87 Original and Review Papers – 319 Research Communications (in International conf/symposia) - 251 Research Communications (in National conf/symposia) – 88

Some recent relevant papers: 1. A comparative analysis of recombinant expression and solubility screening of two phytases in E. coli, MV Ushasree, M

Sumayya & Ashok Pandey, Food Technology and Biotechnology, 48(3), (2011) 2. Adopting structural elements from intrinsically stable phytase - A promising strategy towards thermostable phytases, MV

Ushasree, M Sumayya & Ashok Pandey, Indian Journal of Biotechnology (2011) 3. An improved bioprocess for extracellular L-leucine amino peptidase production using Streptomyces gedanensis, Raji

Rahulan, Ashok Pandey& KM Nampoothiri, Current Microbiology, 62, 1009-1016 (2011) 4. Molecular cloning, over-expression and characterization of the raw starch digesting alpha-amylase of Bacillus

amyloliquefaciens, D Gangadharan, P Ramachandran, P Gunasekaran, Ashok Pandey & KM Nampoothiri, Biologia- Cellular and Molecular Biology, 65(3), 392-398 (2010)

5. Advancement and comparative profiles in the production technologies using solid-state and submerged fermentation for microbial cellulases, Reeta Rani Singhania, RK Sukumaran, AK Patel, C Larroche & Ashok Pandey, Enzyme and Microbial Technology, 46(7), 541-549 (2010)

6. Genome shuffling: A new trend in improved bacterial production of lactic acid, RP John, GS Anisha, Ashok Pandey & KM Nampoothiri, Industrial Biotechnology, 6(3): 164-169 (2010)

7. Molecular cloning, over-expression and biochemical characterization of hypothetical beta lactamases of Mycrobacterium tuberculosis H37Rv, KM Nampoothiri, R Rubex, AK Patel, SS Narayanan, S Krishna, SM Das & Ashok Pandey, Journal of Applied Microbiology,105(1), 59-67 (2008)

8. Direct lactic acid fermentation: Focus on simultaneous saccharification and lactic acid production, RP John, GS Anisha, KM Nampoothiri & Ashok Pandey, Biotechnology Advances, 27, 145-152 (2009)

9. Production and partial purification of α-amylase from a novel isolate Streptomyces gulbargensis, SG Dastager, A Dayanand & Ashok Pandey, Journal of Industrial Microbiology & Biotechnology, 36 (2), 189-194 (2009)

10. Statistical optimization of L-leucine amino peptidase production from Streptomyces gedanensis IFO 13427 under submerged fermentation using response surface methodology, R Rahulan, KM Nampoothiri, G Szakacs, V Nagy &Ashok Pandey, Biochemical Engineering Journal, 43(1), 64-71 (2009)

11. Production of L-leucine aminopeptidase by selected Streptomyces isolates, V. Nagy, KM Nampoothiri, A Pandey, R Rahulan & G Szakacs, Journal of Applied Microbiology, 104, 380-387 (2008)

12. Biosynthesis of silver nanoparticles using aqueous extract from the compactin producing fungal strain, NS Shaligram, M Bule, R Bhambure, RS Singhal, SK Singh, G Szakacs & Ashok Pandey, Process Biochemistry, 44(8), 939-943 (2009)

Research Support Ongoing Research Projects

Title of Project Funding Agency Amount, Rs Date of sanction and Duration

Construction and screening of environmental DNA libraries for novel beta-lactamase inhibitors and lipases

DBT, New Delhi 49.1 lakhs April 2007- March 2011

Development of thermostable and low pH tolerant phytase from Aspergillus niger using site-directed mutagenesis

DBT, New Delhi 38.33 lakhs July 2007-June 2011

Exploration of India’s Rich Microbial Diversity

CSIR, New Delhi 210 lakhs April 2008-March 2012

Creation of Centre for Biofuels

TIFAC & CSIR, New Delhi

632 lakhs July 2008-August 2011

Development of a bioprocess for the production of polyhydroxy butyrate (PHB) from bio-diesel industry generated glycerol

DBT, New Delhi 34.74 lakhs January 2009-December 2011

Place: Trivandrum Signature of PI Date: