Title:
MUTANTS OF L-ASPARAGINE
Document Type and Number:
WIPO Patent Application WO/2012/028945
Kind Code:
A3
Abstract:
The present invention relates to a novel mutant of L-asparaginase enzyme characterized in having high thermostability, pH stability and no glutaminase activity useful for therapeutics and the process of preparing the same. The present invention specifically relates to mutant's MTCC 5580, MTCC 5581 and MTCC 5582 characterized in having higher stability, no glutaminase activity etc., to allow their usage in the form of improved protein therapeutics. A thermostable L-asparaginase from P. furiosus was cloned and expressed in E. coli host. The enzyme was engineered at its active site to create three different mutants based on structural and sequence analysis with a E. coli- derived enzyme homologue. The mutants MTCC 5580, MTCC 5581 and MTCC 5582 were tested for their stability, substrate affinity, optimum pH and temperature of activity and cytotoxicity. Based on the studies, all the three enzymes were found thermostable and with no glutaminase activity as compared to other available enzyme EcA II. MTCC 5579 and the above said three mutants showed the cytotoxicity on the leukemic cell lines. The present study showed that these enzymes are promising candidates for the treatment of leukemia.
Inventors:
KUNDU BISHWAJIT (IN)
BANSAL SAURABH (IN)
MISHRA PRASHANT (IN)
BANSAL SAURABH (IN)
MISHRA PRASHANT (IN)
Application Number:
PCT/IB2011/002018
Publication Date:
July 19, 2012
Filing Date:
September 01, 2011
Export Citation:
Assignee:
INDIAN INST OF TECHNOLOGY DEHLI (IN)
DEPT OF BIOTECHNOLOGY MINISTRY OF SCIENCE & TECHNOLOGY (IN)
KUNDU BISHWAJIT (IN)
BANSAL SAURABH (IN)
MISHRA PRASHANT (IN)
DEPT OF BIOTECHNOLOGY MINISTRY OF SCIENCE & TECHNOLOGY (IN)
KUNDU BISHWAJIT (IN)
BANSAL SAURABH (IN)
MISHRA PRASHANT (IN)
International Classes:
C12N9/82
Domestic Patent References:
| WO2008151807A2 | 2008-12-18 |
Other References:
BANSAL S ET AL: "Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus", BIOCHEMISTRY (MOSCOW), vol. 75, no. 3, March 2010 (2010-03-01), pages 375 - 381, XP002674601
DERST CHRISTIAN ET AL: "Engineering the substrate specificity of Escherichia coli asparaginase II. Selective reduction of glutaminase activity by amino acid replacements at position 248", PROTEIN SCIENCE, vol. 9, no. 10, October 2000 (2000-10-01), pages 2009 - 2017, XP002674602, ISSN: 0961-8368
LI LIANG-ZHU ET AL: "Enhancing the thermostability of Escherichia coli L-asparaginase II by substitution with pro in predicted hydrogen-bonded turn structures", ENZYME AND MICROBIAL TECHNOLOGY, vol. 41, no. 4, September 2007 (2007-09-01), pages 523 - 527, XP022146695, ISSN: 0141-0229
BANSAL SAURABH ET AL: "Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action", FASEB JOURNAL, FED. OF AMERICAN SOC. FOR EXPERIMENTAL BIOLOGY, US, vol. 26, no. 3, 1 March 2012 (2012-03-01), pages 1161 - 1171, XP009158676, ISSN: 0892-6638
DERST CHRISTIAN ET AL: "Engineering the substrate specificity of Escherichia coli asparaginase II. Selective reduction of glutaminase activity by amino acid replacements at position 248", PROTEIN SCIENCE, vol. 9, no. 10, October 2000 (2000-10-01), pages 2009 - 2017, XP002674602, ISSN: 0961-8368
LI LIANG-ZHU ET AL: "Enhancing the thermostability of Escherichia coli L-asparaginase II by substitution with pro in predicted hydrogen-bonded turn structures", ENZYME AND MICROBIAL TECHNOLOGY, vol. 41, no. 4, September 2007 (2007-09-01), pages 523 - 527, XP022146695, ISSN: 0141-0229
BANSAL SAURABH ET AL: "Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action", FASEB JOURNAL, FED. OF AMERICAN SOC. FOR EXPERIMENTAL BIOLOGY, US, vol. 26, no. 3, 1 March 2012 (2012-03-01), pages 1161 - 1171, XP009158676, ISSN: 0892-6638
Attorney, Agent or Firm:
MANDAL, Shuva et al. (FM House A-9 Sector-9, IN)
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