Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
1997-07-23
2002-10-01
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S091200, C435S183000, C514S002600, C514S451000, C514S553000, C530S350000
Reexamination Certificate
active
06458556
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for enhancing enzyme activity at an elevated temperature by using a substance exhibiting chaperone function.
In general, enzymes exhibit lower activity at a temperature above their optimum temperature than the activity at their optimum temperature. It is also known that their activity is lost when they are exposed to a temperature higher than a certain level. Depending on the kind of enzyme, a temperature at which such heat inactivation occurs may vary. However, most of enzymes having optimum temperature of ordinary temperature are inactivated when heated to around 50° C. Enzymes stable at an elevated temperature are also known and such heat-resistant enzymes generally have a higher optimum temperature.
Depending on the conditions where enzymes are used, it is often desirable to use enzymes at an elevated temperature. In such a case, a heat-resistant enzyme as mentioned above is generally used. Examples of such a heat-resistant enzyme include Taq polymerase, which is frequently used for PCR. However, in many cases, a suitable heat-resistant enzyme may not be known, or even if a possible heat-resistant enzyme is known, other conditions to be used may not meet the enzyme.
For example, Superscript II is known as a reverse transcriptase (RNA-dependent DNA polymerase) which can afford a cDNA from a mRNA in vitro. Superscript II is a heat-labile enzyme exhibiting an optimum temperature of 42° C. and completely inactivated at a temperature above 50° C. within 10 minutes. Although Tth DNA polymerase is an enzyme having heat resistance and reverse transcription activity, it requires manganese ions for exerting the enzyme activity. If cDNAs are produced from mRNAs at a higher temperature using the Tth DNA polymerase, mRNAs are fragmented by manganese ions presented in a reaction system and therefore it becomes difficult to obtain full length cDNAs.
Magnesium ions required by a heat-labile reverse transcriptase such as Superscript II mentioned above may also cause the fragmentation of mRNA in a certain buffer or water at an elevated temperature. According to the present inventor's researches, manganese ions exhibit stronger fragmentation activity than magnesium ions and control of the fragmentation due to manganese ions is difficult even using a chelating agent.
Taq polymerase is known as an inherently heat-resistant enzyme. However, it shows reduction of activity during 25 to 30 cycles or more generally used in PCR. Therefore, if the reduction of Taq polymerase activity can be prevented, higher amplification effect and higher cycle number can be realized with fewer units of the enzyme.
In some cases, reverse transcription may be required to be performed at a temperature above 50° C. for some reasons. For example, in order to obtain full length cDNAs, it is desirable to preform reverse transcription while preventing the formation of secondary structure of mRNAs.
However, a heat-resistant enzyme having reverse transcription activity such as Tth DNA polymerase cannot afford full length cDNAs. Therefore, it is necessary to utilize a currently available reverse transcriptase which is used at an ambient temperature.
Similar situation may be frequently found in other enzymes not only polymerases but also restriction enzymes.
For some enzymes, it has been known that an enzyme exhibiting a higher optimum temperature can be obtained by introducing a mutation through genetic engineering. However, such improvement of heat-resistance is not always possible and has not been known so long as reverse transcriptase concerns.
If an enzyme can exhibit higher activity at a higher temperature, its utility may be enhanced even though it is known as a heat resistant enzyme.
Therefore, the object of the present invention is to provide a method for easily and efficiently improving heat resistance of enzyme to obtain high enzyme activity at an elevated temperature.
REFERENCES:
patent: 5614387 (1997-03-01), Shen et al.
patent: 0 035 204 (1981-09-01), None
patent: 0 117 064 (1984-08-01), None
patent: 4-370095 (1992-12-01), None
patent: 5-268952 (1993-10-01), None
patent: 8-131170 (1996-05-01), None
patent: 0 117 064 (1984-08-01), None
patent: 93 16175 (1993-08-01), None
patent: 96 15235 (1996-05-01), None
Thaker et al. ‘Osmolyte Mediated of T7 DNA Polymerase and Plasmid DNA Stability’, Biochemistry, vol. 33, pp. 12255-12259.*
Skowyra et al. ‘TheE. ColiDnak Gene Product, The HSP70 Homolog, Can Reactive Heat Inactivated RNA Polymerase in an ATP Hydrolysis-Dependent Manner’, Cell. vol. 62. pp. 939-944.*
Hottiger et al. ‘The Role of Trehalose Synthesis for the Acquisition of Thermotolerance in Yeast II. Physiological Concentration of Trehalose Increase Thermal Stability of Proteins in Vitro’ Eur. J. Biochem. vol. 219, pp. 187-193.*
Carninci et al., “Thermostabilization and Thermoactivation of Thermolabile Enzymes by Trehalose and Its Application for the Synthesis of Full Length cDNA”Proc. Natl. Acad. Sci. USA, vol. 95, pp. 520-524, Jan. 1998.
Burteau et al., “Stabilisation and Immobilisation of Penicillin Amidase”,FEBS Letters, vol. 258, No. 2, Dec. 4, 1989, Amsterdam NL, pp. 185-189.
Bernier et al., “Stabilization of Beta-Galactosidase by Polyhydric Alcohols”,Journal of Biotechnology, vol. 7, No. 4, 1988, Amsterdam NL, pp. 293-298.
“Enzyme Stabilization”Advances in Biochemical Engineering, vol. 12, Jan. 1, 1979, New York US, pp. 55-67.
Thaker, et al., “Osmolyte Mediation of T7 DNA Polymerase and Plasmid DNA Stability” Biochemistry, 1994, 33, 12255-12259.*
Mahoney, et al., Substrate-induced Thermal Stabilization of Lactase (Escherichia coli) in Milk, in Ann. New York Acad. Sci., 1988, 542, 274-278.*
Coolbear, et al., Proteases from Extreme Thermophiles in Ann. New York Acad. Sci., 1988, 542, 279-281.*
Larreta-garde, et al., “Behavior of Enzymes in the Presence of Additives”, in Ann. New York Acad. Sci., 1988, 542, 294-298.*
Back, et al., “Thermal Stability of Proteins”, Biochemistry, 1979, 18, 5191-5196.*
Buchner, et al., “GroE Facilitates Refolding of Citrate Synthase by Suppressing Aggregation”, Biochemistry, 1991, 30, 1586-1591.
Klibanov, A., “Stabilizationof Enzymes Against Thermal Inactivation” in Adv. Appl. Microbiology, 1983, Academic Press: New York, vol. 29, 1-28.
Colaco, et al., “Extraordinary Stability of Enzymes Dried in Trehalose: Simplified Molecular Biology”, Bio/Technology, 1992, 10 (9), 1007-1011.
Burns Doane Swecker & Mathis L.L.P.
Gupta Anish
Low Christopher S. F.
The Institute of Physical & Chemical Research
LandOfFree
Method for enhancing enzyme activity at elevated temperature does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for enhancing enzyme activity at elevated temperature, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for enhancing enzyme activity at elevated temperature will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2930558