Thermostable esterase and its gene

Details Thermostable esterase and its gene Thermostable esterase and its gene

2001-08-09

2003-02-11

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S252300, C435S320100, C435S471000, C536S023200

Reexamination Certificate

active

06518053

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an esterase having the excellent thermostable property which can be utilized for ester hydrolysis reaction, ester synthesis reaction, ester interchange reaction and the like and its gene.
2. Description of the Related Art
Esterase is an enzyme which hydrolyzes an ester linkage and has ability to catalyze ester synthesis and ester interchange reaction, and has been recently utilized in organic synthesis reaction for manufacturing medicaments, pesticides or intermediates thereof.
It is desirable that the esterase, which is industrially utilized, has high stability to temperature, pH, solvent, pressure and the like. Inter alia, where the esterase has high thermostability, the reaction temperature can be elevated, enabling the reaction rate to be enhanced and an inactivation of the enzyme to be reduced. Accordingly, there is desired the esterase having the excellent thermostability for shortening the reaction time and promoting the reaction efficiency.
DISCLOSURE OF THE INVENTION
Under these circumstances, the present inventors studied hard using the technique of introducing mutation into gene by site-directed mutagenesis and, as a result, found that mutant esterase having the amino acid sequence where the particular amino acid in the wild-type amino acid sequence is substituted shows the excellent thermostability, which resulted in completion of the present invention.
That is, the present invention provides:
1. an esterase (hereinafter referred to as “the present esterase”) which is characterized in that it has at least a partial amino acid sequence necessary for expressing the thermostable esterase activity among the amino acid sequence shown by SEQ ID NO:2 having any one of the following amino acid substitutions:
(1) amino acid substitution where 325th amino acid in the amino acid sequence shown by SEQ ID NO:2 is substituted with isoleucine,
(2) amino acid substitution where 240th amino acid in the amino acid sequence shown by SEQ ID No:2 is substituted with alanine and 288th amino acid is substituted with alanine,
(3) amino acid substitution where 43rd amino acid in the amino acid sequence shown by SEQ ID No:2 is substituted with serine,
2. an esterase which is characterized in that it has at least a partial amino acid sequence necessary for expressing the thermostable esterase activity among the amino acid sequence shown by SEQ ID No:2 having amino acid substitution where 325th amino acid in the amino acid sequence shown by SEQ ID No:2 is substituted with isoleucine,
3. an esterase which is characterized in that it has at least a partial amino acid sequence necessary for expressing the thermostable esterase activity among the amino acid sequence shown by SEQ ID No:2 having amino acid substitution where 240th amino acid in the amino acid sequence shown by SEQ ID No:2 is substituted with alanine, and 288th amino acid is substituted with alanine,
4. an esterase which is characterized in that it has at least a partial amino acid sequence necessary for expressing the thermostable esterase activity among the amino acid sequence shown by SEQ ID NO:2 having amino acid substitution where 43rd amino acid in the amino acid sequence shown by SEQ ID No:2 is substituted with serine,
5. a gene which is characterized in that it encodes the esterase of the above 1 to 4,
6. a plasmid which is characterized in that it contains the gene of the above 5,
7. a microorganism which is characterized in that it contains the plasmid of the above 6,
8. a process for producing an esterase which is characterized by comprising culturing the microorganism of the above 4 and, thereby, allowing the microorganism to produce an esterase having at least a partial amino acid sequence necessary for expressing the thermostable esterase activity among the amino acid sequence shown by SEQ ID No:2 having any one of the following amino acid substitutions:
(1) amino acid substitution where 325th amino acid in the amino acid sequence shown by SEQ ID No:2 is substituted with isoleucine,
(2) amino acid substitution where 240th amino acid in the amino acid sequence shown by SEQ ID No:2 is substituted with alanine and 288th amino acid is substituted with alanine,
(3) amino acid substitution where 43rd amino acid in the amino acid sequence shown by SEQ ID No:2 is substituted with serine.
Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.


REFERENCES:
patent: 7-163364 (1995-06-01), None
patent: 7-213280 (1995-08-01), None
Rollence et al.,CRC Critical Reviews in Biotechnology, vol. 8, No. 3, 1988, pp. 217-224.
Landt, et al.,Gene, vol. 96, 1990, pp. 125-128.

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