Enzyme with acetyl esterase activity

Details Enzyme with acetyl esterase activity Enzyme with acetyl esterase activity

1996-01-04

1998-11-03

Prouty, Rebecca E.

Chemistry: molecular biology and microbiology

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

Hydrolase

435 18, 435101, 435196, 435267, 435274, 435278, C12Q 144, C12N 918, C07G 1700, D21C 300

Patent

active

058307346

DESCRIPTION:

BRIEF SUMMARY
FIELD OF INVENTION

The present invention relates to enzymes with acetyl esterase activity, a method of producing the enzymes, and an enzyme preparation containing one or more of the enzymes.


BACKGROUND OF THE INVENTION

Many polysaccharides can exist in acetylated forms in various biological biological significance of the acetyl groups is not fully understood. It is known that the acetyl group often protects the polysaccharide from degradation by hydrolytic enzymes. Hence, deacetylation of these polysaccharides is necessary in order to achieve partial or complete enzymes for the food industry, primarily in fruit and vegetable processing such as fruit juice production, wine making or pectin extraction, where their ability to modify acetylated polysaccharides to a readily degradable form may be utilised.
It is known that many fungi contain enzymes capable of deacetylating acetylated polysaccharides, which enzymes are commonly designated acetyl the study of these enzymes have been hampered by the lack of well-characterized homogeneous substrates, and by the difficult and time consuming assays for measuring acetate release. Any industrial use of these enzymes has not been described.
WO 92/19728 describes a rhamnogalacturonan acetyl esterase isolated from the fungal species Aspergillus aculeatus. This enzyme is specific for acetylated galacturonic acid residues in hairy regions of pectin. EP 507 369 discloses a DNA sequence encoding an acetyl xylan esterase isolated from Aspergillus niger.
For many purposes, it would be desirable to provide acetyl esterases in a form essentially free from other components. In this way, it would be possible to produce enzyme preparations adapted to specific purposes, such preparations either containing a single acetyl esterase or arbitrary combinations thereof, and optionally containing other polysaccharide degrading enzymes. To serve this end, it is convenient to provide single-component acetyl esterases by recombinant DNA techniques.


SUMMARY OF THE INVENTION

It has now surprisingly been found that the fungal species A. aculeatus, in addition to the above mentioned rhamnogalacturonan acetylesterase, produces a number of novel acetyl esterases with interesting enzymatic activities. Despite the fact that these enzymes are produced in very low amounts (constituting less than 0.1% of the total enzyme production), the present inventors have succeeded in purifying and characterizing the novel enzymes.
Accordingly, the present invention relates to novel enzymes having acetyl esterase activity and in particular to single-component acetyl esterases.
More specifically, in a first aspect the present invention relates to an enzyme with acetyl esterase activity, which enzyme comprises the amino acid sequence shown in SEQ ID No. 1, in which x designates any amino acid residue.
In the present context the term "with acetyl esterase activity" is used to define a group of enzymes, the members of which have the common characteristic of being capable of cleaving the acetyl esterase substrate p-nitrophenol-acetate (PNP-acetate) by the procedure given in the Materials and Methods section below. Furthermore, they may in some cases be able to act on other acetylated non-saccharide substrates. These enzymes are commonly termed acetyl esterases. It will be understood that the natural substrate for each of the acetyl esterases disclosed herein may vary between different acetylated polysaccharides, as exemplified by acetylated mannans, acetylated xylans, acetylated rhamnogalacturonans and acetylated pectins.
In the course of the research leading to the present invention is it was surprisingly found that different acetyl esterases isolated from A. aculeatus comprise the partial amino acid sequence shown in SEQ ID No. 1. The presence of this sequence may be a characteristic feature of acetyl esterases, in particular of acetyl esterases produced by A. aculeatus or by other related organisms. As stated above, x may be any amino acid sequence which in the present context is intended to be understood to c

REFERENCES:
patent: 5681732 (1997-10-01), De Graaff et al.
Tenkanen et al. (1993) Appl Microb Biotech 39:159-165 "Enzymatic Deacylation of Galactoglucomannans".
Khan et al (1990) Enzyme Microb Technol 12:127-137 "Comparison of Natural Hemicellulose and Chemically Acetylated Xylan as Subsyrates . . . . ".
Samson (1994) "Taxonomy-Current Concepts of Aspersillus Systematics" pp. 1-19 in Aspergillus (ed. J. Smith), Plenum Press, New York.

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