Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-03-24
2001-10-30
Prouty, Rebecca E. (Department: 1657)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S254300, C435S320100, C435S212000, C435S326000, C435S254230, C435S256100, C435S254110, C536S023200, C536S023100
Reexamination Certificate
active
06309868
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a new recombinant prolyl-dipeptidyl-peptidase from
Aspergillus oryzae
, a gene encoding this enzyme, recombinant cells expressing this enzyme, and methods for hydrolysing protein containing materials.
BACKGROUND ART
Hydrolysed proteins, which are widely used in the food industry, may be prepared by hydrolysis of protein material with acid, alkali or enzymes. However, on the one hand, acid or alkaline hydrolysis can destroy the essential amino acids produced during hydrolysis thus reducing the nutritional value, whereas enzymatic hydrolysis rarely goes to completion so that the hydrolysed protein contains substantial amounts of peptides.
The filamentous ascomycete
Aspergillus oryzae
is known to secrete a large variety of amylases, proteinases and peptidases, the action of which are essential for the efficient solubilisation and hydrolysis of raw materials (see WO94/25580). Various methods have been used
Aspergillus oryzae
for the preparation of food products, especially methods involving the use of a koji culture.
EP417481 (Nestlé) thus describes a process for the production of a fermented soya sauce, in which a koji is prepared by mixing an
Aspergillus oryzae
koji culture with a mixture of cooked soya and roasted wheat, the koji is then hydrolysed in aqueous suspension for 3 to 8 hours at 45° C. to 60° C. with the enzymes produced during fermentation of the Aspergillus oryzae koji culture, a moromi is further prepared by adding sodium chloride to the hydrolysed koji suspension, the moromi is left to ferment and is then pressed and the liquor obtained is pasteurized and clarified.
EP429760 (Nestlé) describes a process for the production of a flavouring agent in which an aqueous suspension of a protein-rich material is prepared, the proteins are solubilized by hydrolysis of the suspension with a protease at pH6.0 to 11.0, the suspension is heat-treated at pH 4.6 to 6.5, and the suspension is ripened with enzymes of a koji culture fermented by
Aspergillus oryzae.
Likewise, EP96201923.8 (Nestlé) describes a process for the production of a meat flavour, in which a mixture containing a vegetal proteinaceous source and a vegetale carbohydrates containing source is prepared, said mixture having initially at least 45% dry matter, the mixture is inoculated with a koji culture fermented by
Aspergillus oryzae
and by one or more another species of microorganisms involved in the traditional fermentation of meat, and the mixture is incubated until meat flavours are formed.
Depending on the nature of the protein and the enzymes used for proteolysis, the peptides formed can however have extremely bitter tastes and are thus organoleptically undesirable. There is hence a need for methods of hydrolysing proteins leading to high degree of protein hydrolysis and to hydrolysates with excellent organoleptic properties.
In addition, in protein rich materials subjected to enzymatic hydrolysis, a high level of glutaminase is required to convert glutamine into glutamic acid which is an important natural taste enhancer (see WO95/31114). Biochemical analysis of residual peptides in cereals hydrolysed by
Aspergillus oryzae
, i.e. wheat gluten, shows however that a considerable amount of glutamine remains sequestered in proline containing peptides (Adler-Nissen, In: Enzymatic hydrolysis of food proteins. Elsevier Applied Sciences Publishers LTD, p120, 1986). There is hence a need for methods of hydrolysing proteins leading to liberation of high amount of glutamine.
Among the different proteases known from koji molds, two neutral endopeptidase (Nakadai et al., Agric. Biol. Chem., 37, 2695-2708, 1973), an alkaline endopeptidase (Nakadai et al., Agric. Biol. Chem., 37, 2685-2694, 1973), an aspartic protease (Tsujita et al., Biochem. Biophys Acta, 445, 194-204, 1976), several aminopeptidases (Ozawa et al., Agric. Biol. Chem., 37, 1285-1293, 1973), several carboxypeptidases (Nakadai et al., Agric. Biol. Chem., 37, 1237-1251, 1970) have been identified and purified.
More recently a prolyl-dipeptidyl-peptidase activity has been detected in
Aspergillus oryzae
, which is an enzyme providing a high level of hydrolysing specificity towards proteins and peptides starting with X-Pro- thus liberating dipeptides of X-Pro type, wherein X is any amino-acid (Tachi et al., Phytochemistry, 31, 3707-3709, 1992).
SUMMARY OF THE INVENTION
The present invention has for object the new recombinant prolyl-dipeptidyl-peptidase (DPP IV) from
Aspergillus oryzae
comprising the amino-acid sequence from amino acid 1 to amino acid 755 of SEQ ID NO:2 or functional derivatives thereof.
In a second aspect, the invention also provides a DNA molecule encoding the enzyme according to the invention.
In a third aspect, the invention provides a cell expressing the enzyme according to the invention by recombinant technology.
In a fourth aspect, the invention provides an Aspergillus naturally providing a prolyl-dipeptidyl-peptidase activity which has integrated multiple copies of the Aspergillus native promoter which naturally directs the expression of the gene encoding the prolyl-dipeptidyl-peptidase activity.
In a fifth aspect, the invention provides an Aspergillus naturally providing a prolyl-dipeptidyl-peptidase activity which is manipulated genetically so that the dppIV gene is inactivated.
In a sixth aspect, the invention provides a method for producing the enzyme according to the invention, comprising cultivating the cells of the invention in a suitable growth medium under conditions that the cells express the enzyme, and optionally isolating the enzyme in the form of a concentrate.
In a seventh aspect, the invention provides the use of the enzyme or the cells of the invention to hydrolyse protein containing materials.
In another aspect, the invention provides the use of an enzyme and/or a cell providing a prolyl-dipeptidyl-peptidase activity, in combination with at least an enzyme providing a prolidase to hydrolyse protein containing materials.
In a last further aspect, the invention provides a food product comprising a protein hydrolysate obtainable by fermentation with at least a microorganism providing a prolyl-dipeptidyl-peptidase activity higher than 50 mU per ml when grown in a minimal medium containing 1% (w/v) of wheat gluten.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Within the following description, the percentages are given by weight except where otherwise stated, and the amino acid or nucleotide sequences referred as “SEQ ID NO:” are always presented in the sequence listing hereafter.
Likewise, the expression “functional derivative of an enzyme” includes all amino acid sequences which differ by substitution, deletion, addition of some amino acids, for instance 1-20 amino acids, but which keep their original activities or functions. The selection of a functional derivative is considered to be obvious to one skilled in the art, since one may easily creates variants of the DPP IV (having the amino acid sequence SEQ ID NO:2) by slightly adapting methods known to one skilled in the art, for instance the methods described by Adams et al. (EP402450; Genencor), by Dunn et al. (Protein Engineering, 2, 283-291, 1988), by Greener et al. (Strategies, 7, 32-34, 1994), and/or by Deng et al. (Anal. Biochem, 200, 81, 1992).
In particular, a protein may be generally considered as a derivative to another protein, if its sequence is at least 80% identical to the protein, preferably at least 90%, in particular 95%. In the context of the present disclosure, the identity is determined by the ratio between the number of amino acids of a derivative sequence which are identical to those of the DPP IV having the amino acid sequence SEQ ID NO:2 (mature sequence 1-755), and the total number of or amino acids of the said derivative sequence.
In addition, the term “koji” designates the product of the fermentation with a koji mold culture of a mixture of a source of proteins and a source of carbohydrates, especially of a mixture of a leguminous plant or of a cooked oleagginous plant and of a
Affolter Michael
Doumas Agnes
Monod Michel
Van Den Broek Peter
Monshipouri M.
Nestec S.A.
Prouty Rebecca E.
Winston & Strawn
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