Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1993-02-18
2003-07-01
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S203000, C435S209000, C435S252300, C435S325000, C536S023740, C536S023200, C530S324000, C426S549000, C426S592000, C426S660000, C426S656000, C426S496000
Reexamination Certificate
active
06586209
ABSTRACT:
This invention lies in the field of recombinant DNA technology and is directed at a cell having a certain function in a process, containing recombinant DNA encoding at least one enzyme. The invention is directed especially at a cell having a function in the field of food processing and also at cells with a function in processes in which a cellulose-containing raw material is used, such as processes for preparing beer, paper, starch, gluten etc, and processes for decomposing cellulose-containing waste such as agricultural waste, waste from paper rills etc.
In particular the invention is directed at cells having a function in the process of fermentation, more especially at cells with a function in the process of preparing bakery products.
The cell according to the invention is characterized in that the cell becomes polyfunctional for the process in which it has a function, upon expression of the recombinant DNA encoding at least one enzyme. In the case of a fermentation process for example, such as the preparation of bread, yeast is used as a cell with a particular function in said process. A yeast cell according to the invention does not only have its normal function, i.c. a function that a yeast lacking the recombinant DNA can also carry out, but also has another function in said process of bread preparation. An example of such an additional function is the expression and secretion of a bread improving enzyme.
The present invention is directed in particular at a cell with a function in the preparation of bakery products. Cells containing recombinant DNA encoding enzymes selected from the group of enzymes with amylolytic and/or hemicellulolytic and/or cellulolytic activity are suitable.
The invention is also directed at a process for the production of at least one enzyme by a polyfunctional cell as described above comprising culturing such a polyfunctional cell in a suitable nutrient medium and optionally isolating the resulting enzyme form. In such a process, said enzyme is preferably selected from the group of enzymes with amylolytic and/or hemicellulolytic and/or cellulolytic activity. A suitable medium for carrying out the process according to the invention can consist of the medium in which the process for which the cell is polyfunctional is carried out. In the process of the preparation of a bakery product for example said medium can be the dough that it to be baked. Naturally the other usual media for culturing cells can also be used. The choice of media will depend on whether the enzyme is to be used in situ or has to be isolated. In some cases it will suffice to use the medium containing said enzyme and in other cases the enzyme will have to be isolated from the medium.
The invention is also directed at an enzyme encoded by the recombinant DNA in said polyfunctional cell, whereby said enzyme is obtainable from such a polyfunctional cell via the afore mentioned process for producing an enzyme. The invention is further directed at the use of such a polyfunctional cell or such an enzyme, for example in the processes described above, such as food processing and processes using a cellulose containing raw material, preferably in a process for the preparation of a bakery product.
Flour, yeast, water and salt are the basic ingredients of bread and other bakery products. For centuries materials having a positive effect on the manageability of the dough or the quality of the baked product have been added in the manufacture of bread and similar bakery products, for the sake of convenience further referred to as bread making. Said additives, referred to as “bread improvers”, contain enzymes from malt or of microbial origin which play an important part in the different phases of bread making, namely, the preparation of the bread batter, fermentation, baking, and storage of the bread product.
One of the relevant characteristics of bread that is influenced by adding specific enzymes is the so-called bread volume. In order to obtain a high bread volume in practice compositions containing cellulolytic, hemicellulolytic and/or amylolytic enzymes are added. The commercially available compositions of microbial origin, mostly originating from a fungus o; one of the genera Aspergillus and Trichoderma, are substantially unpurified complex mixtures of different enzyme activities, whereby it is not exactly known which enzymes are present in the composition and which have a bread improving activity. This lack of knowledge impedes further bread improvement and especially impedes the control of the different dough processing and bread properties, such as the bread volume.
Further investigation into the process of preparation of bakery products resulted in the discovery that, in addition to &agr;-amylase, at least a xylanase enzyme is also of importance for the bread volume. A xylanase is an enzyme that catalyzes the degradation of xylans occurring in the pentosan part of starch “tailings”. The term “tailings” is directed at a fraction of, e.g., wheat starch consisting of water-insoluble hemicellulose (pentosans and arabinoxylans) and damaged starch. This fraction is formed as the intermediate or top layer of the starch pellet during centrifugation of a dough suspension obtained by washing dough to remove the gluten fraction.
Different xylanases have already been described in the literature, including xylanases of the bacterial species
Bacillus pumilus
(Panbangred et al., Mol. Gen. Genet. 192, 335-341, 1983, and Fukusaki et al., FEBS Lett. 321, 197-201, 1984),
Bacillus subtilis
(Paice et al., Arch. Microbiol. 144, 201-206, 1986), and
Bacillus circulans
(Yang et al., Nucl. Acids Res. 16, 7187, 1988), of the yeast Aureobasidium (Leathers, Biotech. Lett. 12, 775-780, 1988) and of the fungus
Aspergillus niger
(Fournier et al., Biotechnology and Bioengineering 27, 539-546, 1985).
It is known from European patent application EP-A-0338452 that the properties of dough and the quality of bread can be improved by adding different enzyme compositions to the dough, including an enzyme composition having hemicellulose degrading or xylanase activity, the origin of which is not further specified. Such a hemicellulolytic enzyme composition is a relatively undefined enzyme mixture which may contain different hemicellulolytic enzymes having various effects on the dough and bread properties. The presence of xylanases having bread improving activity to a smaller or greater extent is the coincidental result of the manner in which the enzyme composition that is intended as a bread improver has been obtained. A controlled further optimization of bread improvers, however, was not possible due to the lack of the required knowledge and of suitable recombinant DNA constructs encoding a xylanase having broad improving activity that could be used for a high production of such a xylanase.
For the purpose of this invention, “bread improving activity” is generally taken to mean a favourable effect on any property of the prepared bakery product (including bread) or of the dough from which the bakery or bread product is made, and is particularly taken to mean a favourable effect on the bread volume.
The investigation on which the invention is based has extended to the identification and cloning of a gene (xylA) encoding an enzyme, xylanase having bread improving activity, originating from a fungus of the species
Aspergillus niger
var.
awamori,
as well as to the transformation of different species of host cells in such a manner that the gene is expressed or can be expressed in said host cells. The invention, however, comprises all xylanase genes originating from fungi and especially from fungal strains from the same genus and therefore the invention is not limited to the actually cloned gene.
The present invention is therefore also directed at recombinant DNA material comprising DNA with a nucleotide sequence encoding at least a ripening form of a xylanase of fungal origin.
The term “ripening form” refers to the different forms in which the enzyme may occur after expression of the associated gene. More in particular, i
Hessing Johanna G. M.
Maat Jan
Roza Martinus
van Gorcom Robert F. M.
Verbakel Johannes Maria A.
Eyler Yvonne
Quest International B.V.
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