Invented by Peter Stougaard, Ole Cai Hansen, DuPont Nutrition Biosciences ApS
The DuPont Nutrition Biosciences ApS invention works as followsA method for producing hexose oxidease using recombinantDNA technology, recombinanthexoseoxidase, and the use thereof, in particular in manufacturing food products like doughs and dairy products as well as pharmaceuticals, cosmetics, and dental care products. The enzyme can be derived from marine algal species such as Euthoracristata, Chondrus crispus and Iridophycus Flaccidum. The recombinant Hexose Oxase can be used in useful embodiments by Pichia pastoris or Saccharomyces cerevisiae. coli.
Background for Recombinant Hexose Oxase: A method of producing it and the use of such enzyme.
Hexose O2-oxidoreductase (D?hexose) is an enzyme that, in the presence oxygen, is capable of oxidizing D?glucose as well as several other reducing sugars, including maltose or lactose, to their respective lactones and then hydrolyzing the respective aldobionic acid. Hexoseoxidase is able to use a wider range of sugar substrates than glucose oxidase, which can only convert D?glucose. You can see the hexose-oxidase catalyzing oxidation as follows: be illustrated as follows:\nD-Glucose+O2—>?-D-gluconolactone+H2O2, or\nD-Galactose+O2—>?-D-galactogalactone+H2O2
At present, hexose oxidase (in this case also referred as HOX) was obtained by isolating the enzymes from various red algal species like Iridophycus Flaccidum (Bean et al. 1956) and Chondrus Crispus (Sullivan et. al. 1973). 1973). The algal species Euthora Cristata has also been proven to produce hexose oxide.
It has been reported that certain foods could be made using hexose oxidease obtained from natural sources. Hexose oxide from Iridophycus flaccidum was shown to convert lactose to milk and produce the aldobionic acids. It has also been suggested that it could be used as an acidifying agent in milk (e.g. To replace the acidifying microbial culture for this purpose (Rand 1972). In this respect, hexoseoxidase has been referred to as an interesting enzyme rather than glucose oxidase. This enzyme cannot be used in milk or other food products that do not contain glucose. Although glucose may be made available in this way as a substrate to the glucose-oxidase enzyme, it is clear that only half of the lactose end products can be used as substrates by the glucose-oxidase. Consequently, glucose oxidase cannot be used as an effective acidifying agent in natural milk and dairy products.
The ability of oxygen oxidoreductases, including that of Hexose Oxase, to produce hydrogen peroxide has been used to increase the storage stability for certain food products such as cheese, butter, and fruit juice, as disclosed in JP – B-73/016612. The possibility exists that oxidoreductases could be useful in oxygen scavengers and antioxidants in food products has been also suggested.
Oxidizing agents are used in the baking and milling industries. For improving the baking performance of flour, oxidizing agents such as e.g., peroxides and ascorbic acid, Kbromate, azodicarbonamide are used to improve its stretchability and provide a desired strength and stability. This is because flour proteins such as e.g. Gluten in wheat flour has thiol group which, when they are oxidized, form disulphide bond. This results in better quality dough and improved volume and crumb structure for baked goods.
However such use of many of the currently available oxygenizing agents is objected by consumers or not allowed by regulatory bodies. Therefore, it was attempted to find alternative flour and dough additives. The prior art suggests the use glucose oxidase to accomplish the above purpose. Thus, U.S. Pat. No. No. 2,783,150 describes the addition of glucose oxide to flour in order to improve the rheological properties of dough. CA 2,012,723 describes bread-improvement agents that include cellulolytic enzymes, glucose oxidase, and JP-A-8484848 suggests bread-improving compositions with glucose oxidase/lipase.
However, glucose oxidase is a bread and dough improving additive. This enzyme needs glucose as a substrate to work in a dough system. Generally, however, the glucose content of cereal flours are low. In wheat flour, glucose is found in a range of 0.04 to 0.4% w/w. Some flours may not contain any glucose. The absence of glucose or low glucose content in doughs may limit the ability to use glucose oxidase to improve the dough. The content of maltose in freshly prepared dough is much higher than that of the past. Further maltose forms in the dough because of the activity of??-amylase, which is either in the flour or added to it.
The only source for hexose oxidese at the moment is partially or crude enzyme preparations that have been extracted from marine algal species. It is obvious that this method of producing hexose oxidease is too laborious and expensive to justify a commercial production. It is also difficult to provide a sufficient amount of pure enzyme at a reasonable price.
It is a significant industrial need to find an alternative source for this valuable industrial enzyme that is more economical than a natural one. The enzyme must also be provided in its purest form. Without any contaminating enzyme activity or any other undesirable contaminants including algal pigments or environmental pollutants that may be present in marine areas where the hexose oxide-producing algal species are found.
Furthermore the industrial availability in sufficient quantities and at affordable prices of food-grade hexose oxide will undoubtedly open up new applications for that enzyme in the food industry as well as in other industrial sectors, as will be discussed in detail in the following. The novel use of recombinant Hexose Oxase in food industry includes its use as a dough improver. Another example is the use of the active polypeptide of hexose oxide or a recombinant organism that produces the polypeptide in manufacturing lactones.
The invention, which uses recombinantDNA technology, has made it possible for the first-time to provide hexose oxide active polypeptides at industrially acceptable quantities and at a high quality and purity level. This makes the invention highly suitable to any industrial purpose, including the manufacture of food products and pharmaceuticals.
According to the invention, the first aspect relates to a method for producing a polypeptide with hexose oxide activity. This involves isolating or synthesizing the DNA fragment and introducing it into a host organism. The DNA fragment is then combined with an expression signal for the polypeptide. Finally, the host organism is grown under conditions that allow the polypeptide to be expressed in the cultivation medium.
In another aspect, the invention refers to a polypeptide isolated having hexose oxide activity. It comprises at least one amino acids sequence chosen from the group consisting
In yet further aspects, the invention refers to a fragment of DNA that codes for a polypeptide with hexose-oxidase activity. It also pertains to a cell containing such a modified DNA molecule.
In addition, the invention relates to the use the above hexose oxide active polypeptide or the microbial cells expressing such a protein in the manufacture of a food or animal feed, and the manufacturing a pharmaceutical, cosmetic, or tooth care product.
In addition to useful aspects, there is a method for reducing the sugar content in a food product. This involves adding to the food product a certain amount of the polypeptide/the microbial cells as disclosed herein. A method of making a baked product out of dough. This includes adding the hexose-oxidase active protein polypeptide, or a microbial expressing such a monopeptide to the dough. Also, the invention provides a method for improving the polypeptide/the microbial cells according to the invention, at least one traditional dough component.
Another aspect of the invention is the use of the polypeptide, or a microcellular cell according to the invention, as an analytical reagent to measure the sugar content.
The invention provides an interesting feature: the invention allows the use of a polypeptide, or a microcellular cell, according to the invention, in the manufacture of a lactone. The polypeptide/or the microcellular cell is applied to reactors containing a carbohydrate that can be oxidized using the polypeptide, and the reactor is operated under conditions that the carbohydrate is oxygenated.
DETAILED DESCLOSURE OF THE INTELLION
Many marine algal species naturally produce hexose oxideases. These species are called i.a. These species are found in the family Gigartinaceae, which is part of the order Gigartinales. Chondrus crispus, Iridophycus Flaccidum and Chondrus crispus are two examples of hexose oxidase-producing algal species that belong to Gigartinaceae. According to the invention, algal species belonging to Cryptomeniales, including Euthora Cristata, could also be sources of the hexose oxide active polypeptide. These algal species could be useful sources of hexose oxide and DNA coding for hexose-oxidase active peptides. The term “hexose-oxidase active proteinpeptide” is used herein. denotes an enzyme that at least oxidizes D.glucose or D.galactose, D.mannose, maltose (lactose), and cellobiose
When using natural sources to isolate native hexose oxidase as it has been done previously and in the present invention, with the purpose of identifying Algal material that could be used in the construction cDNA, as well as as as the starting point for constructing primers for synthetic DNA oligonucleotides, enzymes are typically extracted from the algal start material using an aqueous extraction media.Click here to view the patent on Google Patents.