Recombinant bacterial phytases and uses thereof

Details Recombinant bacterial phytases and uses thereof Recombinant bacterial phytases and uses thereof

1999-05-25

2001-02-06

Achutamurthy, Ponnathapu (Department: 1652)

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

C435S196000, C536S023200

Reexamination Certificate

active

06183740

ABSTRACT:

CONTENTS
1. FIELD OF THE INVENTION
2. BACKGROUND
2.1—General Overview of the Problem to Be Solved
2.1.1—Brief Summary
2.1.2—Nutritional Concerns
2.1.3—Ex Vivo Processing Concerns
2.1.4—Medical Concerns
2.1.5—Environmental Concerns
2.1.6—Financial Concerns
2.2—General Overview of Phytate & Phytate Hydrolysis
2.2.1—Phytate Hydrolysis Leads to Release of Nutrients
2.2.2—Microbial Enzymes Can Hydrolyze Phytate
2.3—Solving the Problem of Insufficient Phytate Hydrolysis
2.3.1—Enzyme Additives in Commercial Applications
2.3.2—Optimization of Enzyme Additives Is Needed
3. SUMMARY OF THE INVENTION
4. BRIEF DESCRIPTION OF THE DRAWINGS
5. DEFINITIONS OF TERMS
6. DETAILED DESCRIPTION OF THE INVENTION
6.1—Novel Phytase
6.1.1—General Overview
6.1.2—Phytase Polypeptides
6.1.3—Phytase Polynucleotides
6.1.4—Methods of Isolation
6.1.5—Determination of Activity
6.2—Production Of Novel Phytase
6.2.1—General Overview
6.2.2—Recombinant Expression
6.2.3—Use Of Transgenic Plants And Plant Organs
6.2.4—Examples of Serviceable Plants
6.2.5—Plant Transformation Methods
6.2.6—Methods for Dicots
6.2.7—Methods for Monocots
6.2.8—Methods for Expression in Plants
6.2.9—Dual Expression of Novel Phytase & Other Molecules
6.2.10—Soluble Preparation of Novel Phytase & Stabilized Liquid Formulations Thereof
6.3—Use Of Novel Phytase
6.3.1—General Overview
6.3.2—Administration to Organisms
6.3.3—Steeping Of Cereals
6.3.4—Preparation Of Bread Dough
6.3.5—Production Of Soybean-Containing Foodstuffs
6.3.6—Production Of Liquid Foodstuffs Including Sake
6.3.7—Production Of Mineral Absorbefacient
6.3.8—Use In Combination With Other Phytases &/Or Acid Phosphatases
6.3.9—Use In Combination With Enzymes That Act On Polysaccharides (e.g. Xylanases)
6.3.10—Use In Combination With Vitamin D
6.3.11—Use In Combination With Lactic Acid-Producing Bacteria
6.3.12—Solubilization Of Proteins In Combination With Proteases
6.3.13—Triple Treatment Of Compost Using Novel Phytase, Saponin, & Chitosan
6.3.14—Use As Hybridization Probes & Amplification Templates
6.3.15—Use in Directed Evolution
6.3.16—Use in antibody production
EXAMPLE 1—Isolation, Bacterial Expression And Purification Of Phytase
7. LITERATURE CITED
CLAIMS
ABSTRACT
1. FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention have been identified as phytases and in particular, microbial enzymes having phytase activity.
2. BACKGROUND
2.1—General Overview of the Problem to Be Solved
2.1.1—Brief Summary: Minerals are essential elements for the growth of all organisms. Dietary minerals can be derived from many source materials, including plants. E.g., plant seeds are a rich source of minerals since they contain ions that are complexed with the phosphate groups of phytic acid molecules. These phytate-associated minerals satisfy the dietary needs of some species of farmed organisms, such as multi-stomached ruminants. Accordingly, ruminants do not require dietary supplementation with inorganic phosphate and minerals because microorganisms in the rumen produce enzynes that catalyze conversion of phytate (myo-inositol-hexaphosphate) to inositol and inorganic phosphate. In the process, minerals that have been complexed with phytate are released. The majority of species of farmed organisms, however, are unable to efficiently utilize phytate-associated minerals. Thus, for example, in the livestock production of monogastric animals (e.g., pigs, birds, and fish), feed is commonly supplemented with minerals &/or with antibiotic substances that alter the digestive flora environment of the consuming organism to enhance growth rates.
As such, there are many problematic burdens—related to nutrition, ex vivo processing steps, health and medicine, environmental conservation, and resource management—that are associated with an insufficient hydrolysis of phytate in many applications. The following are non-limiting examples of these problems:
1) The supplementation of diets with inorganic minerals is a costly expense.
2) The presence of unhydrolyzed phytate is undesirable and problematic in many ex vivo applications (e.g. by causing the presence of unwanted sludge).
3) The supplementation of diets with antibiotics poses a medical threat to humans and animals alike by increasing the abundance of antibiotic-tolerant pathogens.
4) The discharge of unabsorbed fecal minerals into the environment disrupts and damages the ecosystems of surrounding soils, fish farm waters, and surface waters at large.
5) The valuable nutritional offerings of many potential foodstuffs remain significantly untapped and squandered.
2.1.2—Nutritional Concerns: Many potentially nutritious plants, including particularly their seeds, contain appreciable amounts of nutrients, e.g. phosphate, that are associated with phytate in a manner such that these nutrients are not freely available upon consumption. The unavailability of these nutrients is overcome by some organisms, including cows and other ruminants, that have a sufficient digestive ability—largely derived from the presence of symbiotic life forms in their digestive tracts—to hydrolyze phytate and liberate the associated nutrients. However, the majority of species of farmed animals, including pigs, fish, chickens, turkeys, as well as other non-ruminant organisms including man, are unable to efficiently liberate these nutrients after injestion.
Consequently, phytate-containing foodstuffs require supplementation with exogenous nutrients and/or with a source of phytase activity in order to ammend their deficient nutritional offerings upon consumption by a very large number of species of organisms.
2.1.3—Ex vivo Processing Concerns: In yet another aspect, the presence of unhydrolized phytate leads to problematic consequences in ex vivo processes including—but not limited to—the processing of foodstuffs. In but merely one exemplification, as described in EP0321004-B1 (Vaara et al), there is a step in the processing of corn and sorghum kernels whereby the hard kernels are steeped in water to soften them. Water-soluble subtances that leach out during this process become part of a corn steep liquor, which is concentrated by evaporation. Unhydrolized phytic acid in the corn steep liquor, largely in the form of calcium and magnesium salts, is associated with phosphorus and deposits an undesirable sludge with proteins and metal ions. This sludge is problematic in the evaporation, transportation and storage of the corn steep liquor. Accordingly, the instantly disclosed phytase molecules—either alone or in combination with other reagents (including but not limited to enzymes, including proteases)—are serviceable not only in this application (e.g., for prevention of the unwanted slugde) but also in other applications where phytate hydrolysis is desirable.
2.1.4—Medical Concerns: The supplementation of diets with antibiotic substances has many beneficial results in livestock production. For example, in addition to its role as a prophylactic means to ward off disease, the administration of exogenous antibiotics has been shown to increase growth rates by upwards of 3-5%. The mechanism of this action may also involve—in part—an alteration in the digestive flora environment of farmed animals, resulting in a microfloral balance that is more optimal for nutrient absorption.
However, a significant negative effect associated with the overuse of antibiotics is the danger of creating a repository of pathogenic antibiotic-resistant microbial strains. This danger is imminent, and the rise of drug-resistant pathogens in humans has already been linked to the use of antibiotics in livestock. For example, Avoparcin, the antibiotic used in animal feeds, was banned in many places in 1997, and animals are now being given another antibiotic, virginiamycin, which is very similar to the new drug, Synercid, used to replace vancomycin in human beings. H

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