Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1999-11-09
2001-01-30
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S243000, C435S245000
Reexamination Certificate
active
06180390
ABSTRACT:
FIELD OF INVENTION
This invention relates to the isolation of thermophilic phytase-producing microorganisms, method for producing phytase using such microorganisms, phytase obtained therefrom, and usage of the phytase to hydrolyze phytic acid or phytate. In particular, this invention relates to phytase-producing microorganisms, which belong to Streptomyces sp., Pseudonocardia sp. or Microbispora sp., and which produce phytase available for recovery in an efficient and practical manner.
BACKGROUND OF THE INVENTION
Phytic acid (myoinositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) is known as one of the major component of plant-derived food. It is the primarily source of inositol and the main storage form of phosphorus in plant seeds that are used as animal feed ingredients (oilseed meals, cereal grains, and legumes). Approximately 75% of the total phosphorus in cereals, legumes and seeds exist as phytic acid phosphorus. When one or more of the acidic protons of the phophate groups in phytic acid are replaced by a counterion, the compound is usually referred to as a phytate salt. The name phytin is used for the calcium-magnesium salt of phytate derived from plant seeds (a discontinued product of Ciba-Geigy).
Phytic acid plays an important role in the dormacy and germination stages of plant seeds. It was believed that phosphorus was liberated on germination and incorporated into ATP. Recent studies have established the role of inositol phosphate intermediates in the transport of materials into cells and their role in transport as secondary messengers and in signal transduction in plant and animal cells is a very active area of research.
There are many applications of phytic acid, including industrial use as a corrosion inhibitor on metals, a rust remover and an additive to lubricating greases, use as a food additive, and medical applications, including use in the prevention of dental caries, use as an imaging agent for organ scintography and an X-ray enhancement contrasting agent, use as a hypocholestromic agent, use to reduce gastric secretion for treatment of gastritis, gastroduodenitis, gastric duodenal ulcers and diarrhea, use as an antidote for toxic metal absorption, therapeutic uses in the prevention and dilution of calcium deposits associated with various diseases and for reducing calcium concentration in urine (thus checking the formation of renal calculi), use as a preventive agent against severe poisoning with pressurized oxygen and preventing thirst during exercise, use as a taste-improving agent in orally administered antibiotics, and use in the treatment of multiple sclerosis (see U.S. Pat. No. 5,217,959 issued to Robert Sabin). For further discussions of industrial applications of phytic acid, see Graf, JAOCS 60, 1861-1867, 1983.
Phytic acid may be prepared in pure form from various plant sources, such as wheat, corn, soybeans, sesame seeds, peanuts, lima beans, barley, oats, wild rice and sunflower seeds, and it can be extracted with dilute hydrochloric acid at room temperature, precipitated with various reagents including ferric chloride, bicarbonates, potassium hydroxide, sodium hydroxide, ammonium hydroxide, calcium hydroxide, magnesium hydroxide or alcohol. It is then further purified by conventional chemical techniques. Hydrolyis of phytic acid and phytates may be carried out by partial acid or basic hydrolysis or by hydrolysis using phytase, and the resultant products include phosphate, inositol and various inositol phosphate intermediates.
Phytic acid phosphorus has anti-nutritive properties as it is essentially poorly metabolized by monogastric animals, such as pourltry and swine, because these animals produce little or no phytase in their digestive tracts. In addition, phytic acid forms complexes with proteins and divalent cations, such as calcium, iron, zinc, magnesium, manganese, copper and molybdenum. Phytic acid also binds to starch and influences the digestibility and solubility of starch. Phytic acid excreted in the manure of feed animals is enzymatically hydrolyzed by soil and water microorganisms. The released phosphorus is transported into rivers and lakes and if introduced in high quantities causes eutrophication. The anti-nutritive properties and its valus as a possible phosphorus source, have stimulated researchers to develop a method to remove phytic acid in a manner that is economically competitive with mineral supplementation.
Hydrolyzing phytic acid is thought to be a useful way of increasing the nutritional value of many plant foodstuffs. The enzymes that catalyze the conversion of phytic acid to inositol and inorganic phosphate are known as phytases. Phytase is found to be distributed in the seeds and pollens of plants, and also some microorganisms. The mass production of phytase from plant origin is not economic since preliminary treatment is necessary and the production procedure becomes time-consuming, troublesome and expensive. Therefore, the production of phytase from microbial origin is of greater potential in development. The feeding of microbial phytase to monogastric animals alter the phytic acid complexes and increase the bioavailability of phosphorus, calcium and probably proteins to monogastric animals.
The research of phytase spans more than 87 years from its discovery by Suzuki et al. (Tokyo Imp. Univ. Coll. Agr. Bull., 7: 503-512, 1907) until its commercialization in Europe in 1993-1994 by Gist-brocades. The international Union of Biochemistry (1979) lists two phytase: a 3-phytase, EC 3.1.3.8, which hydrolyzes the ester bond at the 3-position of myoinositolhexakus phosphate to D-myoinositol 1,2,4,5,6-pentakisphosphate+orthophosphate, and a 6-phytase, EC 3.1.3.26, which first phdrolyzes the 6-position of myoinositolhexakus phosphate to D-myoinositol 1,2,3,4,5-pentakis phosphate+orthophosphate. Subsequent ester bonds in the substrate are hydrolyzed at different rates. The 6-phytase dephosphorylates phytic acid completely, whereas the aforesaid 3-phytase does not hydrolyze the phosphomono ester.
Currently, phytase-producing microorganisms include bacteria, such as
Bacillus subtilis
and
Escherichia coli
; yeasts, such as
Saccharomyces cerevisias
and
Schwannoiomyces castellii
; and fungi, such as
Aspergillus niger, A. oryzae, A. ficuum, Penicillium simplicissimum.
Yeasts produce phytase intracellularly and, hence, it is difficult and less efficient to recover phytase with a high yield.
Of all the microorganisms surveyed,
Aspergillus niger
(syn.
A. ficuum
) NRRL 3135 produces phytase extracellularly, and the phytase produced thereby is known to be most active and has been commercialized. The microorganism is subjected to solid state fermentation (SSF) together with cereal grains, legume beans or foodstuffs so as to substantially remove or reduce phytic acid therefrom.
A thorough review of the research and development of phytase is provided by Rudy J. Wodzinski et al., in “Phytase,”
Advances in Applied Microbiology
, Vol. 42, p. 263-303, 1996, and other relevant documentary references are cited in the Reference List accompanying the Specification.
However, since during fermentation, the temperature within the fermenter normally will rise to 55° C. or higher, the efficiacy of removing phytic acid by microbial fermentation using non-thermophilic microorganisms is significantly decreased. In addition, fungal cells grow slowly and hence, it takes longer time (about 10 days) for the fungal cells to produce phytase having high enzymatic activity.
There thus still exists a need to produce phytase by a phytase-producing microorganism which is thermophilic and which maintains the ability to remove phytic acid during solid state fermentation. The industrial application of such a phytase-producing microorganism is promising as the phytase produced thereby is thermostable and fermentation using the same is efficient and energy saving.
SUMMARY OF THE INVENTION
An object of this invention is to isolate a thermophilic microorganism which produced phytase extracellularly and which can be used in large-scale solid state fer
Chu Jaw-Shiow
Chu Wen-Shen
Chung Su-Fang
Tseng Min
Wen Chiou-Yen
Food Industry Research & Development Institute
Lee Li
Smith Lynette R. F.
Sughrue Mion Zinn Macpeak & Seas, PLLC
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