High specific activity phytase gene and its efficient expression

A high-efficiency expression, phytase technology, applied in the direction of plant gene improvement, genetic engineering, hydrolase, etc., can solve the problems of difficult phytase products and low phytase expression, and achieve the effect of huge economic benefits

Active Publication Date: 2007-10-03
北京昕大洋科技发展有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Although phytase has good feeding effect (Ware J.H.et al., US Patent No.3297548,1967; Nelson T.S.et al., J.Nutrition 101:1289-1294,1971; Nelson T.S.et al., Poult Sci. 47:1842-1848, 1968), but it has not been widely used in the feed industry so far. The fundamental reason is that the expression level of phytase in natural microorganisms is too low, so it is difficult to obtain cheap phytic acid in large quantities Enzyme products cannot meet the requirements of the development of the feed industry (Han Y.W., Animal Feed Sci.Technol., 24:345-350, 1989). With the development of high and new technologies such as biotechnology and genetic engineering, people have realized that through genetic engineering Means, utilize bioreactor to efficiently express phytase gene, it is expected to reach the purpose (Conneely O.M., Biotechnologyin the Feed Industry, T.P.Lyons (Ed), Alltech Technical Publications.Nicholasville of greatly improving phytase output, reducing production cost , K Y.57-66, 1992)

Method used

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  • High specific activity phytase gene and its efficient expression
  • High specific activity phytase gene and its efficient expression
  • High specific activity phytase gene and its efficient expression

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Embodiment Construction

[0028] Experimental conditions

[0029]1. Strains and vectors Escherichia coli strain E.coli DH5a, plasmid pET-22b(+), etc. were purchased from Promega Company, yeast strain Pichia pastoris GS115(His-Mut+), and plasmid pPIC9 were donated by Dr. D.Luo, University of Alberta, Canada (Invitrogen company's product).

[0030] 2. Enzymes and kits Restriction enzymes and ligases are products of Boehringer.

[0031] 3. Biochemical reagents IPTG, X-Gal, SDS and sodium phytate are products of Sigma. TEMED, ammonium persulfate, acrylamide and methylene bisacrylamide are products of Promega.

[0032] 4. Culture medium Escherichia coli culture medium is LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). Yeast complete medium is YPD (1% yeast extract, 2% peptone, 2% glucose); yeast transformation medium is RDB [18.6% sorbitol, 2% glucose, 1.34% Yeast Nitrogen Base W / O amino acids (YNB ), 0.00004% Biotin, 0.005% glutamic acid, 0.005% methionine, 0.005% lysine, ...

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Abstract

The invention relates to a height ratio active phytase and the coding gene. The total length of the gene phyQ is 1299bp, and has 432 amino acids comparing to the reporting phytase the most homology is phytase APPA from bacteria coli. The invention also supplies the rebuilding yeast cell containing the phytase gene of the invention. It could be used as the feed additive.

Description

technical field [0001] The invention relates to a high specific activity phytase and its coding gene. The invention also relates to a recombinant yeast cell containing the phytase gene and capable of highly expressing the phytase. Background technique [0002] 50-70% of phosphorus in plants exists in the form of phytic acid phosphorus (phytic acid) (Lolas M.Etal. Food Sci. 42: 1094-1097, 1977; Nelson T.S. Poultry Sci. 47: 862-871, 1967) , cannot be directly utilized by monogastric animals and people (Cromwell G.L.Biotechnology in the Feed Industry, Lyons T.P.ed.Alltech Technical Publication, Nicholasville, KY, 133-145), resulting in waste of phosphorus sources and increased feed costs. At the same time, phytate phosphorus cannot Utilized by animals and directly excreted from the body, it will also cause phosphorus pollution in soil and water. In addition, phytic acid can also chelate with the trace elements and proteins necessary for animals, so that these nutrients cannot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/81C12N15/866C12N15/82
Inventor 刘大庆王福江
Owner 北京昕大洋科技发展有限公司
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