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Heat stability improvement and efficient expression of phytase with high specific activity

A high-efficiency expression and thermal stability technology, applied in the field of improved phytase and its encoding gene, can solve the problems of poor thermal stability and large loss

Inactive Publication Date: 2005-12-14
安徽固源生物工程有限责任公司
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  • Abstract
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  • Application Information

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Problems solved by technology

[0007] The optimal temperature of APPA is 60°C, but its thermal stability is poor. The remaining enzyme activity is only 40% when it is incubated at 60°C for 30 minutes. 15% or less, so that it loses too much in animal feed pelleting

Method used

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  • Heat stability improvement and efficient expression of phytase with high specific activity
  • Heat stability improvement and efficient expression of phytase with high specific activity
  • Heat stability improvement and efficient expression of phytase with high specific activity

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Embodiment Construction

[0024] Experimental conditions

[0025] 1. Strains and vectors Escherichia coli strain E.coli DH5a and plasmid pET-22b(+) were purchased from Promega, and yeast strain Pichia pastoris GS115(His-Mut+) and plasmid pFF01 were kindly donated by Dr. D.Luo, University of Alberta, Canada.

[0026] 2. Enzymes and kits Restriction enzymes, ligases, Taq enzymes, and DNAaseI are products of Boehringer. PCR Kits were purchased from Promega.

[0027] 3. Biochemical reagents DNA synthesis reagents are products of Milipore Company. Primers were synthesized with ABI Cyclone DNA synthesizer. IPTG, X-Gal, SDS and sodium phytate are products of Sigma. TEMED, ammonium persulfate, acrylamide and methylene bisacrylamide are products of Promega.

[0028]4. Medium The culture medium for Escherichia coli 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...

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Abstract

The present invention provides one kind of improved high specific activity phytase and its coding gene, and the improved phytase has even higher heat stability. The present invention also provides recombinant yeast cell containing the phytase gene of the present invention. The recombinant yeast of the present invention has phytase expressing amount up to 6.5E6 U in each ml of fermented liquid. The phytase of the present invention may be used widely in feed and food industry.

Description

technical field [0001] The invention relates to an improved 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] Phytase (EC.3.1.3.8) is an enzyme that can hydrolyze phytic acid. It degrades phytate phosphorus into inositol and phosphoric acid. Phytase widely exists in microorganisms, such as Aspergillus in fungi (Yamada K. et al. Agric. Biol. Chem. 32: 1275-1282, 1986; Van Gorcom R.F.M. et al., US Patent No. 5436156, 1995) , Yeast (Nayini N.R.et al.Lebensm Wiss.Technol.17:24-26,1984); Bacillus subtilis (Paver, V.K.J., Bacteriol.151:1102-1108,1982) in bacteria, Pseudomonas (Cosgrove D.J., Austral.J.Biol.Sci.23:1207-1220, 1970), Lactobacillus (Shirai K., Letters Appl.Biol.Sci.19:366-369, 1994), Escherichia coli (Greiner R., Arch Biochem. Biophys. 303:107-113, 1993) et al. [0003] Phytase can be widely used in monogastric animal feed (War...

Claims

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Application Information

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IPC IPC(8): C12N9/16
Inventor 孙跃军孙齐军
Owner 安徽固源生物工程有限责任公司
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