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Production for phytase with high living rate high temp. resisting by pichia

A technology of Pichia pastoris and phytase, applied in the field of phytase gene, can solve the problems of long time required, easy degradation of mutant strains, loss of other functions of strains and the like

Inactive Publication Date: 2007-02-28
STAR LAKE BIOSCI CO INC ZHAOQING GUANGDONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the early stage of phytase development, the original strains were used for fermentation and production. However, the expression of the original strains was low and the production cost was high. The strains with increased enzyme production could be obtained by chemical mutagenesis or physical mutagenesis. However, mutations easily lead to The loss of other functions of the strain, in order to obtain a strain with high expression of the target gene and other functions are not affected, long-term observation and screening is required, which takes a long time, in addition, the mutagenized strain is easy to degenerate

Method used

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  • Production for phytase with high living rate high temp. resisting by pichia
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  • Production for phytase with high living rate high temp. resisting by pichia

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Experimental program
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Effect test

Embodiment 1

[0060] Embodiment 1: the chemical synthesis of phytase gene

[0061] 1.1 The phytase gene of Aspergillus fig was synthesized by serial extension PCR. The length of the primer is 70-90bp, 20 oligonucleotide primers are synthesized, the primers are connected by 20-30bp overlapping sequences, and the Tm value is 60-66. Add all primers to the reaction system, the amount of primers in the middle is 10-20ng, and the amount of primers on both sides is 100-200ng. The PCR reaction system is 100 μL. The PCR amplification conditions were 94°C, 30s; 65°C, 30s; 72°C, 2min. A total of 35 cycles were performed.

[0062] 1.2 The Escherichia coli phytase gene was synthesized by continuous extension PCR. The length of the primer is 70-90bp, 20 oligonucleotide primers are synthesized, the primers are connected by 20-30bp overlapping sequences, and the Tm value is 60-66. Add all primers to the reaction system, the amount of primers in the middle is 10-20ng, and the amount of primers on both ...

Embodiment 2

[0063] Embodiment 2: DNA molecular rearrangement (DNA Shuffling) of phytase gene

[0064]2.1PCR amplification of acid phytase gene and recovery

[0065] The high temperature resistant phytase gene was used as a template, and FphyZ1 and FphyF1 were used as primers to amplify the high temperature resistant phytase gene, FphyZ1: (5'-TTGGATCCTCCAAATCCTGCGACACCGTTGACTTG-3'); FphyF1: (5'-CGAGCTCTTAGGAGAAGCATTCACCCAGTTACCAC-3').

[0066] Use the high specific activity phytase gene as a template, phyiZ1, phyiF1 as primers to amplify the high specific activity phytase gene, phyiZ1: (5'-TTGGATCCTTGGCTGTCCCAGGCTTCCAGAAAC-3');

[0067] phyiF1: (5'-CGAGCTCTTAAGCGAAGCATTCAGCCCAGTCAC-3').

[0068] The phytase gene of Aspergillus figus was used as a template, and phyiZ1 and phyiF1 were used as primers to amplify the phytase gene, phynZ1: (5'-TTGCCTATTCCTGTCCAAAACACTTC-3'); phynF1: (5'-GAGCTCATTATTCGGAAGGAACGAAACCGCAC-3').

[0069] Using the high specific activity phytase gene as a template,...

Embodiment 4

[0083] Example 4: Yeast expression phytase vector construction

[0084] Design primers according to the sequences at both ends of the high-temperature-resistant and high-specific activity gene obtained by screening, and add an XhoI cutting point and a signal peptide cutting sequence at the 5' end of the gene. Add NotI cutting point: the primer is: PHY1F (5'-AACGCGGCCGCTTAAGCGAAGCATTCAGCCAGTCACCACCGGTAC-3'). After the amplified fragment was cloned, it was digested with XhoI and NotI, and inserted into pPIC9 vector (product of Invitrogen company) directionally to construct the yeast expression vector pPphyXH of PhyXH (Figure 1).

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Abstract

The invention discloses utilization of pichia pastoris to produce high-specific activity high temperature- resistant phytase, adopts many isogenous phytase genes, using DNA enzyme to make partial enzyme mediation, recovering all the DNA degraded fragments, in vitro rearranging DNA molecules, making enzyme cutting on all the rearranged DNA molecules, then composing them in an expression carrier, electrically shocking them in Escherichia coli, screening mutant phytase gene by phytase expression and activity, and finally obtaining high-specific activity high temperature-resistant phytase gene Phy XH, which is constructed into phytase gene expression carrier pPhy XH, electrically shocking and transferring in pichia pastoris, and selecting recombinant high-phytase expression pichia pastoris.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering. Specifically, a plurality of homologous phytase gene fragments are used for in vitro recombination, and directional screening is used to obtain a phytase gene with high specific activity and high temperature tolerance, which can be efficiently expressed in Pichia pastoris Express. Background technique [0002] Phytase widely exists in plants, animals and microorganisms. Phytases produced by different species have great differences in specific activity, optimum reaction pH, heat resistance and other properties. Many microorganisms can produce phytase with high specific activity. In 1968, Shien et al. investigated 2000 strains from 68 soil samples and found that 21 of all 22 black mold strains could produce phytase. The first isolated and purified phytase is from Aspergillus terreus NO.9A-1, its optimal pH is 4.5, the optimal reaction temperature is 70°C, and the enzyme can be stably...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N1/19C12N15/81C12R1/84
Inventor 姚泉洪彭日荷熊爱生吴伟刘承训
Owner STAR LAKE BIOSCI CO INC ZHAOQING GUANGDONG
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