Cloning and expression of a novel phytase

一种植酸酶、植酸酶活性的技术，应用在新的植酸酶的克隆和表达领域，能够解决分离植酸酶手段有限、耗费时间和劳力、昂贵等问题

Active Publication Date: 2009-05-06
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In addition, the means of isolating phytase in the prior art are limited, mainly by constructing a genome library, or directly starting from purified protein
However, both of these methods are time-consuming and labor-intensive, very inefficient, and very expensive
Moreover, the similarity of phytase in different species is relatively low, so it is difficult to obtain a sequence, which is also a challenge for using existing methods to obtain a new gene

Method used

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  • Cloning and expression of a novel phytase
  • Cloning and expression of a novel phytase
  • Cloning and expression of a novel phytase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Isolation of Phytase-producing Bacteria

[0066] Phytase-producing strains were isolated from permafrost samples from China No. 1 Glacier (Xinjiang). Specifically, in order to isolate phytase-producing strains, permafrost samples from China's No. 1 Glacier (Xinjiang) were collected in April 2005 and stored in a freezer for several days until they could be processed in the laboratory. Frozen soil was suspended in sterile water and diluted, then, the suspensions of different samples were inoculated into agar-free LB medium, followed by incubation at 4°C, 10°C, 15°C, and 30°C for 1–2 days, respectively. Then, the ferrous sulfate molybdenum blue method (see the following example 4 for details) was used to measure the phytase activity at different temperatures, in the culture fluid and cell disruption fluid of different samples. Phytase activity was detectable in the cell lysate, showing that these strains had intracellular phytase activity. Firstly, the samples...

Embodiment 2

[0067] Embodiment 2: analyze the characteristic of phytase producing bacterium H-27

[0068] The H-27 bacteria isolated in Example 1 were determined to be Gram-negative bacteria by the Gram staining method. This bacterium is a typical bacillus with a similar size to E. coli under the microscope. Some of its physiological and biochemical properties were further studied. The results showed that the bacteria were Gram-negative, facultative, anaerobic microorganisms, which could grow in anaerobic or aerobic environments. The optimal growth temperature of this bacterium is 30°C, and we also analyzed the 16s rRNA sequence of this bacterium. Using the Promega Taq kit, the 16SrDNA sequence (SEQ ID NO.1) of the bacterium was amplified by PCR, and the 16SrDNA sequence of the bacterium was compared with the reference sequence in GenBank by using BLAST and the multiple sequence alignment program CLUSTAL W . Results The base sequence of 16s rDNA showed 99.5% identity with Yersinia inte...

Embodiment 3

[0070] Embodiment 3: Cloning of phytase gene

[0071] Homologous cloning is very efficient and simple when the protein's gene is a known member of a multigene family.

[0072] Apparently, according to the classification of phytases, most phytases of bacterial origin belong to the histidine acid phosphatase (HAP) family. Through multiple sequence alignment programs CLUSTAL W and BLOCKS (http: / / blocks.fhcrc.org / blocks / make_blocks.html) to analyze the various sequences of the HAP family, we found that there are two conserved regions in the HAP enzyme, namely RHGXRXP and HD area. Based on these two conserved regions, a pair of degenerate primers can be designed to amplify part of the phytase sequence from Yersinia intermedia genomic DNA by PCR.

[0073] Acquisition of Partial Phytase Gene Sequence

[0074] Degenerate primers were designed based on the two conserved regions and synthesized using a DNA synthesizer. PCR amplification was performed using Yersinia intermedia genomi...

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Abstract

The present invention relates to a novel phytase enzyme, a novel isolated nucleic acid molecule coding the enzyme, and a novel Yersinia intermedia having phytase activity. Particularly, the present invention relates to the phytase having (a) Theoretical molecular weight 45.5 kDa, (b) high specific activity 3960+-248 U / mg, (c) high stability at high temperature and wide pH, (d) optimal pH of 4.0-5.0, (e) optimal temperature of 50-60 DEG C, (f) high resistance to pepsin and trypsin. The phytase is very suitable to be used in feed of monogastrics as feed additive. The present invention also relates to a recombinant vector comprising said nucleic acid molecule, a recombinant host cell (e.g., Pichia pastoris ) harboring said recombinant vector, and a method for producing phytase using the recombinant host cell. The present invention further provides a feed additive comprising said phytase and / or host cells expressing a phytase as effective ingredient. In addition, the present invention provides a novel method for isolating phytase from a target organism.

Description

technical field [0001] The present invention relates to a kind of novel phytase gene, the phytase protein encoded by the gene, the intermediate type Yersinia producing the phytase, and a method for expressing phytase in host cells (such as Pichia pastoris) , and a feed additive using the phytase as an active ingredient. In addition, the present invention also provides a simple and rapid method for isolating the phytase gene from the target organism. Background technique [0002] The content of phytate phosphorus accounts for 50-70% of the total phosphorus in animal diets. However, monogastric animals such as chickens and pigs lack digestive enzymes to hydrolyze inorganic phosphorus from phytic acid molecules, resulting in very low utilization of phosphorus. Phytate phosphorus that has not been digested is excreted through the digestive tract. This leads to an increased phosphorus ecological burden on land and water resources. Additionally, since phytic acid chelates seve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N9/52C12N15/55C12N15/63C07K14/195C12P21/00A23K1/165C12R1/01
CPCA23K1/1653Y10S530/825C12N9/16A23K1/1833C07K14/195A23K20/189A23K50/00
Inventor 姚斌罗会颖黄火清王亚茹袁铁铮史秀云柏映国孟昆杨培龙
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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