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Novel neutral phytase

A technology of phytase and phytase protein, applied in hydrolytic enzymes, plant genetic improvement, botany equipment and methods, etc., can solve problems such as difficult extraction production and low phytase content

Inactive Publication Date: 2010-03-03
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the content of phytase in natural sources is too low to be extracted and produced in large quantities

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 produces the screening of the Citrobacter of phytase

[0028] Soil was obtained from high-temperature irradiation environment in Xinjiang for microbial enrichment culture. The medium uses LB medium, and its ingredients are: 0.5% yeast powder, 0.3% peptone, 0.5% sodium chloride, and pH 7. Culture conditions: Weigh an appropriate amount of soil and put it into LB medium, 37°C, 200r / min, and cultivate for 3-6 days. Draw a small amount of culture solution every day for gradient dilution (10-2-10-10), spread it on a solid medium containing sodium phytate, and culture it at 37°C for 2-3 days. Solid medium composition: sodium phytate 1%, NaNO30.3%, MgSO4·7H2O 0.5%, KCl 0.5%, K2HPO40.1%, FeSO4·7H2O 0.001%, agar 1%, pH6.0. If there are obvious bacterial circles around the colonies grown in the medium, it can be preliminarily judged that the bacteria can produce phytase, and the colonies that produce the largest bacterial circles in the medium can be selected.

Embodiment 2

[0029] Example 2 Extraction and Identification of Citrobacter sp. Genome

[0030] Pick a single colony of the bacteria in the selected medium and put it into LB liquid medium for culture, and take the bacterial liquid for the extraction of the genomic DNA of the bacteria. Using the TIANamp Bacteria DNA Kit Bacterial Genomic DNA Extraction Kit from Tiangen Company, the genomic DNA of the bacteria was extracted according to the operating instructions of the kit provided by the company. In order to identify the genus, the 16s rDNA sequence of the strain was amplified by polymerase chain reaction (PCR) using genomic DNA as a template.

[0031] Design of primers for bacterial 16S rDNA: F 27:5′-agagtttgatcatggctcag-3′

[0032] R 1492: 5′-tacggttacccttgttacgactt-3′

[0033] The length of the PCR product is about 1.4kb. After sequencing, it is compared with the DNA sequence in the GenBank database. It is found that the sequence is 98% similar to the 16s rDNA g...

Embodiment 3

[0034] Cloning of Phytase Gene in Example 3 Citrobacter Genome

[0035] Find the phytase gene sequence in other Citrobacter from Genbank, and design degenerate primers, as follows:

[0036] phyA F(EcoR I): 5′- GAATTC ATGAGTACATTCATCATTC-3′

[0037] phyA R(Not I): 5′- GCGGCCGC TTATTCCGTAACTGCACAC-3′

[0038] The underlined part is EcoRI (upstream primer F) and NotI (downstream primer R) recognition sites.

[0039] After the PCR product was subjected to 0.8% agarose gel electrophoresis, the observed length was about 1.3Kb. T4 DNA ligase was used to connect to the T-vector (pMD18-T simple vector, Takara Company). After sequencing, the deduced amino acid sequence is shown as SEQ ID NO:2.

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Abstract

The invention discloses a novel neutral phytase gene obtained from Citrobacter braakii, which has a nucleotide sequence shown as SEQ ID NO:1. The gene can be utilized to industrially produce neutral phytase with high enzymatic activity.

Description

technical field [0001] The invention relates to a novel neutral phytase, in particular to a gene sequence for encoding the neutral phytase. Background technique [0002] Phytase is an enzyme that hydrolyzes phytic acid into inositol and phosphate. Adding phytase to feed can hydrolyze phytic acid and phytate into inositol and phosphate, which can be absorbed and utilized by animals, thereby improving phosphorus utilization and bone mineralization, saving phosphorus resources and reducing feed costs. However, the content of phytase in natural sources is too low to be extracted and produced in large quantities. [0003] Therefore, finding a new type of phytase, cloning the phytase gene by genetic engineering means, and then expressing the phytase gene efficiently in a bioreactor can make the preparation of phytase industrialized. Contents of the invention [0004] The purpose of the present invention is to provide a new phytase gene. After the gene is cloned and expressed, ...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/16C12N15/81C12N1/19C12R1/84
Inventor 平淑珍马鑫赵仲麟林敏陆伟陈明张维燕永亮
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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