High specific activity phytase gene and application thereof

A residue and amino acid technology, applied in high specific activity phytase and its application field, can solve the problem of unsatisfactory enzyme activity

Inactive Publication Date: 2008-09-10
SICHUAN HEBEN BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Moreover, among the various phytases found so far, the enzymatic activity of most phytases is not ideal enough, and the highest activity is about 8000U / ml (fermentation titer)

Method used

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  • High specific activity phytase gene and application thereof
  • High specific activity phytase gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment approach

[0068] As an embodiment of the present invention, the gene encoding the AppA-M protein of the present invention can be used to prepare a transgenic plant, and the obtained transgenic plant expresses the AppA-M protein. Using the transgenic plant as a feed ingredient for animals can provide a stable source of phytase in the feed, thus no additional addition of phytase is required.

[0069] In addition, the gene encoding the AppA-M protein can also be introduced into the animal body, so that the animal itself can express the AppA-M protein, making the AppA-M protein an endogenous enzyme. This saves a lot of extra work from external sources.

[0070] The present invention also provides a composition, which contains a safe and effective amount of the AppA-M protein of the present invention and a food or feed acceptable carrier or excipient. Such carriers include, but are not limited to: buffer, dextrose, water, glycerol, ethanol, acetic acid, wheat bran, corn cob, or combinations...

Embodiment 1

[0076] Embodiment 1: Cloning of high specific activity phytase gene

[0077] 1. Collection of samples

[0078] Collect a large number of soil samples or specimens in the rumen of animals from forest areas where dead leaves have accumulated for many years. Because these places have a large amount of plant-based phytic acid, they can be screened to obtain bacteria with a strong metabolic ability for phytic acid, thereby A phytase gene with high specific activity properties was isolated.

[0079] 2. Isolation of community-level total DNA from soil samples using the rabbit culture method

[0080] Weigh 2 g of the sample, add 0.6 g of fine glass beads (d<0.11 mm), and shake twice at 4000 rpm. Add 300μl 2% SDS + 12% phenol Tris buffer (pH8.0) solution on ice for 1 hour, add an equal amount of phenol Tris buffer, pH8.0 (about 700ml), mix well, and centrifuge at 4°C, 13,000rpm 5 minutes. Add 0.1 volume of 3M NaAc pH 5.2 to the upper layer solution, mix well, add 0.6 volume of isop...

Embodiment 2

[0087] Embodiment 2: Transformation and artificial synthesis of high specific activity phytase gene

[0088] According to the gene sequence encoding the mature protein of Escherichia coli phytase, according to the codon preference of Pichia pastoris, sequence modification was carried out without changing its amino acid sequence, and at the same time, AT sequences (ATTTA, AATAA, The emergence of AATTAA, etc.). And according to the multi-cloning site design of the cloning vector used, adding suitable restriction endonuclease sites, the whole gene is divided into A (161bp), B (287bp), C (325bp), D (225bp), E ( 235bp) into 5 parts, and then each part is divided into single-stranded nucleotide fragments with a length of no more than 60 nucleotides for artificial synthesis.

[0089] After removing the 66bp signal peptide coding sequence at the N-terminal, the gene has a full length of 1233bp. According to the codon bias of Pichia pastoris, without changing the amino acid sequence,...

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Abstract

The invention discloses a novel AppA-M protein and application of the same. The AppA-M protein is a phytase with high specific activity and can be used for decomposition of phytic acids or phytates. The invention also discloses a gene for encoding the AppA-M protein, and a carrier and a host cell which comprise the gene. The AppA-M protein has wide application prospect in fields such as feedstuff additives and so on.

Description

technical field [0001] The invention belongs to the field of biotechnology, more specifically, the invention relates to a high specific activity phytase and application thereof. Background technique [0002] Phosphorus is an essential mineral element for animals. Although most of the phosphorus present in animal feed can be absorbed and utilized by the body, its high price limits its use in feed. Most of the phosphorus in plant feed exists in the form of phytic acid (ie phytic acid) or phytate, while monogastric animals lack phytase (Phytase, which catalyzes phytic acid and phytic acid) to decompose phytic acid. Salt hydrolysis enzyme), resulting in only 1 / 3 or lower phosphorus utilization rate in the feed, in order to supplement the lack of available phosphorus, inorganic phosphate must be added to the feed, which will inevitably lead to waste of phosphorus sources and lead to excessive phosphorus excretion. By adding phytase to the feed of monogastric animals, the utiliz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/63A23K1/165A23L1/305C12N15/82A23K20/147A23K20/189A23L33/17
Inventor 王小行李钢邓运涛蒋彦杨滔
Owner SICHUAN HEBEN BIOENG
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