Phytase from aspergillus fumigatus

A technology of phytase and phytase activity, applied in the field of phytase, can solve the problems of low expression level of phytase, enzymatic performance can not meet the application of feed processing, etc., to improve daily weight gain and feed-to-meat ratio , the effect of reducing pollution

Active Publication Date: 2014-03-26
QINGDAO VLAND BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the expression level of phytase in natural materials is low, and the enzymatic properties cannot meet the application of feed processing. Using genetic engineering means can not only improve the enzyme activity of phytase, but also improve the enzyme activity by constructing engineering strains. Some properties make it more suitable for the conditions of industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Cloning of Aspergillus fumigatus phytase gene

[0016] Aspergillus fumigatus ( Aspergillus fumigatus ) The total genomic DNA is used as a template, and the upstream and downstream primers are used for amplification.

[0017] PCR amplification conditions were 95°C for 4min; 94°C for 30S; 55°C for 40S, 72°C for 1min for 30 cycles; 72°C for 7min. The PCR amplification products were recovered using a gel extraction kit for sequencing analysis. The results showed that the nucleotide sequence of the amplified product was SEQ ID NO:2, and the encoded amino acid sequence was SEQ ID NO:1. After NCBI Blast comparison, it was found that the amino acid sequence was 89% similar to the phytase sequence of Aspergillus fumigatus, which was a new allele.

Embodiment 2

[0018] Example 2 Construction of expression vector

[0019] Connect the amplified product recovered in Example 1 to the pMD18-T vector to obtain the cloning vector pMD-TR plasmid, then perform double digestion with NcoI and KpnI to recover the TR fragment; take 2 μl of the recovered product and double digest with NcoI and KpnI The pKDN-5 vector was ligated and introduced into Escherichia coli DH5α overnight to obtain the recombinant expression plasmid pKDN-TR.

Embodiment 3

[0020] Example 3 Transformation and screening

[0021] 3.1 Protoplast preparation

[0022] Inoculated with Trichoderma reesei ( Trichoderma reesei ) mycelia were grown on PDA plates for 4 days; colonies with a diameter of about 3 cm were cut out and placed in about 60 ml of YEG (0.5% yeast powder, 1% glucose) liquid medium, shaken at 30°C and 200 rpm overnight; Collect the mycelia by gauze filtration; place the mycelia in 10-20 ml lyase solution (Sigma L1412) for enzymolysis for 2-3 hours; take out the enzymolysis solution, add 0.7 M NaCl solution, shake gently, and pour it into three layers Filter with sterilized lens tissue, collect the filtrate, centrifuge at 3000 rpm for 10 min; discard the supernatant, add 10-20 ml STC solution (20% sucrose, 50mM Tris-Cl, 50mM CaCl 2 ) suspension, 3000 rpm, centrifuged for 10 min; add an appropriate amount of STC suspension (150 μl / tube, 10 8 pieces / ml).

[0023] Conversion and Validation

[0024] Take 2 μg pKDN-TR DNA and add...

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Abstract

The invention provides phytase from aspergillus fumigatus, and creates a trichoderma reesei engineering strain capable of recombining and expressing the phytase by introducing phytase genes into trichoderma reesei. The engineering strain can efficiently express the phytase, and the shake-flask fermenting enzyme activity is 880 U/mL. The phytase prepared by the invention can be widely used in feedstuff, can greatly improve the average daily weight gain and the feed conversion ratio in each stage of cultivated animals, and reduces the phosphorus excretion by 13.3%, thereby facilitating increase of cultivation benefits and reducing environmental contamination.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering, and in particular relates to a phytase derived from Aspergillus fumigatus and its application. Background technique [0002] Phytase (Myo-i-nositol hexaphosphate phosphohydrotase) is a general term for a class of enzymes that catalyze the hydrolysis of phytic acid and phytate into inositol and phosphoric acid (or phosphate). Phytase has a special spatial structure, which can sequentially separate phosphorus in phytic acid molecules, degrade phytate into inorganic phosphorus and inositol, and release other nutrients bound by phytate. It can be widely used as a feed additive. At present, the effect of feeding monogastric animals with phytase has been verified. After adding phytase to the feed, the amount of inorganic phosphorus can be reduced by 5-70%, and the discharge of phosphorus in manure can be reduced by more than 30-40%. It can effectively reduce environmental pollution, so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55A23K1/165C12R1/68
CPCA23K20/189C12N9/16
Inventor 李佩佩张青王华明黄亦钧
Owner QINGDAO VLAND BIOTECH GRP
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