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Bacillus subtilis neutral phytase phyC gene

A technology of Bacillus subtilis and phytase, applied in plant genetic improvement, genetic engineering, hydrolytic enzymes, etc., can solve the problems of low enzyme activity and limited application range of acid phytase

Inactive Publication Date: 2009-07-29
SOUTHWEST UNIVERSITY
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0004] The report that obtains phytase from microorganism has following three kinds at present: (1) mainly derive from the acid phytase (phyA) of Aspergillus niger, it has higher enzymatic activity, and the pH value effective range of its enzymatic reaction is in Between 4.5 and 6.0, suitable for monogastric animals and a few fish with acidic gastric pH, not suitable for carps with neutral digestive tract, which limits the application range of acid phytase
(2) Neutral phytase (phyC) derived from Bacillus, which has higher thermal stability, causes less loss of enzyme activity during feed pelleting or puffing, and the optimum pH of the enzymatic reaction is 7.0~ 7.5, suitable for carps feed, can make up for the deficiency of acid phytase, but its enzyme activity is not high

Method used

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Embodiment

[0057] Example: First, primers were designed according to the neutral phytase phyC gene reported on NCBI, the upstream is: 5'TACgTA ATg AAT CAT TCA AAA ACA CTT 3', the downstream is: 5'gCA CTA gTT TAT TTT CCg CTTCTg TCA; Add the SnaBI restriction enzyme site in the upstream; expand the culture of Bacillus subtilis and extract its genome, and then use it as a template to amplify with the designed specific primers to obtain the target band. After the PCR product is recovered, it is combined with pGEM- The T vector was ligated and transformed into Escherichia coli DH5α competent cells, and the positive clones were obtained and sent to Shanghai Invitrogen Company for sequencing. The sequencing results showed that the amplified sequence was consistent with the expected results; the T clone containing the target fragment and the pPIC9K empty plasmid were respectively used with SnaBI Carry out double enzyme digestion with NotI, recover the target fragments respectively, connect them w...

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Abstract

The invention relates to neutral phytase gene phyC which comes from microorganism, has enzymolysis phytic acid, phytate and a complex compound thereof and releases mineral nutrition element activity of phosphorus and the like, the optimal pH in enzymatic reaction of an expression product thereof is neutral and is suitable for genetic transformation researches on crops such as mulberry tree, corn and rice and other crops with neutral harvested products. Therefore, with the phyC gene transferred to the mulberry tree, the optimal pH in enzymatic reaction of the expression product is consistent with the pH of the mulberry leaf, in addition, the enzymatic reaction of the expression product of the phyC gene can carry out enzymolysis on the phytic acid, the phytate and the complex compound thereof to release the phosphorus, contributes to absorption and application of the phosphorus by the mulberry tree, reduces fertilization of phosphate fertilizer and pollution of the phosphate on the environment, promotes growth of the mulberry tree; in addition, neutral phytase gene phyC of the invention features relatively high enzymatic activity, approximately 10% higher than the activity of the phytase of the same kind according to measurement.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a neutral phytase gene phyC derived from microorganisms which has the activity of enzymatically decomposing phytic acid, phytate and complexes thereof, and releasing mineral nutrients such as phosphorus. Background technique [0002] Phytic acid (phytic acid), also known as myo-inositol hexakisphosphate, can be used as storage phosphorus for the plant itself, and is gradually hydrolyzed and utilized when the seeds germinate. But most of it exists in the form of double salt, which is an anti-nutritional factor. Like trypsinogen inhibitors, prothrombin, saponin, tannins, oxalic acid, glucosinolates, gossypol, anti-fat-soluble vitamins, anti-B vitamins and other anti-nutrients, it is used in feed by livestock After the poultry eats, it affects the digestion and absorption of mineral nutrients and protein by livestock and poultry. And non-ruminants are physiologically unavailable for p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/16
Inventor 余茂德李镇刚王茜龄金筱耘黎其友吴存容
Owner SOUTHWEST UNIVERSITY
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