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Phytase mutant with improved specific activity

A technology of mutants and acid enzymes, applied in the fields of host cells, vectors, and DNA molecules, can solve the problems of not being able to obtain a large amount of phytase products, polluting the environment with high-phosphorus feces, and being difficult to meet the needs of the development of the feed industry.

Pending Publication Date: 2021-11-30
QINGDAO VLAND BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, phosphorus in the form of phytate phosphorus is difficult to be utilized due to the lack of enzymes that can decompose phytic acid in monogastric animals, and its utilization rate is only 0% to 40%, which causes many problems: first, it causes waste of phosphorus On the one hand, the phosphorus source in the feed cannot be effectively utilized; on the other hand, in order to meet the demand of animals for phosphorus, inorganic phosphorus must be added to the feed, which increases the cost of feed; secondly, the formation of high-phosphorus feces pollutes the environment
[0005] The expression level of phytase in natural microorganisms is too low, and cheap phytase products cannot be obtained in large quantities, and it is difficult to meet the needs of the development of the feed industry

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0372] Example 1 Screening of high ratios

[0373] The applicant is mutated with 10 sites of wild-type phytase APPA (amino acid sequence as SEQ ID NO: 1, which encoded nucleotide sequence as SEQ ID NO: 2) (W46E, Q62W, G70E, A73P, T114H, N137V) , D142R, S146E, R159Y, Y255d) obtain phytase mutant APPA-T0, and the amino acid sequence is SEQ ID NO: 3, and a synthesis of a nucleotide sequence is SEQ ID NO: 4 with reference to this sequence. Compared to phytase APPA, the heat resistance of mutant APPA-T0 is significantly increased, and after 5 min at 75 ° C, the phytase APPA residual enzyme is less than 10%, while the residual enzyme activity of mutant APPA-T0 is high. 85%.

[0374] In order to further improve the specific activity of the heat resistant phytase mutant APPA-T0, the applicant is analyzed by the protein structure of its gene, which has two domains: 134 amino acid residues of the N-terminal and the C-terminal 152 amino acid residues. A common composition of structural domai...

Embodiment 2

[0383] Example 2 Expression of phytase mutants in Pichia

[0384] The gene sequence of APPA-T0 gene sequence SEQ ID NO: 4, and mutant gene sequences were optimized in accordance with the password preference of Pichia, and EcoRI and NOTII were plus EcoRI and NOTII at both ends of the synthetic sequence 5 'and 3', respectively. Enzyme cutting point.

[0385] 2.1 Construction of the expression vector

[0386] The gene sequences of the synthesized APPA-T0 and mutants were respectively digested, respectively, and then connected to the PPIC-9K vector after digestion of the same enzyme 16 ° C overnight, and transformed E. coli DH5A, coated to LB + AMP Flat, 37 ° C inverted culture, after the transformant appeared, colony PCR (reaction system: monoclonal picked by template, RTAQDNA polymerase 0.5 ul, 10 × buffer 2.0 μL, DNTPS (2.5mm) 2.0 μL, 5'AOX primer (10M): 0.5μL, 3'AOX primer: 0.5μL, DDH 2 O14.5μL, reaction procedure: 95 ° C for 5 min, 30 cycles: 94 ° C 30sec, 55 ° C 30SEC, 72 ° C, 7...

Embodiment 3

[0393] Example 3 Expression of phytase mutants in Rirish wooden mile

[0394] According to the codon preference of the wooden mile, the gene sequence of the APPA-T0 is optimized, and the gene sequence of the mutant is optimized, and KPNI and Mlui are added to both the synthetic sequences 5 'and 3'. Two enzyme dug points.

[0395] 3.1 Construction of expression vector

[0396] The synthesized phytase gene fragment was purified by restriction endonuclease KPNI and MLUI (Fermentas) using a PSC1G vector, purified by the gel purified kit, and used T4 DNA ligase (Fermentas) The above-mentioned phytase gene was attached to the enzyme-digestive product of the PSC1G vector, and E. coli TRANS5α (transgen) was transformed, selection was performed with ampicillin and sequencing the cloning. After the sequencing is correct, recombinant plasmid containing the phytase gene is obtained.

[0397] 3.2 Construction of a recombinant strain

[0398] (1) Original branium

[0399] Take the Subsence of S...

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PUM

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Abstract

The invention relates to the field of biotechnology, in particular to a phytase mutant, a preparation method and application thereof, a DNA molecule for encoding the phytase mutant, a carrier and a host cell. The mutant provided by the invention comprises a substitution of an amino acid at at least one position selected from the group consisting of 67, 72, 79, 115, 121, 295, 300, 307, 318, 329, 360, 376 and 385. The heat resistance of the mutant is remarkably improved, so that the phytase can be widely applied to feeds.

Description

Technical field [0001] The present invention relates to the field of biological technology, and Background technique [0002] The phytase is a phosphatase that can hydrolyze acid. It can degrade phosphoric phosphoric acid (hexagonal acid inositol) into an inositol and inorganic phosphate. This enzyme is divided into two categories: 3 - phytase (EC. 3. 1. 3. 8) and 6 - phytase (EC. 3. 1. 6). The phytase is widely present in plants, animals and microorganisms, such as corn, wheat and other higher plants, Bacillus Bacillus, Pseudikarium, Lactobacillus, Escherichia coli, etc. . [0003] Among the crop seeds such as cereals, beans and oil, the basic storage form of phosphorus is phosphorus phosphoric acid, with a content of up to 1% to 3%, which accounts for 60% to 80% of total phosphorus in plants. However, the phosphorus existing in the form of phosphoric acid is difficult to be utilized due to the lack of enzyme that can decompose phytic acid in the body, and the utilization is onl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/81C12N15/80C12N1/15C12N1/19A23K20/189C12R1/84C12R1/885
CPCC12N9/16C12N15/80C12N15/815A23K20/189
Inventor 程斯达吴秀秀康丽华李馨培李宾张静静郭瑞黄亦钧
Owner QINGDAO VLAND BIOTECH GRP
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