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Phytase mutants ykappa-l396v, yeappa-l396v and their coding genes and applications

A technology of phytase and mutants, applied in the field of genetic engineering, can solve problems such as reducing the activity of biological functions

Active Publication Date: 2019-03-26
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently discovered HAP phytase generally has the highest activity at pH 1.3-5.5 and 45-70°C, but strong acid and high concentration of protease in the digestive system often reduce its biological function activity

Method used

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  • Phytase mutants ykappa-l396v, yeappa-l396v and their coding genes and applications
  • Phytase mutants ykappa-l396v, yeappa-l396v and their coding genes and applications
  • Phytase mutants ykappa-l396v, yeappa-l396v and their coding genes and applications

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Obtaining of mutant genes

[0036] Mutants YkAPPA-L396V and YeAPPA-L396V of YkAPPA and YeAPPA were produced by Overlap PCR method. The method uses recombinant plasmids pEASY-T3-YkAPPA and pEASY-T3-YeAPPA containing wild phytase gene as templates, and introduces mutations through two rounds of PCR reactions. The upstream and downstream primers for amplifying the complete coding sequence of the mutant gene have EcoR I and Not I recognition sequences, respectively YkAPPA Forward: 5'-cgcgaattcgcaccgcttgcagcacaatctac-3' and YkAPPA Reverse: 5'-gatgcggccgcttaaatatggcaggctggctcG-3'; YeAPPA Forward: 5' -cgcgaattcgcaccgcttgcagcacaatctac-3' and YeAPPA Reverse: 5' -gatgcggccgcttaaatggcaggctggctcg-3'. The upstream and downstream primers for introducing mutations at specific positions are YkAPPA-L396V Forward: 5’-AGGTG G TGAATTTTTCGGCCTCACCTTA-3’, YkAPPA-L396V Reverse:5’-TAAGGTGAGGCCGAAAAATTCA C CACCT-3', YkAPPA-L396A Forward:5'-CAATGCAGATAAG GCA GATTTGAAAAAc-3', Y...

Embodiment 2

[0037] Example 2: Expression and purification of mutant phytase and wild enzyme in bacteria

[0038] Remove the wild enzyme and mutant enzyme of the signal peptide sequence, insert it into the expression vector pET-22b (+) and transform it into Escherichia coli BL21 (DE3) cells, and inducer IPTG (isopropyl-β- D-galactoside) induces the expression of phytase. The crude enzyme solution was purified by Ni-NTA (nickel-nitrilotriacetic acid) column and DEAE (diethylaminoethyl) column. Both the purified wild enzyme and the mutant enzyme showed a specific protein band of about 46 kDa in SDS-PAGE electrophoresis (data not shown).

Embodiment 3

[0039] Embodiment 3: the protease resistance comparison of mutant phytase and wild enzyme

[0040] Wild enzymes YkAPPA and YeAPPA and mutant enzymes YkAPPA-L396V and YeAPPA-L396V were treated with different concentrations of pepsin (pH2) and trypsin (pH7) at 37°C for 2 hours, respectively, and the remaining enzyme activities in the treated samples were analyzed to study protease Effect on phytase activity. The ratio of protease to phytase is between 1 / 1000 and 1 / 20. Phytase activity was determined using the ferrous sulfate molybdenum blue method. Use 1.5mmol / L sodium phytate as substrate, react at 37°C for 30min, stop the reaction with TCA, and then use chromogenic solution (1% ammonium molybdate tetrahydrate, 3.2% concentrated sulfuric acid, 7.32% ferrous sulfate) color. The control is to add TCA and mix to denature the enzyme before adding the enzyme solution, and the others are the same. Enzyme activity was calculated according to the 700nm light absorbance after color ...

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Abstract

The invention relates to the field of gene engineering, in particular to phytase mutants YkAPPA-L396V and YeAPPA-L396V and an encoding gene and application thereof. The phytase mutants YkAPPA-L396V and YeAPPA-L396V are obtained by mutating leucine at the 396th site of phytase with the amino acid sequence as shown in SEQ ID NO.1 or 3 into valine. Compared with a wild type, the two phytase mutants YkAPPA-L396V and YeAPPA-L396V have pepsin resistance remarkably improved and are beneficial to feed enzyme development and application.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to phytase mutants YkAPPA-L396V, YeAPPA-L396V, coding genes and applications thereof. Background technique [0002] Phytate phosphorus is the most common storage form of phosphorus in plant feed. It is difficult to absorb due to the lack of phytase to hydrolyze phytic acid and its salts in monogastric animals, thus causing serious environmental pollution. In addition, mineral iron and protein often form insoluble complexes with phytic acid and cannot be absorbed. Therefore, the mineralization of phytic acid by phytase is an effective strategy to increase the bioavailability of nutrient elements, reduce production costs and protect the environment in monogastric animal production. [0003] Since the discovery of the first phytase in 1907, numerous phytases have been identified in bacteria, fungi, plants, and some animals. According to the catalytic mechanism, phytase is divided i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55A23K20/189
CPCC12N9/16
Inventor 杨培龙牛灿芳姚斌闻治国李秀梅王亚茹罗会颖马锐
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES