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A kind of low-temperature acid protease and its coding gene and application

A technology of acid protease and encoding gene, which is applied to low-temperature acid protease and its encoding gene and application field to achieve the effect of high-efficiency protein degradation activity

Active Publication Date: 2020-11-24
TECHNICAL INST OF PHYSICS & CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, the ACE inhibitory peptide generated in the previous stage is inevitably affected by heat treatment and partially degraded

Method used

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  • A kind of low-temperature acid protease and its coding gene and application
  • A kind of low-temperature acid protease and its coding gene and application
  • A kind of low-temperature acid protease and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The acquisition of embodiment 1 low-temperature acid protease gene and its expression

[0035] 1. Establishment of low temperature acid protease gene transcription library

[0036] The adult Antarctic fish (Notothenia rossii) was vivisectioned, and the gastric mucosa samples were taken, quickly frozen in liquid nitrogen, and stored at -80°C for later use. After the tissue samples were ground with liquid nitrogen, total RNA was extracted with the RNAiso Plus (Takara, Dalian) kit. RNA samples were digested with RNase-free DNase I to remove genomic DNA contamination, then detected by agarose gel electrophoresis and UV spectrophotometer, and the concentration was adjusted to 500 ng / μL. Using the above RNA sample as a template, it was reverse-transcribed into cDNA by AMV Reverse Transcription Kit (Promega), and stored at -20°C.

[0037] 2. Acquisition of low temperature acid protease gene

[0038] With the cDNA obtained in step 1 as template, utilize primer 1 shown in Seq...

Embodiment 2

[0046] The activity detection of embodiment 2 low-temperature acid protease (hemoglobin hydrolysis method)

[0047] 1. Principle: The target substrate, bovine hemoglobin, is hydrolyzed by acid protease under appropriate buffer conditions to release free tyrosine or small peptides containing tyrosine into the reaction solution. Afterwards, trichloroacetic acid was added to the reaction system to precipitate macromolecular proteins (including acid protease and unreacted substrate hemoglobin), and the content of tyrosine in the supernatant was determined by colorimetry, which could indirectly reflect the activity level of acid protease.

[0048] 2. Experimental methods and results:

[0049] 2.1 Test sample

[0050] Low temperature acid protease lpepA1 (derived from Example 1); commercially available porcine pepsin (pepA (Swine)).

[0051] In the test of the above samples, the reaction test tubes of "experimental group" and "blank control group" were respectively set up to measu...

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Abstract

The invention discloses low-temperature acidic protease as well as a coding gene and application thereof. The invention firstly discloses low-temperature acidic protease. The low-temperature acidic protease is a protein shown as follows (a) or (b): (a) protein consisting of an amino acid sequence shown by a sequence list SEQ ID NO.1; and (b) protein with an amino acid sequence shown in the sequence list SEQ ID NO.1, having a protein degradation activity at a low-temperature acidic condition and obtained by substituting and / or deleting and / or inserting one or more amino acid residues. The invention further discloses application of the low-temperature acidic protease in degrading the protein under the low-temperature acidic condition. The low-temperature acidic protease still has high-efficient protein degradation activity at low temperature of 4 to 10 DEG C and has relatively low thermal inactivation temperature.

Description

technical field [0001] The present invention relates to the field of enzyme engineering. More specifically, it relates to a low-temperature acid protease and its coding gene and application. Background technique [0002] Aspartic protease, represented by pepsin, can act on the peptide bonds formed by the amino acids of aromatic amino acids or acidic amino acids in the target protein under acidic (pH1.5-pH5) conditions to achieve the hydrolysis of the target protein . A typical aspartic endopeptidase contains two aspartic acid residues in its active center, the catalytic residues are located within the conserved Asp-Thr / Ser-Gly motif and form the enzyme active site. Mammalian aspartic proteases are synthesized as zymogens, which are then activated to a functionally active form. The main functions of aspartic proteases in organisms are to degrade proteins, antigens and promote the activation of other enzymes. [0003] At present, acid protease is widely used in light chemi...

Claims

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Application Information

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IPC IPC(8): C12N9/64C12N15/57C12N15/63C12P21/06
CPCC12N9/6402C12P21/06
Inventor 郭燕川邵明非
Owner TECHNICAL INST OF PHYSICS & CHEMISTRY - CHINESE ACAD OF SCI
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