A kind of thermostable cysteine ​​protease and its coding gene and application

A cysteine ​​protease, coding gene technology, applied in the field of enzyme engineering, to achieve the effect of high-efficiency catalytic activity and high-efficiency protein degradation activity

Active Publication Date: 2020-11-06
TECHNICAL INST OF PHYSICS & CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, large-scale automated cooking production operations require enzyme preparations to maintain high catalytic activity in a long-term high-temperature environment, and the existing papain is difficult to meet the production needs of this aspect

Method used

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  • A kind of thermostable cysteine ​​protease and its coding gene and application
  • A kind of thermostable cysteine ​​protease and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Acquisition of Thermostable Cysteine ​​Protease Gene and Its Recombinant Expression

[0034] 1. Extraction of total DNA samples of microbial communities containing thermostable cysteine ​​protease

[0035] A gelatin wastewater high-temperature anaerobic digestion (UASB process) sludge sample was quickly frozen in liquid nitrogen after sampling and stored at -80°C for later use. After sludge samples were ground with liquid nitrogen, total DNA was extracted according to the FAST DNA Spin kit for Soil (Qbiogene, USA) kit. DNA samples were detected by agarose gel electrophoresis and ultraviolet spectrophotometer, the concentration was adjusted to 100ng / μL, and stored at -20°C.

[0036] 2. Acquisition of thermostable cysteine ​​protease gene

[0037] Using the total DNA obtained in step 1 as a template, use the primer 1 shown in the sequence table SEQ ID NO.3 and the primer 2 shown in the sequence table SEQ ID NO.4 to carry out PCR reaction to amplify the thermos...

Embodiment 2

[0046] The activity detection of embodiment 2 thermostable cysteine ​​protease (casein hydrolysis method)

[0047] 1. Principle:

[0048] The target substrate casein is hydrolyzed by cysteine ​​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 cysteine ​​protease and unreacted substrate casein), and the content of tyrosine in the supernatant was determined by the Folin method, which can indirectly reflect cysteine The definition of the activity level of acid protease: 1 μg of tyrosine produced by hydrolyzing casein per minute at 75°C is defined as 1 protease activity unit (1U).

[0049] 2. Experimental methods and results:

[0050] 2.1 Test sample

[0051] Cysteine ​​protease kospCl (from Example 1); commercially available papain. Before the test, the protease dry powder was...

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Abstract

The invention discloses a heat-resistant cysteine protease, and coding genes and applications thereof. The heat-resistant cysteine protease is a protein selected from proteins: a, composed of amino acid sequences represented by SEQ ID NO.1; b, which possess protein degradation activity under high temperature conditions and are obtained via substitution and / or deletion and / or inserting of one or aplurality of amino acid residues of amino acid sequences represented by SEQ ID NO.1. The invention also discloses applications of the heat-resistant cysteine protease in degrading proteins under hightemperature conditions. The heat-resistant cysteine protease possesses high efficiency protein degrading activity as a temperature as high as 75 DEG C, and the high efficiency catalytic activity may last for 30min or longer in a solution of a temperature as high as 85 DEG C.

Description

technical field [0001] The present invention relates to the field of enzyme engineering. More specifically, it relates to a thermostable cysteine ​​protease and its coding gene and application. Background technique [0002] Cysteine ​​proteases (EC3.4.22) are a large class of important proteolytic enzymes whose catalytic active sites contain nucleophilic Cys residues. Currently, the cysteine ​​proteases used in large-scale commercial applications are mainly derived from plants, such as papain and bromelain. According to the structural study of papain, a representative species of C1 family cysteine ​​proteases, its precursor contains two structural domains of similar size, and the active site is located at the bottom of the gap formed by the two structural domains. Papain contains a catalytic tribody consisting of conservative amino acid residues: Cys25-His159-Asn175, and the Gln residue in the peptide chain also plays an important role in the enzyme activity. Papain can h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/50C12N15/57C12P21/06
CPCC12N9/50C12P21/06
Inventor 郭燕川邵明非
Owner TECHNICAL INST OF PHYSICS & CHEMISTRY - CHINESE ACAD OF SCI
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