Fungus-derived acid protease g412 as well as gene and application thereof

An acid protease, ppic9-g412 technology, applied in the field of genetic engineering, can solve the problems of low enzyme activity, reduced catalytic efficiency, and restrictions on the application of acid protease, and achieve the effect of easy fermentation production and high reaction temperature

Active Publication Date: 2017-11-24
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the properties of most acid proteases are not satisfactory, and the enzyme activity is not very high, which brings great waste to industrial production and food processing, and also limits its application range to a certain extent.
Due to the difference between the optimal action conditions of the enzyme itself and the catalyzed environmental conditions (such as pH, temperature, etc.), the catalytic efficiency of the enzyme is reduced, and its industrial application is limited.
Most of the acid proteases used in industrial production are fungal acid proteases. The optimum pH value of such enzymes is about 3.0. When the pH value increases, the enzyme activity of acid proteases will decrease significantly, and these enzymes are not heat-resistant. It is very unstable when the temperature reaches above 50°C, which limits the application of acid protease

Method used

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  • Fungus-derived acid protease g412 as well as gene and application thereof
  • Fungus-derived acid protease g412 as well as gene and application thereof
  • Fungus-derived acid protease g412 as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Cloning of protease encoding gene g412

[0048] Extract Talaromyces leycettanus JCM 12802 genomic DNA and place it at -20°C for use.

[0049] Design cloning primers g412F and g412R, and use Talaromyces leycettanus JCM 12802 genomic DNA as template for PCR amplification. The PCR reaction parameters are: 95℃5min; 94℃30sec, 60℃30sec, 72℃2min, 35 cycles, 72℃10min. A fragment of about 1800 bp was obtained, and the fragment was recovered and sent to Ruibo Biotechnology Co., Ltd. for sequencing.

[0050] Table 1 Primers needed for this experiment

[0051]

[0052] Extract Talaromyces leycettanus JCM 12802 total RNA using Oligo(dT) 20 And reverse transcriptase to obtain a strand of cDNA, and then design the primers g412F and g412R (see Table 1) to amplify the open reading frame, amplify the single-stranded cDNA, obtain the cDNA sequence of the protease, and send the amplified product after recovery Sequencing by Ruibo Biotechnology Co., Ltd.

[0053] After comparing the geno...

Embodiment 2

[0054] Example 2 Construction of Protease Engineering Strain

[0055] (1) Construction of expression vector and expression in yeast

[0056] Using the correctly sequenced protease g412 cDNA as a template, primers F and R with SnaB I and Not I restriction sites (see Table 1) were designed and synthesized to amplify the coding region of the mature protein of g412. The PCR product was digested with SnaB I and NotI and ligated into the expression vector pPIC9 (Invitrogen, San Diego). The mature protein sequence of protease g412 was inserted downstream of the signal peptide sequence of the above expression vector to form a correct reading frame with the signal peptide. The yeast expression vector pPIC9-g412 was constructed and transformed into E. coli competent cells Trans1. The positive transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used to prepare large quantities of recombinant plasmids. Linearized expression plasmid vector DNA ...

Embodiment 3

[0060] Example 3 Preparation of recombinant protease

[0061] (1) The protease gene g412 is expressed in large quantities at the shake flask level in Pichia pastoris

[0062] The transformants with higher enzyme activity were selected and inoculated into 300mL BMGY liquid medium in a 1L Erlenmeyer flask, cultured with shaking at 30℃, 220rpm shaker for 48h; centrifuged at 5,000rpm for 5min, gently discard the supernatant, and then add 100mL containing 0.5% methanol BMMY liquid medium was induced and cultured at 30°C and 220 rpm for 72 hours. During the induction culture, add methanol solution once every 24 hours to compensate for the loss of methanol and keep the methanol concentration at about 0.5%; (3) Centrifuge at 12,000×g for 10 minutes, collect the supernatant fermentation broth, detect the enzyme activity and perform SDS-PAGE protein Electrophoresis analysis.

[0063] (2) Purification of recombinant protease

[0064] The supernatant of the recombinant protease expressed in the...

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Abstract

The invention relates to the field of genetic engineering. In particular, the invention relates to a fungus-derived acid protease g412 as well as a gene and application thereof, and the amino acid sequence of the fungus-derived acid protease g412 is shown as SEQ ID NO.1 or SEQ ID NO.2. The acid protease disclosed by the invention has good properties, and can be applied to industries such as food, feed and pharmacy. According to the technical solution of the invention, a genetic engineering means can be utilized to produce the protease with excellent properties which is suitable for industrial application.

Description

Technical field [0001] The invention relates to the field of genetic engineering. Specifically, the present invention relates to a fungus-derived acid protease g412, its gene and application. Background technique [0002] Protease is a type of enzyme that catalyzes the hydrolysis of protein, and it is widely used in food, washing, tanning and other industries. Microbial proteases have become an important source of current proteases. There are many ways to classify proteases. According to their pH, they are divided into acid proteases, alkaline proteases and neutral proteases; proteases can be divided into four categories according to their active centers: serine proteases, aspartic proteases, and cysteine Proteases and metalloproteinases. Aspartic proteases are a class of proteolytic enzymes that are active at acidic pH. [0003] Proteases are widely used in food, brewing, fur and leather, medicine and feed industries. The addition of acid protease in feed can improve the dige...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/58C12N15/57C12N15/81C12N1/19C12R1/84
CPCC12N9/58C12N15/815
Inventor 姚斌罗会颖郭玉杰涂涛王苑黄火清柏映国苏小运王亚茹孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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