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Alkaline protease PA3 and its coding gene and application thereof

A protease and alkaline technology, applied in the field of agricultural biology, can solve problems such as differences in amino acid results

Inactive Publication Date: 2019-10-18
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

When using Pichia pastoris to express exogenous genes, the codon optimization of the gene is carried out according to the preference of Pichia pastoris. At present, although the prior art calculates the codon preference in Pichia pastoris according to different methods and proposes the preference codon and Rare codons, but there are differences in the results of preferred codons for some amino acids, so when choosing, it is necessary to combine the specific conditions of the foreign gene and the host cell type for specific analysis

Method used

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  • Alkaline protease PA3 and its coding gene and application thereof
  • Alkaline protease PA3 and its coding gene and application thereof
  • Alkaline protease PA3 and its coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 obtains alkaline protease gene pa3 and constructs expression vector

[0053] According to the protein sequence published in Genbank (serial number: CEJ93023.1), codon optimization was performed on it, and the optimized gene was obtained by chemical synthesis, numbered pa3.

[0054] The gene pa3 is connected to the pUC57 carrier, and the present invention realizes the connection of the target gene pa3 and the expression vector pPIC9r through the method of homologous recombination, and completes the construction of the expression vector.

[0055] First, through the PCR method, specific primers are used to introduce homologous segments into the target gene.

[0056] Table 1 pa3-specific primers

[0057]

[0058] Note: The underline marks the overlapping sequence homologous to the vector pPIC9r

[0059] Using the recombinant plasmid pUC57-pa3 as a template, carry out PCR amplification reaction through specific primers; at the same time, use restriction end...

Embodiment 2

[0062] The construction of embodiment 2 alkaline protease PA3 engineering strains

[0063] A large number of recombinant pPIC9-pa3 plasmids were extracted, and linearized using restriction endonuclease BglII to linearize the system.

[0064] After digestion at 37°C for 2 hours, 10 μL of the digestion product was subjected to agarose gel electrophoresis to confirm whether the digestion was complete, and then the digestion product was recovered and purified using the Omega Gel Extraction kit. The purified and recovered linearized product was electrotransformed into a competent Pichia pastoris (electrotransformer parameters: Fungus, PIC). The bacteria solution after electrotransfer was spread on the MD plate and incubated at 30°C for 48h. After transformants grew on the MD plate, 36 single clones were picked from the MD plate with a sterilized toothpick, placed on the milk double-layer screening plate, and cultured at 30° C. for 72 hours. According to whether there are hydrolys...

Embodiment 3

[0065] Embodiment 3 prepares recombinant alkaline protease PA3

[0066] (1) Shake flask horizontal expression of recombinant alkaline protease PA3

[0067] The selected transformants with higher enzyme activity were inoculated in 30 mL of YPD liquid medium, and cultured at 30° C. and 200 rpm in a shaker for 48 hours to obtain seed liquid. Then the seed solution was transferred to 300mL BMGY medium according to the inoculation amount of 1%, and cultivated in a shaker at 30°C and 200rpm for 48h, and then the bacteria were transferred to 200mL BMMY medium, and cultured in a shaker at 30°C and 200rpm After culturing for 48 hours, 0.5% (V / V) methanol was added every 24 hours during this period.

[0068] (2) Purification of recombinant alkaline protease PA3

[0069] After the fermentation process, the bacterial liquid was centrifuged at 12000rpm for 10 minutes to remove the bacterial cells and other insoluble matter, and the supernatant was collected, which was the crude enzyme li...

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Abstract

The invention belongs to the technical field of agro-ecology, and particularly relates to alkaline protease PA3 and its coding gene and an application thereof. The present invention provides an alkaline protease PA3 derived from Torrubiella hemipterigena, an amino acid sequence of which is shown as SEQ ID NO. 1 or SEQ ID NO. 2, the alkaline protease PA3 has good properties and can be applied to many fields such as food, feed, leather production, washing, medicine and the like. According to the technical solution of the present invention, the alkaline protease PA3 which is excellent in properties and suitable for industrial application can be produced by genetic engineering means.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to alkaline protease PA3 and its encoding gene and application. Background technique [0002] Protease refers to a class of enzymes that hydrolyze proteins by cleaving peptide bonds. They have a wide range of sources and are widely distributed in animals, plants, and microorganisms. Compared with animal and plant-derived proteases, microbial-derived proteases have the characteristics of convenient cultivation, simple operation, and high enzyme production, which are convenient for industrialized mass production. Therefore, microbial-derived proteases have become an important source of proteases. Protease plays a vital role in the process of cell metabolism. A large number of physiological activities rely on protease. In addition, protease also has industrial application characteristics. As one of the most important industrial enzyme preparations, it is widely use...

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 罗会颖任雅馨涂涛王亚茹黄火清苏小运柏映国王苑张杰姚斌
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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