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Recombinant beauveria brongniartii proteinase K mutant PK-M1 and preparation method thereof

A technology of Beauveria bassiana and PK-M1, which is applied in the field of genetic modification and protein engineering, can solve the problems of large-scale industrial application limitations, poor stability of enzyme activity, etc., and achieve large-scale production, increase yield, increase yield effect

Active Publication Date: 2019-01-29
大连博格林生物科技有限公司
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Problems solved by technology

However, in practical application, it was found that the recombinant Beauveria bassiana proteinase K solution had poor enzyme activity stability when stored at 4°C, and the enzyme activity decreased rapidly, which limited large-scale industrial application.

Method used

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  • Recombinant beauveria brongniartii proteinase K mutant PK-M1 and preparation method thereof
  • Recombinant beauveria brongniartii proteinase K mutant PK-M1 and preparation method thereof
  • Recombinant beauveria brongniartii proteinase K mutant PK-M1 and preparation method thereof

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Embodiment Construction

[0028] For the experimental methods that do not indicate specific conditions in the examples of the present invention, generally, conventional conditions can be used, such as the conditions described in the "Molecular Cloning Experiment Guide" written by J. Sambrook (Sambrook), or according to the manufacturer's suggestion conditions. The terminology involved in the present invention and relevant measuring method are explained as follows:

[0029] 1. Determination method of protease activity: adopt the national standard protease preparation method of the People's Republic of China (GB / T25327-2009).

[0030] 2. Definition of enzyme activity unit: 1g of solid enzyme powder (or 1mL of liquid enzyme), under certain temperature and pH conditions, hydrolyzes casein for 1min to produce 1μg of tyrosine, which is 1 enzyme activity unit, expressed in U / g (U / mL) said.

[0031] 3. Protease K uses the Folin method to determine the activity of protease. The solutions used include: Folin s...

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Abstract

The invention discloses a recombinant beauveria brongniartii proteinase K mutant PK-M1 and a preparation method of the recombinant beauveria brongniartii proteinase K mutant PK-M1. According to the technical scheme, starting from transforming the molecular structure of proteinase K of recombinant beauveria brongniartii, the proteinase K mutant PK-M1 with the activity and stability both improved isobtained, and the amino acid sequence is as shown in SEQ ID NO:1; an expression system is optimized, by utilizing the yeast cell external secretion expression technology, the recombinant proteinase Kmutant can be fermented in large scale and efficiently expressed, and further, the yield of proteinase K is improved; and with the protein purification manner realizing efficient affinity chromatography, the yield of the proteinase K in the purifying process is improved, and thus the large-scale production of the proteinase K is realized.

Description

technical field [0001] The invention relates to the technical fields of genetic modification and protein engineering, in particular to a recombinant Beauveria bassiana protease K mutant PK-M1 with high enzyme activity, good stability, simple operation and suitable for large-scale production and a preparation method. Background technique [0002] Proteinase K ( Proteinase K , EC 3.4.21.64) is an important serine protease first described in 1974 in Candida albicans Limberella ( Tritirachium album limber ) found in the extract. Proteinase K has extremely high enzymatic activity and broad substrate specificity, and has a broad-spectrum and efficient cutting ability for natural proteins. The enzyme prefers to cut the carboxy-terminal peptide bonds of aliphatic amino acids and aromatic amino acids, and can preferentially decompose the ester bonds and peptide bonds adjacent to the C-terminals of hydrophobic amino acids, sulfur-containing amino acids, and aromatic amino acids. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/58C12N15/81C12R1/84
CPCC12N9/58C12N15/815C12Y304/21064
Inventor 岳敏李民强
Owner 大连博格林生物科技有限公司
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