Keratinase mutant with improved low-temperature enzymolysis performance, and application thereof

A technology of keratinase mutation and keratinase, which is applied in the field of keratinase mutants to achieve the effects of reducing costs, reducing heating process requirements, and avoiding loss of vitality

Active Publication Date: 2021-04-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the wild-type keratinase shake flask fermentation broth has high enzyme activity at lower reaction temperature compared with other keratinase in shake flask level fermentation broth in the existing reports, its low temperature enzyme activity still has a very great improvement space

Method used

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  • Keratinase mutant with improved low-temperature enzymolysis performance, and application thereof
  • Keratinase mutant with improved low-temperature enzymolysis performance, and application thereof
  • Keratinase mutant with improved low-temperature enzymolysis performance, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Construction of keratinase error-prone PCR mutants

[0049] Specific steps are as follows:

[0050] 1. Construction of recombinant plasmid pP43NMK-kerB

[0051] (1) Chemically synthesized nucleotide sequence as shown in SEQ ID NO.1 can be used to produce the gene of keratinase (by the gene of coding signal peptide, the gene of coding leader peptide, 6×His tag and the gene of coding keratinase in sequence obtained in series); and the obtained gene was ligated with the pP43NMK plasmid using a homologous recombination kit (Clonexpress II One Step Cloning Kit) to obtain a ligation product.

[0052] (2) Transform the ligation product into Escherichia coli JM109, spread the transformed product on LB solid medium, culture at 37°C for 12-14 hours, pick 4 transformants on the LB solid medium, insert them into LB liquid medium for culture, After culturing at 37°C for 12 hours, the plasmid was extracted, and the extracted plasmid was verified by enzyme digestion and s...

Embodiment 2

[0070] Embodiment 2: Construction of recombinant bacteria and screening of mutants

[0071] Specific steps are as follows:

[0072] (1) The recombinant plasmid containing the mutant gene obtained in Example 1 and the recombinant plasmid pP43NMK-kerB containing the wild-type enzyme were respectively transformed into Bacillus subtilis (Bacillus subtilis) WB600 to obtain transformation products respectively, and respectively coated the transformation products on the additive Kanamycin (final concentration 50mg L -1 ) LB solid medium, cultivated at 37°C for 8h, respectively inoculated a large number of single colonies obtained into 96 deep-well plates containing fermentation medium, cultivated at 37°C and 220rpm for 24h, and obtained the fermentation broth containing mutants and Fermentation broth containing wild-type keratinase;

[0073] (2) Centrifuge the fermentation broth obtained in step (1) at 4° C. and 4000 rpm for 20 min to obtain fermentation supernatants respectively. ...

Embodiment 3

[0080] Embodiment 3: Separation and purification of keratinase mutant

[0081] Specific steps are as follows:

[0082] Purification of keratinase: AKTAavant protein purifier was used for purification of recombinant protein. Since the keratinase mutants are all added with a histidine tag, they can be separated and purified using a nickel ion affinity chromatography purification column, and the specific steps are as follows:

[0083] (1) Equilibration: equilibrate the purification column with 5 times the volume of 20mmol / L pH 7.4 Tris-HCl buffer;

[0084] (2) Sample loading: the pre-treated sample is loaded at a flow rate of 0.5ml / min, and the sample loading volume is generally not more than 5 times the column volume;

[0085] (3) Elution: including elution of unadsorbed substances, miscellaneous proteins and target proteins, the flow rate is 2.0mL / min, the eluent is 20mmol / L Tris-HCl buffer solution with pH 7.2 containing 50mM imidazole, and the elution is carried out with 10...

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Abstract

The invention discloses a keratinase mutant with improved low-temperature enzymolysis performance, and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. Error-prone PCR mutation is carried out on propeptide of a keratinase gene and a main enzyme, low-temperature enzymolysis activity mutant recombinant bacteria are obtained through construction and screening, and a keratinase mutant T31 / V45D / S100D is obtained. Research results show that the residual enzyme activity of the keratinase mutant T3I / V45D / S100D at 20 DEG C is improved to 2.28 times of that of wild parent keratinase, and the residual enzyme activity at 30 DEG C is improved to 1.95 times of that of the wild parent keratinase.

Description

technical field [0001] The invention relates to a keratinase mutant with improved low-temperature enzymolysis performance and application thereof, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Keratinase is a specific keratinase that can degrade keratin substrates (such as cutin, dander, feathers, etc.), and is produced by various microorganisms such as fungi, actinomycetes, and bacteria when they grow with keratin as a single carbon source . Keratinase, as a protease with broad substrate specificity and strong hydrolysis catalytic ability, is widely used in daily chemical industry, animal husbandry industry, feed industry, leather industry, and pharmaceutical industry, and has great research and application value. [0003] However, most of the wild keratinases found in current research are high-temperature alkaline proteases, and their optimum reaction temperature is generally around 60°C, and their enzyme ac...

Claims

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Application Information

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IPC IPC(8): C12N9/54C12N15/57C12P21/06C14C1/06
Inventor 张娟李江华周冠宇陈坚堵国成冒鑫哲
Owner JIANGNAN UNIV
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