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Keratinase mutant with improved thermal stability and application

A kind of keratinase mutation, keratinase technology, applied in the application, hydrolase, microorganism-based method and other directions, can solve the problem of time-consuming and laborious, the protein structure-activity relationship is not completely resolved and other problems, and achieve the effect of good application value and prospect.

Active Publication Date: 2021-10-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional transformation methods, such as directed evolution, are time-consuming and labor-intensive; completely rational design has limitations. Although the amount of protein structure data information that can be applied is increasing rapidly and the level of computer technology is also increasing, protein structure-activity relationship has not yet been established. fully resolved

Method used

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  • Keratinase mutant with improved thermal stability and application
  • Keratinase mutant with improved thermal stability and application
  • Keratinase mutant with improved thermal stability and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Construction of keratinase homology model

[0035] According to the amino acid sequence of the keratinase gene kerBp, the homology comparison was carried out in the NCBI database, and three crystal structures with high homology (greater than 60%) were selected as templates, and the 3D three-dimensional structure of the keratinase was simulated by EasyModeller software, and SAVES was used. The Verify 3D function of the online analysis server (http: / / servicesn.mbi.ucla.edu) evaluates the quality of the built model. The relevant analysis operation of the three-dimensional structure was performed by PyMOL and DiscoveryStudio software.

Embodiment 2

[0036] Example 2: Calculation and Analysis of Highly Flexible Loop Area of ​​Keratinase

[0037] The sequence (SEQ ID NO.2) and structure of keratinase were analyzed by using FoldUnfold and IUPred protein disorder region structure prediction tools. Combined with the homologous sequence comparison analysis and the visual analysis of the structure, three highly flexible Loop regions were determined, namely Loop158-171, Loop265-272 and Loop290-305 ( figure 1 ). Further, the B-Factor and RMSF of the amino acids in the above three Loop regions were predicted and calculated by the prediction software PredyFlexy, and 22 amino acid residues with higher B-Factor and RMSF were screened out as the objects of site-directed mutation. Based on the 22 mutation sites on the 3 highly flexible loops screened above, this experiment judged whether the mutant type was stable by calculating the difference between the Gibbs free energy change (ΔΔG) between the wild type and the mutant type, and fur...

Embodiment 3

[0038] Example 3: Prediction and analysis of the calcium ion binding region of keratinase

[0039] The amino acid sequence of keratinase was analyzed by the online prediction tool IonCom, and 37 potential amino acid residues related to calcium ion binding were predicted, and these amino acids were further positioned in the homology model structure of keratinase and divided into three parts from the spatial position 4 groups ( figure 2), by observing the characteristics of the three-dimensional structure of the above four groups, a conserved calcium ion binding site CaI composed of amino acids D149, L183, D184, N185, T186, I187, G188, V189 and L190 was determined. According to the analysis of the CaI region of the conserved calcium ion binding site of keratinase, D149, L183, I187, V189 and N185 are the conserved amino acids of the calcium ion binding site of keratinase, in which the calcium ion directly interacts with residues D149, L183, I187 and V189 Coordination, while oth...

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Abstract

The invention discloses a keratinase mutant with improved thermal stability and application, and belongs to the technical field of gene engineering and enzyme engineering. According to the present invention, the mutants comprise A165E, Q170T, N292F, L190Q, H92N and S171G, the thermal stability of the mutants is improved to different degrees on the basis of well maintaining the catalytic activity, the Q170T mutant has the optimal thermal stability, and the half-life period at the temperature of 60 DEG C is improved to 38.5 min from 17.3 min of the wild type. The half-life period of the six-site combined mutant is remarkably prolonged to 66.1 min, and the optimal reaction temperature is increased to 60 DEG C from 40 DEG C. The mutant has better catalytic activity and thermal stability, and has higher application value and potential than wild keratinase. The protein thermal stability improving method based on computer-aided analysis can become a general way for modifying the thermal stability of industrial enzymes.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, in particular to a keratinase mutant with improved thermostability and its application. Background technique [0002] Keratinase has a specific function of degrading natural keratin and is a protease with strong biological activity. Most of them belong to extracellular serine proteases, and a small part belong to conjugating enzymes or intracellular proteases. The keratinase-producing microorganisms reported in the literature are mainly derived from bacteria, fungi or actinomycetes, and most of these microorganisms are isolated from feathers, bird feathers or hair waste accumulations. Compared with other proteases, keratinase can treat raw materials containing keratin more gently and efficiently, and at the same time, it can improve the nutritional value of keratin after treatment. At present, keratinase is widely used in medicine, chemical industry, feed, tex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/54C12N15/57C12N15/70C12N1/21C12P21/06C14C1/06C12R1/19
CPCC12N9/54C12N15/70C12P21/06C14C1/065
Inventor 史劲松苏畅郑可欣许正宏龚劲松钱建瑛江佳宇秦安琪何纪萌
Owner JIANGNAN UNIV
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