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Keratinase mutant subjected to thermal stability modification

A kind of keratinase mutation and keratinase technology, which is applied in the field of keratinase mutants, and can solve the problem that the thermal stability of strains is not suitable for industrial applications and the like

Active Publication Date: 2018-12-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the thermostability of this strain is not yet suitable for industrial application

Method used

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  • Keratinase mutant subjected to thermal stability modification
  • Keratinase mutant subjected to thermal stability modification
  • Keratinase mutant subjected to thermal stability modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: site-directed mutagenesis and construction of recombinant bacteria

[0031] The highly flexible loop region is often the part of the enzyme molecular structure that is unstable to heat and is prone to unfolding first. In this study, the amino acid sequence was selected as SEQ ID NO: 2. Eight key amino acid sites on the highly flexible loop region of keratinase (S78L, L96T, N118S, N123Y, S173R, N218S, M222A, S236C) for mutation transformation research.

[0032] Construction of keratinase site-directed transformation mutants by one-step reverse PCR method, figure 1 Shown are the PCR verification results of each mutant colony, and the band at 1800 bp in the figure is consistent with the size of the target fragment amplified with the plasmid universal primers. After further sequencing verification, the correctly constructed plasmid for site-directed mutagenesis was transformed into host cells.

Embodiment 2

[0033] Embodiment 2: thermal stability of mutant recombinant bacteria

[0034] Stratify the correctly constructed recombinant bacteria at each mutation site to isolate a single colony, pick a single colony and culture it in LB medium (Kan) for 8-12 hours, and inoculate it into TB fermentation medium (Kan) with 2% inoculum After 30 hours of fermentation in medium, the fermentation supernatant, i.e. each mutant recombinant enzyme, was heat-treated at 60° C. for 15 minutes, and the enzyme activities before and after heat treatment were detected respectively. The activity of the original enzyme that was not mutated and not heat-treated at 60°C was taken as 100%. The result is as figure 2 As shown, only the three mutants, N118S, N218S, and S236C, had the same enzyme activity levels as the initial control bacteria, and the rest showed a decrease in enzyme activity. After heat treatment, the remaining enzyme activities of N118S and N218S mutant enzymes were higher than those of th...

Embodiment 3

[0037] Embodiment 3: keratinase degradation feather degradation situation analysis

[0038]Ferment the recombinant bacteria with improved thermostability of the modified keratinase to produce enzyme under the condition of optimized medium, and prepare 1000U / mL feather degrading enzyme solution, then take 60mL feather degrading enzyme solution and add it to 0.6g feather meal In a 250mL Erlenmeyer flask, the reaction system was placed at 40°C and 220rpm for reaction, and the degradation of the feathers was observed at regular intervals, and samples were taken to detect the components of the reaction solution. The results showed that after 48 hours of reaction, keratinase showed a good feather degradation effect, gradually degrading from the initial intact feathers to fine fibers, and the stems of feathers were difficult to degrade, but were obviously thinner than the initial stems. Observed by scanning electron microscope, the situation before and after feather degradation is as...

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Abstract

The invention discloses a keratinase mutant subjected to thermal stability modification and belongs to the technical field of industrial biotechnologies. Based on a keratinase three-dimensional structure model, aiming at eight key amino acid residue sites in a zymoprotein Loop region of keratinase, the thermal stability mutant recombinant bacteria are constructed by adopting one-step inverse PCR (Polymerase Chain Reaction). The thermal stability research results show that the thermal stability of the mutant N218S keratinase at 60 DEG C is improved by 3 times or more. The keratinase is used forperforming feather-degrading study. The feather surface is observed to be split and roughened within 48 hours after keratinase treatment, the feather-degrading rate reaches 25%, and the total contentof amino acids in the feather degradation liquid is increased by 1.82mg / mL. The study lays a foundation for degrading the feather to prepare high-quality feed and degrading hard keratin wastes.

Description

technical field [0001] The invention relates to a thermostability modified keratinase mutant, which belongs to the field of industrial biotechnology. Background technique [0002] Keratin has a complex structure, strong rigidity, and rich disulfide bonds, so it is difficult to be degraded by common proteases. Keratinase is a special protease that can specifically degrade feathers, wool, hair, nails and other rich keratin. It firstly opens the complex disulfide bonds in keratin through disulfide bond reduction, and then realizes the degradation of keratin through proteolysis. The particularity of keratinase enables it to be widely used in animal husbandry, feed industry, leather industry, and pharmaceutical industry. [0003] Summarizing the existing studies, the enzymatic activity and thermal stability of keratinase are generally poor. The problem of insufficient thermal stability makes it necessary to accelerate the reaction by heating in practical applications, which in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C12N15/57C12N15/70C12N15/74C12N15/81C12N15/80C12N1/21C12N1/15C12N1/19C12P21/06
CPCC12N9/50C12N15/70C12N15/74C12N15/80C12N15/81C12P21/06
Inventor 龚劲松陶丽妍苏畅许正宏史劲松
Owner JIANGNAN UNIV
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