A prolyl endopeptidase mutant with improved catalytic activity and specific enzyme activity

A technology of prolyl endopeptidase and Aspergillus oryzae prolyl endopeptidase, which is applied in the field of enzyme engineering, can solve the problems of catalytic efficiency and low specific enzyme activity, and achieve the effect of improving catalytic efficiency and specific activity

Active Publication Date: 2020-04-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the defects of the existing Aspergillus oryzae prolyl endopeptidase in catalytic efficiency and low specif

Method used

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  • A prolyl endopeptidase mutant with improved catalytic activity and specific enzyme activity
  • A prolyl endopeptidase mutant with improved catalytic activity and specific enzyme activity
  • A prolyl endopeptidase mutant with improved catalytic activity and specific enzyme activity

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Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Selection of mutation sites

[0029] According to previous reports, the flexibility of the loop loop, an irregular area near the enzyme active center, plays a key role in the entry of the substrate into the enzyme active center and the substrate specificity, that is, the increase in the flexibility of the loop loop increases the catalytic activity of the enzyme. , combined with the three-dimensional structure obtained by homology modeling, the analysis shows that the loopA and loopB loops in the simulated structure of Aspergillus oryzae prolyl endopeptidase are both close to the surface of the active pocket ( figure 1 ), and before the substrate enters the active site, these two loops play a role in blocking the substrate from entering the active center, so these amino acids may be key amino acids. Therefore, it is possible to try to replace the hydrophilic amino acid Thr on the loop ring with other small molecular weight amino acids or hydrophobic amino acid...

Embodiment 2

[0032] Embodiment 2: the construction of the recombinant bacterium expressing mutant

[0033] Construct the expression mutant recombinant bacteria according to the following method:

[0034] (1) Construction of a recombinant plasmid with a histidine tag containing the parent Aspergillus oryzae prolyl endopeptidase: the parent Aspergillus oryzae prolyl endopeptidase gene (amino acid sequence such as SEQ ID NO.1, nucleotide The sequence is shown in SEQ ID NO.2) was cloned into pPIC9K to obtain the recombinant plasmid pPIC9K-AO-PEP, and then pPIC9K-AO-PEP was used as a template, and the F and R with 6*His tag added as primers were used for PCR, and the correct The target fragment was digested with SnaBI and NotI, and pPIC9K was ligated to obtain an expression plasmid containing 6*his tag named pPIC9K-AO-PEP-6his.

[0035] (2) Using pPIC9K-AO-PEP-6his as a template, using the primers in Table 1, using the one-step plasmid PCR-mediated site-directed mutagenesis method to construct...

Embodiment 3

[0040] Example 3: Fermentative expression and mutant purification of recombinant bacteria

[0041] Fermentative expression of recombinant bacteria and purification of mutants were carried out as follows.

[0042] (1) Fermentative expression of recombinant bacteria:

[0043] Pick a single colony of recombinant Pichia pastoris to inoculate a 250ml Erlenmeyer flask filled with 25ml BMGY medium, and culture at 30°C until the logarithmic phase OD 600 =2-6(16-18h); collect the cells by centrifugation, discard the supernatant, transfer the cells to a 500ml Erlenmeyer flask filled with 50ml of BMMY medium, place them on a shaker at 28°C and 250r / min to continue culturing, and take samples every 24h , and supplemented with 100% methanol to make the final concentration reach 1%, induced culture for 4 days, centrifuged to collect the bacteria, and carried out enzyme activity determination and SDS-PAGE electrophoresis analysis.

[0044] (2) Enzyme purification:

[0045] Take 60ml of fe...

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Abstract

The invention provides a prolyl endopeptidase mutant with increased catalytic activity and enzyme specific activity, and belongs to the field of enzyme engineering. The invention replaces hydrophobic amino acids Thr370 and/or Thr516 on a loop near a catalytic center of a parent Aspergillus oryzae prolyl endopeptidase with other small molecular weight amino acids or hydrophobic amino acids, or mutates Cys508 and/or Gln512 near catalytic key site group histidine His507 into His508 and/or Arg512 to obtain a series of mutants.

Description

technical field [0001] The invention relates to a prolyl endopeptidase mutant with improved catalytic activity and specific enzyme activity, belonging to the field of enzyme engineering. Background technique [0002] Proline (Pro) is the only amino acid with an imino structure, and its ring structure affects the spatial conformation when forming peptide bonds with other amino acids. The proline-specific endoprotease has strong specificity for the position of Pro in the polypeptide and the configuration of the bond, and can specifically hydrolyze the peptide bond of the carboxy-terminal of proline in small molecular weight polypeptides. Therefore, it can be used in food industry, medicine, polypeptide industry and other fields; at the same time, it can be used as a molecular biology tool enzyme for protein sequence determination, peptide mapping analysis, enzyme cleavage at specific sites, modification and processing of peptide chains Wait. [0003] Especially in the food i...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N1/21C12N15/10C12N9/58
CPCC12N9/58C12N15/10
Inventor 喻晓蔚徐岩康超
Owner JIANGNAN UNIV
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