Gene engineering bacterium for efficiently expressing Aspergillus oryzae prolyl endopeptidase and application thereof

A high-efficiency expression and gene technology, applied in the field of bioengineering to achieve good tolerance and increase yield

Active Publication Date: 2014-02-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Further, through the BLASTp similarity search on NCBI, it was found that the gene automatically annotated by the machine in Aspergillus oryzae RIB40, which has been reported for whole genome sequencing

Method used

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  • Gene engineering bacterium for efficiently expressing Aspergillus oryzae prolyl endopeptidase and application thereof
  • Gene engineering bacterium for efficiently expressing Aspergillus oryzae prolyl endopeptidase and application thereof
  • Gene engineering bacterium for efficiently expressing Aspergillus oryzae prolyl endopeptidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0041] The acquisition of embodiment 1A.oryzae proline-specific endoprotease gene

[0042] Based on Aeromonas (Genbank: AF065429), Pseudomonas capsular (Genbank: AB010298), Myxococcus flavum (Genbank: ABF89794) and Aspergillus fumigatus (Genbank: XM744168) and Aspergillus niger (Genbank: AX458699) sources The proline-specific endoprotease protein sequence design degenerate primers are as follows:

[0043] g1:5-RASMTWSRYATYASGGRA-3;

[0044] g2: 5-SVBYCBCYMBRGRKMANRNTB-3;

[0045] Using Aspergillus oryzae cDNA as a template and g1 and g2 as primers for PCR, a 1000bp product fragment was obtained. After the product was sequenced and compared with NCBI, it was found that it was all related to the A. oryzae protease gene (Sequence ID: XM_001825944.2 ) with high conserved sequence homology.

[0046] Furthermore, we redesigned primers and performed PCR based on the sequence of the A. oryzae protease gene (Sequence ID: XM_001825944.2) published in the GenBank database. The steps a...

Embodiment 2

[0066] Example 2 Obtaining the Predicted Crystal Structure of A.oryzae Proline-Specific Endoprotease and Its Amino Acid Similarity with Other Aspergillus Proline-Specific Endoproteases Using "Homologous Modeling" Method

[0067] The total length of the Aspergillus oryzae proline-specific endoprotease gene S2 is 1743bp, and the predicted open reading frame of the new protein is located at nucleotides 64-1743, encoding 558 amino acid residues and a molecular weight of 65kDa.

[0068] According to SignalP prediction, the possibility of the N-terminus of the protein being a signal peptide is 86.4%, and the cleavage site of the signal peptide is located between amino acids 21 and 22 (see figure 2 ).

[0069] Submit the amino acid sequence of A. oryzae proline-specific endoprotease to the SWISS-MODEL protein online modeling server (http: / / swissmodel.expasy.org / ) for homology modeling, and then use Discovery studio software to analyze Aspergillus oryzae Proline-specific endoproteas...

Embodiment 3

[0071] Example 3 Construction of Aspergillus oryzae proline-specific endoprotease eukaryotic expression vector, recombinant expression and protein expression thereof

[0072] 1. Construction of eukaryotic expression vector

[0073] 1) Primer design: design primers starting from the mature peptide sequence after the signal peptide

[0074] g5:5'-CGG TACGTA TTGGGGT TGTTTAGAGG-3';

[0075] g6:5'-CC GCGGCCGC CTACATCACCGCCCCCTTTG-3';

[0076] 2) PCR reaction, using the cloning vector pMD-19T-S2 as a template, annealing at 55-62°C, 35 cycles.

[0077] 3) SnaBI and NotI double-digest the PCR product of S2 and plasmid pPIC9

[0078] Element

Usage amount

Purification of PCR products / plasmids

30μl

10*quitcut buffer

5μl

QuitCut SnaBI

1μl

Quit Cut Not I

1μl

wxya 2 o

13μl

total capacity

50μl

[0079] Digest at 37℃ for 2hr

[0080] 4) Ligate, transform, and identify by double enzyme digestion ac...

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Abstract

The invention discloses a gene engineering bacterium for efficiently expressing Aspergillus oryzae prolyl endopeptidase and application thereof. A gene S2 is introduced into Pichia pastoris to perform efficient expression. The gene S2 codes a new proline-specific endoprotease, and the nucleotide sequences are disclosed as 1) or 2): 1) nucleotide sequences disclosed as SEQ ID NO.1 and SEQ ID NO.2; and 2) nucleotide sequences of proline-specific endoprotease with coding activity obtained by performing base deletion, substitution, insertion or mutation on the defined nucleotide sequence. The obtained recombinase has the advantages of proline-specific endoprotease activity, acid environment resistance and the like, has obvious effects on enhancing the stability of beer, wine, fruit juice beverages and other non-living things, and has huge application potential.

Description

technical field [0001] The invention relates to a yeast engineering bacterium for highly expressing proline-specific endoprotease and its application, belonging to the technical field of bioengineering. Background technique [0002] Protease is a large class of enzymes that catalyze proteolysis to generate peptones, hydrazones, polypeptides, and amino acids. Proteases can be divided into internal and external peptidases according to the mode of action; according to the active center, they can be divided into serine proteases, sulfhydryl proteases, metalloproteases and carboxyl proteases; according to the source of proteases, they can be divided into plant, animal and microbial proteases. Among them, serine protease is a kind of important proteolytic enzyme with serine as the active center, which plays an important and extensive physiological role in biological organisms. The members of the serine protease family are very large and are divided into 81 categories, such as dig...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N9/62C12H1/15A23L2/84C12R1/84C12R1/69
Inventor 喻晓蔚徐岩康超
Owner JIANGNAN UNIV
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