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Novel proline specific endoprotease gene and application thereof

An endoprotease, proline technology, applied in the application, genetic engineering, plant gene improvement and other directions, can solve the problems of the proline specific endoprotease coding sequence and preparation method, etc., to improve beer abiotic Effects of stability, increased yield, and large application potential

Active Publication Date: 2014-02-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, even if there is a Japanese patent in 1995 to show proline-specific endoprotease activity from the soy sauce-producing Aspergillus oryzae strain, the relevant Aspergillus oryzae proline-specific endoprotease coding sequence and preparation at home and abroad The report of the method has not yet been seen

Method used

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  • Novel proline specific endoprotease gene and application thereof
  • Novel proline specific endoprotease gene and application thereof
  • Novel proline specific endoprotease gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Acquisition of A.oryzae proline-specific endoprotease gene

[0047] 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:

[0048] g1:5-RASMTWSRYATYASGGRA-3

[0049] g2:5-SVBYCBCYMBRGRKMANRNTB-3

[0050] 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.

[0051] 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 are as fol...

Embodiment 2

[0072] 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"

[0073] 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.

[0074] 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 ).

[0075]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 endoprotease protei...

Embodiment 3

[0077] Example 3 Construction of Aspergillus oryzae proline-specific endoprotease eukaryotic expression vector, recombinant expression and its protein expression

[0078] 1. Construction of eukaryotic expression vector

[0079] 1) Primer design: design primers starting from the mature peptide sequence after the signal peptide

[0080] g5:5′-CGG TACGTA TTGGGGT TGTTTAGAGG-3';

[0081] g6:5'-CC GCGGCCGC CTACATCACCGCCCCCTTTG-3';

[0082] 2) PCR reaction, using the cloning vector pMD-19T-S2 as a template, annealing at 62°C, 35 cycles.

[0083] 3) SnaBI and NotI double digestion PCR product of S2 and plasmid pPIC-9

[0084] 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

[0085] Digest at 37℃ for 2hr

[0086] 4) Ligate, transform, and identify by double enzyme digestion accordi...

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Abstract

The invention discloses a novel proline specific endoprotease gene and an application thereof. The gene is characterized in that a nucleotide sequence of the gene is represented by 1) or 2): 1) the nucleotide sequence of the gene is a nucleotide sequence represented by SEQ ID NO.1; and 2) on the basis of the nucleotide sequence limited by 1), the nucleotide sequence of coded and activated proline specific endoprotease is formed through absence, substitution, insertion or mutation of a basic group. A recombinant enzyme obtained through the gene has the advantages of proline specific endoprotease activity, resistance to acid environment and the like, has an obvious effect on the improvement of the non-biological stability of beer, wine, juice and the like and has the huge utilization potentiality.

Description

technical field [0001] The invention relates to a proline-specific endoprotease gene, more particularly a proline-specific endoprotease gene derived from A.oryzae and A.flavus and its expression and 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. Serine protease family members are very lar...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/60C12N1/19C12N15/81C12H1/12C12R1/84
Inventor 徐岩喻晓蔚康超
Owner JIANGNAN UNIV
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