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A new proline-specific endoprotease gene and its application

An endoprotease and specific 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, and improve the non-biological stability of beer. performance, increased yield, good tolerance

Active Publication Date: 2016-05-18
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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  • A new proline-specific endoprotease gene and its application
  • A new proline-specific endoprotease gene and its application
  • A new proline-specific endoprotease gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

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

[0047] Based on Aeromonas (Genbank: AF065429), Pseudomonas capsularis (Genbank: AB010298), Myxococcus aureus (Genbank: ABF89794) and Aspergillus fumigatus (Genbank: XM744168) and Aspergillus niger (Genbank: AX458699) Degenerate primers were designed for the proline-specific endoprotease protein sequence as follows:

[0048] g1: 5-RASMTWSRYATYASGGRA-3

[0049] g2: 5-SVBYCBCYMBRGRKMANRNTB-3

[0050] Using the Aspergillus oryzae cDNA as the template and g1 and g2 as the primers for PCR, a 1000bp product fragment was obtained. After the product was sequenced and compared at NCBI, it was found that it was all related to the A.oryzae protease gene (SequenceID: XM_001825944.2) The conserved sequence homology is high.

[0051] Furthermore, we redesigned the primers according to the sequence of the A.oryzae protease gene (SequenceID: XM_001825944.2) published in the GenBank database, and carried out PCR. T...

Embodiment 2

[0072] Example 2 Obtaining the predicted crystal structure of A.oryzae proline-specific endoprotease by "homology modeling" method and its amino acid similarity with other Aspergillus proline-specific endoprotease

[0073] The full 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, with a molecular weight of 65kDa.

[0074] According to SignalP prediction, the probability of the N-terminal of the protein being a signal peptide is 86.4%, and the signal peptide cleavage site 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 Discoverystudio software to analyze Aspergillus oryzae Amino acid-specific endoprotease protein h...

Embodiment 3

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

[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 TTGGGGTTGTTTAGAGG-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

QuitCut NotI

1μl

ddH 2 O

13μl

total capacity

50μl

[0085] Digest at 37℃ for 2hr

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

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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] Proteases are 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; serine proteases, sulfhydryl proteases, metalloproteases and carboxyl proteases according to the active center; plant, animal and microbial proteases according to the source of the protease. Among them, serine proteases are a kind of important proteolytic enzymes with serine as the active center, which play an important and extensive physiological role in biological organisms. The members of the serine protease family are very large and are divided into 81 catego...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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