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Aspergillus niger alpha-glucosidase gene and high-efficiency expression method thereof

A technology of glucosidase and Aspergillus niger, which is applied in the field of enzyme engineering and can solve problems such as insufficient expression level to meet production needs.

Active Publication Date: 2013-06-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In the previous research of the inventor, the total RNA of A. niger CICIM F0620 was used as a template, and the α-glucosidase cDNA was obtained by RT-PCR, and expressed in P. pastoris KM71. Type and other genetically engineered bacteria have been significantly improved, but the expression level is still not enough to meet production needs

Method used

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  • Aspergillus niger alpha-glucosidase gene and high-efficiency expression method thereof
  • Aspergillus niger alpha-glucosidase gene and high-efficiency expression method thereof
  • Aspergillus niger alpha-glucosidase gene and high-efficiency expression method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Example 1: This example illustrates the codon optimization process for alpha-glucosidase.

[0017] 1. Optimization and synthesis of AGL gene:

[0018] Based on the α-glucosidase sequence of A. niger CBS513.88 in the NCBI database (NCBI accession number: 4991096), its codon usage frequency was analyzed by Graphical Codon Usage Analyzer (http: / / gcua.schoedl.de / ) , using DNAWorks software to optimize the Aspergillus niger α-glucosidase gene sequence. DNAMAN was used to analyze the restriction endonuclease sites of the target gene. The optimized gene was synthesized by Shanghai Sangon Biotechnology Co., Ltd. Our strategy is to replace rare codons with favored codons. A total of 728 bases were changed, involving 222 codons. The GC content decreased from 50.0% to 44.2%. The homology between the synthetic sequence and the original sequence was 74.0%.

[0019] Optimized α-glucosidase gene aglu OP The nucleotide sequence is shown as SEQ ID No1.

[0020] 2. Cloning and scree...

Embodiment 2

[0025] Example 2: This example illustrates recombinant P. pastoris KM71 / pPIC9K-aglu OP Strain 3L tank fermentation culture

[0026] Draw 200μL of the glycerol tube bacteria solution and inoculate it into a 500mL Erlenmeyer flask containing 50mL of seed medium, cultivate at 30°C and 200r / min for 16-20h, and insert the above culture solution with 10% inoculum volume into 0.8L A 3L fermenter (BIOFLO, America), controlled pH5 with 25% ammonia water, cultured at 30°C, and maintained dissolved oxygen at about 20% by coupling with stirring speed and adjusting ventilation. When dissolved oxygen rose rapidly, it indicated that the culture medium The glycerol in the medium has been exhausted, and the glycerol feeding solution is started to be fed exponentially. When the bacterial cell concentration reaches 115.2g / L, the feeding is stopped, and the dissolved oxygen rebounds again. Set the induction temperature at 26°C and add 2% methanol. After the recombinant P. pastoris adapts to meth...

Embodiment 3

[0027] Example 3: This example illustrates the co-expression of the P. pastoris disulfide isomerase pdi gene

[0028] The recombinant expression vector pPICZA-pdi constructed earlier in our laboratory was linearized by Sac I digestion and then electroporated into P. pastoris KM71 / pPIC9K-aglu OP In , the transformants were able to grow on basal medium because the cells already contained the G418 resistance gene and the pPIC9K vector integrated into the chromosome. In order to improve the screening efficiency, spread the above-mentioned competent cells on the MD plate containing 0.5 mg / mL G418 resistance concentration, and then pick a single colony and inoculate it on the YPD plate containing gradient Zeocin resistance. The Zeocin resistance gradient was 0.25mg / mL, 0.5mg / mL, 1mg / mL, 2mg / mL. Pick a single colony on a 2 mg / mL Zeocin-resistant plate with a toothpick and inoculate it in 10 mL of YPD medium, culture at 30°C overnight, and store in a glycerol tube. Then take 1mL of ...

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Abstract

The invention discloses an aspergillus niger alpha-glucosidase aglu<OP> and a high-efficiency expression method of the aspergillus niger alpha-glucosidase aglu<OP> in pichia pastoris. The nucleotide sequence of the optimized alpha-glucosidase gene is shown in SEQ ID No.1. The gene is constructed into a pichia pastoris expression vector, and the pichia pastoris expression vector and disulphide bond isomerase are transformed into the pichia pastoris for co-expression, and the enzyme activity of the alpha-glucosidase gene can be up to 10.1U / mL after induction is carried out for 110 hours in a 3L tank, and is 2.9 times of the enzyme cavity before optimization. The optimized alpha-glucosidase gene can be used for industrial production of the alpha-glucosidase.

Description

technical field [0001] The invention relates to an Aspergillus niger α-glucosidase gene and a high-efficiency expression method thereof, belonging to the technical field of enzyme engineering. technical background [0002] α-glucosidase (EC3.2.1.20), also known as α-D-glucoside hydrolase, has a wide range of sources and can catalyze the hydrolysis of oligosaccharides to generate glucose. It plays an important role in the carbohydrate metabolism of animals, plants and microorganisms. physiological function. Among them, the α-glucosidase (AGL) derived from Aspergillus niger not only has hydrolysis activity, but also has good transglycoside ability, that is, it cuts the α-1,4 glycosidic bond from the non-reducing end of maltose and frees The resulting glucose residues are transferred to other sugar substrates to form α-1,6 glycosidic bonds, thereby obtaining non-fermentable isomaltooligosaccharides (IMOs for short, mainly including isomaltose, panose, isomaltotriose, etc.). B...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/81C12N9/30C12R1/685C12R1/84
Inventor 吴敬刘旭吴丹陈坚
Owner JIANGNAN UNIV
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