Novel glucoamylase VGA (video graphics array), gene thereof and application thereof

A saccharification enzyme, A-VGA technology, applied in the field of genetic engineering, can solve the problems of narrow scope of action, large loss of enzyme activity, inactivation, etc.

Active Publication Date: 2012-03-21
湖南康捷生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The tolerance temperature of the glucoamylase used in industry is generally below 85°C. If the temperature exceeds this temperature, the enzyme activity will be greatly lost, or even completely inactivated. In addition, the currently produced and used glucoamylase generally has a narrow pH range, generally 4.5~ 6.5, these all limit the application of glucoamylase to a certain extent

Method used

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  • Novel glucoamylase VGA (video graphics array), gene thereof and application thereof
  • Novel glucoamylase VGA (video graphics array), gene thereof and application thereof
  • Novel glucoamylase VGA (video graphics array), gene thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1, the enzyme production property test of aspergillus niger (Aspergillus niger)

[0103] Using the screened Aspergillus niger (preservation number: CGMCC No.4235) for producing saccharification enzyme to carry out the shake flask fermentation experiment, its fermentation medium is as follows:

[0104] Corn starch 10%, bean cake powder 2%, corn steep liquor 1.5%, MgSO 4 0.1%, K 2 HPO 4 0.1%

[0105] The fermentation medium was sterilized at 121°C and 0.1 MPa for 20 minutes, inoculated after cooling, and cultured at 32°C and 200 rpm for 2-3 days.

[0106] After the fermentation is over, the fermentation broth is centrifuged, and the supernatant is taken to test the enzymatic properties of the glucoamylase such as reaction temperature, heat resistance, and pH action range. The test results show that the optimum reaction temperature of the glucoamylase produced by the mold is about 40°C, and it has good heat resistance. After being treated in an aqueous sol...

Embodiment 2

[0108] Embodiment 2, the cloning of Aspergillus niger (Aspergillus niger) glucoamylase VGA gene

[0109] Use the RNA extraction kit to extract the total RNA of Aspergillus niger, according to the reverse transcriptase SuperScript TM III Reverse Transcriptase Instructions Synthesis of first-strand cDNA. Using cDNA as a template, design glucoamylase primers for PCR amplification, double-digest the PCR product with EcoRI and XbaI, and then connect it to the Pichia pastoris expression vector pPICzalphaA that has undergone the same digestion, and transform the ligated product into Escherichia coli Topl0 competent After the cells were screened by the antibiotic Zeocin, positive clones were obtained. Plasmids of positive clones were extracted. The samples were sent to Shanghai Yingjun Biological Co., Ltd. for sequencing. The sequencing results showed that the obtained cloned DNA insert contained the complete open reading frame of the glucoamylase gene. The glucoamylase gene VGA h...

Embodiment 3

[0115] Embodiment 3, the construction of the Pichia pastoris engineered bacterium that comprises novel glucoamylase gene VGA

[0116] The optimized new glucoamylase gene VGA was double-digested with EcoRI and XbaI, and reconnected to the vector pPICzαA after the same double-digestion to obtain the recombinant expression vector pPICzαA-VGA. Then, SacI was used for linearization, and the linearized recombinant vector was electroporated to transform Pichia pastoris X33, and after electroporation, it was spread on a YPD (Zeo+) plate for screening to obtain a recombinant strain of Pichia pastoris.

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Abstract

The invention relates to the field of genetic engineering, in particular to a novel glucoamylase VGA (video graphics array), a gene thereof and application thereof. The novel glucoamylase VGA is provided with an amino acid sequence as indicated in SEQ (sequence) ID (identification) NO.1. The invention further provides a gene encoding the novel glucoamylase VGA, a recombinant vector comprising thegene, a recombinant strain comprising the gene and application of the gene, and a nucleotide sequence of the gene is indicated as SEQ ID NO.2. The enzyme activity of the novel glucoamylase VGA is stable under the condition of 2.0-7.5 pH (potential of hydrogen), and the novel glucoamylase VGA is remarkable in amylolysis effect. In addition, the glucoamylase has better high-temperature resistance, and loss of the enzyme activity of the glucoamylase is lower than 15% after the glucoamylase is treated in 85 DEG C water liquor for 10min, so that the glucoamylase completely meets various industrialrequirements and is suitable for being applied as a novel high-temperature-resistant glucoamylase.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a novel glucoamylase VGA and its gene and application. Background technique [0002] Glucoamylase is an enzyme with the largest output and the widest application range in the world. It has a long history of application. my country began to use saccharification koji to make wine as early as 1500 years ago. [0003] The full name of glucoamylase is glucoamylase (GAcoamylase, EC3.2.1.3), also known as γ-amylase, referred to as glucoamylase. Glucoamylase is an amyloglucosidase that can use starch, dextrin, etc. as substrates to hydrolyze α-1,4 glucosidic bonds sequentially from the non-reducing end of starch under certain conditions to produce glucose. [0004] Glucoamylase can be used in various industries such as alcohol, brewing, glucose, fructose syrup, antibiotics, lactic acid, organic acid, monosodium glutamate, cotton spinning mills, etc. It has a wid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/34C12N15/56C12N15/81C12N1/19C12R1/84
Inventor 罗长财李阳源钟开新
Owner 湖南康捷生物科技有限公司
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