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Method for enhancing glucose conversion rate of Aspergillus niger saccharifying enzyme

A technology of Aspergillus niger and conversion rate, which is applied in the field of genetic engineering to achieve the effect of reducing production costs and increasing the conversion rate of starch and sugar

Active Publication Date: 2015-10-07
NANJING BESTZYME BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no record in the patent that the gene deletion of isomaltose synthase will make the target protein produced by it can significantly increase the DX value when it is used for glucose conversion

Method used

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  • Method for enhancing glucose conversion rate of Aspergillus niger saccharifying enzyme
  • Method for enhancing glucose conversion rate of Aspergillus niger saccharifying enzyme

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The construction of embodiment 1 pHphtk plasmid

[0023] The plasmid consists of the following 3 parts, constructed by GenScript company, see the plasmid map figure 1 .

[0024] (1) 2305bp fragment obtained after XbaI-PciI double digestion of pUC57 plasmid;

[0025] (2) hph gene expression cassette, the sequence is shown in SEQ ID NO.4;

[0026] (3) HSV-tk expression cassette, the sequence is shown in SEQ ID NO.5.

Embodiment 2

[0027]Example 2 Construction of integrated plasmids pAgdAhphtk and pAgdBhphtk

[0028] The integrated plasmid used for agdA gene knockout is called: pAgdAhphtk, and its construction method is as follows:

[0029] The pHphtk plasmid was linearized by vector-F and vector-R primers; at the same time, agdA-5'-F and agdA-5'-R, agdA-3'-F and agdA-3'-R were used as primers, respectively, to Aspergillus niger genomic DNA was used as a template, and a 2kb DNA fragment flanking the 5' end of agdA and a 2kb DNA fragment flanking the 3' end of agdA with a 40bp recombination arm were amplified by PCR; , 2kb DNA fragment flanking the 3' end of agdA by Gibson Master Mix Kit (E2611, New England Biolabs) was recombined to obtain the integrated plasmid pAgdAhphtk, see the plasmid map figure 2 .

[0030] The integrated plasmid used for agdB gene knockout is called: pAgdBhphtk, and its construction method is as follows:

[0031] The pHphtk plasmid was linearized by vector-F and vector-R pri...

Embodiment 3

[0035] Embodiment 3 Transformation of pAgdAhphtk / pAgdBhphtk plasmid

[0036] The starting strain of this example is CBS513.88, which was purchased from the Netherlands Fungal Culture Collection (CBS-KNAW Fungal Biodiversity Centre).

[0037] Using the protoplast transformation method, the specific steps are as follows:

[0038] Preparation of protoplasts: Aspergillus niger mycelium was cultured in nutrient-rich TZ liquid medium (0.8% beef extract powder; 0.2% yeast extract; 0.5% peptone; 0.2% NaCl; 3% sucrose; pH5.8). The mycelium was filtered from the culture medium through mira-cloth (Calbiochem Company) and washed with 0.7M NaCl (pH 5.8). Sigma) and lysozyme (Sigma) 0.2% enzymatic hydrolysis solution (pH5.8), 30° C., 65 rpm for 3 h. Then place the enzymatic solution containing protoplasts on ice and filter with four layers of lens-cleaning paper. After the filtrate is centrifuged at 3000rpm, soft & 4°C for 10min, the supernatant is discarded; the protoplasts attached to t...

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Abstract

The invention discloses a method for enhancing glucose conversion rate of an Aspergillus niger saccharifying enzyme. The composite of an agdB-gene-inactivated Aspergillus-niger-secreted saccharifying enzyme and a debranching enzyme is utilized to convert starch sugar. The alpha glucosaccharase agdB gene is inactivated to lower the saccharifying enzyme fermentation activity of Aspergillus niger, and the saccharifying enzyme can be directly compounded with the debranching enzyme to convert glucose, thereby obviously enhancing the DX value.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a method for improving the glucose conversion rate of Aspergillus niger glucoamylase. Background technique [0002] Glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) is an enzyme that catalyzes the release of D-glucose from the non-reducing ends of polysaccharide molecules such as starch or oligosaccharides. Commercially, glucoamylase is produced by several filamentous fungi and yeasts including Aspergillus niger and Aspergillus oryzae. Filamentous fungi are well known as cell factories for the production of valuable products such as enzymes, among which Aspergillus niger and Aspergillus oryzae are widely used to express host for the production of food additives. [0003] Existing high-concentration glucose syrup and fructose syrup are all obtained by enzymatically hydrolyzing starch. Specifically, after the starch is liquefied by a medium- or high-temperature amylase, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/14C12R1/685
Inventor 白挨玺卞芙蓉王彩梅张垠孙艳李峰
Owner NANJING BESTZYME BIO ENG CO LTD
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