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Vitreoscilla hemoglobin gene expression box and method for improving yield of saccharifying enzyme produced by aspergillus niger

A gene expression cassette and hemoglobin technology, which are applied in the field of Vibrio vibrio vitreous hemoglobin gene expression cassette and the field of improving the yield of Aspergillus niger saccharifying enzyme

Inactive Publication Date: 2011-05-18
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on improving the oxygen-dissolving ability of Aspergillus niger species under oxygen-limited conditions by using hemoglobin of Vitiligo hyaline, and then improving its ability to produce glucoamylase

Method used

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  • Vitreoscilla hemoglobin gene expression box and method for improving yield of saccharifying enzyme produced by aspergillus niger
  • Vitreoscilla hemoglobin gene expression box and method for improving yield of saccharifying enzyme produced by aspergillus niger
  • Vitreoscilla hemoglobin gene expression box and method for improving yield of saccharifying enzyme produced by aspergillus niger

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of the Vitella hyaline hemoglobin gene (vhb) expression cassette Pgpd-vhb-Tgpd

[0033] (1) Strain, plasmid and culture medium

[0034] Aspergillus niger (Aspergillus niger) was purchased from China Industrial Microorganism Culture Collection Center, CICC No. 40125; the plasmid pPICZαA-vhb containing the vhb gene, its construction process can be found in Figure 8 . Plasmid pPICZαA was purchased from Invitrogen.

[0035] Cultivation of Aspergillus niger:

[0036] The solid resuscitation adopts PDA medium, and the culture temperature is 28°C; the liquid expansion culture adopts YPD or corn steep liquor powder medium (1% corn steep liquor powder, 12% maltodextrin, 2% ammonium sulfate.), and the culture temperature is 28°C. The rotational speed is 200rpm.

[0037] Aspergillus niger fermented to produce glucoamylase medium formula: 8% corn starch, 4.3% medicinal medium, 1% wheat bran. Among them, corn starch needs to be liquefied by high-temperat...

Embodiment 2

[0051] Embodiment 2 prepares the recombinant Aspergillus niger containing Pgpd-vhb-Tgpd expression cassette

[0052] (1) Preparation of Aspergillus niger protoplasts

[0053] 1) Draw 1.5mL of sterile water from the fresh and mature Aspergillus niger spores on the PDA culture plate, and prepare them at a concentration of 0.8~1.0×10 8 Each / mL spore suspension was inoculated in 40-50 mL of YPD / Mandels liquid basal medium, and cultured at 28°C and 250 rpm for about 11 hours.

[0054] 2) Take the bacterial solution cultured for about 11 hours and observe it under an optical microscope. When most of the spores have germinated, transfer the culture solution to a 25mL large centrifuge tube, centrifuge at 8000rpm for 7min, remove the supernatant, and wash with 1M MgSO 4 10mL was washed twice, centrifuged at 8000rpm for 10min, and the supernatant was removed.

[0055] 3) Preparation of enzymatic hydrolysis solution and enzymatic hydrolysis reaction: Accurately weigh 100 mg of lytic en...

Embodiment 3

[0068] Example 3 Verification of the ability of recombinant Aspergillus niger to produce saccharification enzymes

[0069] (1) Fermentation

[0070] Ferment the recombinant Aspergillus niger obtained in Example 2 and the original control strain Aspergillus niger HE01 respectively, and measure the glucoamylase enzyme activity of the recombinant bacteria and the original control bacteria under normal conditions and anaerobic conditions to detect VHB in anaerobic conditions. The effect of different conditions on the enzyme activity of glucoamylase produced by recombinant bacteria.

[0071] The oxygen-poor condition is controlled by adjusting the rotating speed of the shaker, and the specific experimental method is as follows:

[0072] Strain seed culture stage: Inject glycerin-preserved recombinant bacteria and control original strains into Aspergillus niger seed medium for activation for about 3-4 days, at 28-30°C, 200r / min. When the strains grow vigorously, re-insert into the...

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Abstract

The invention relates to the field of genetic engineering, and discloses a vitreoscilla hemoglobin gene expression box and a method for improving yield of saccharifying enzyme produced by aspergillus niger. The expression box comprises a transparent vitreoscilla hemoglobin gene, and a promoter and a terminator of a glyceraldehyde-3-phosphate dehydrogenase gene. In the invention, aspergillus brasiliensis is subject to molecular modification, the problem of oxygen depletion of the aspergillus niger under the oxygen-limiting condition is solved from the molecular level and the energy consumption is reduced in a process of producing the saccharifying enzyme by the deep fermentation of the aspergillus niger liquid.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a hemoglobin gene expression cassette of Vitella hyaline and a method for improving the yield of glucoamylase produced by Aspergillus niger. Background technique [0002] Glucose is an indispensable nutrient for metabolism in organisms, and the heat released by its oxidation reaction is an important source of energy for human life activities. Glucoamylase is generally used in industrial production to produce glucose. Glucoamylase is one of the main enzymes in the process of converting starch into glucose. It belongs to an enzyme preparation commonly used in food industry production, also known as Glucoamylase (Glucoamylase , EC.3.2.1.3.) or amyloglucosidase (Amyloglucosidase), Y-amylase (Y-amylase). Glucoamylase is an enzyme with exolytic activity, which hydrolyzes the non-reducing ends of 1, 4 linkages on carbon chains such as starch, starch dextrin, and glycogen to obtain th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/31C12N1/15C12N15/63C12R1/685
Inventor 王娟余少文胡萍
Owner SHENZHEN UNIV
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