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Glucamylase

A kind of technology of glucoamylase and glucose, applied in glucoamylase and its application field

Active Publication Date: 2014-04-02
山东中科生物创新产业园管理有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no research report on Glioma pink glucoamylase gene at home and abroad. The present invention uses molecular cloning and genetic engineering techniques to clone the glucoamylase gene from Glioma pink and transfer it into Aspergillus niger G1 have been expressed efficiently

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Screening of glucoamylase gene

[0020] Through comparative proteomics, the pre-established glucoamylase sequence of our laboratory (Protein Expression and Molecular Biology Laboratory) and the predicted protein sequence of the whole genome of Glioma rosea were locally blasted to obtain some sequences with high homology . These highly homologous sequences were subjected to NCBI online Blastp and the comparison results were analyzed to determine the most likely glucoamylase sequence. Use SignalP4.1Server to analyze these possible sequences, and use the sequence containing the signal peptide as the target gene for cloning.

[0021] Cloning of the glucoamylase gene

[0022] The target gene was obtained by PCR amplification using the whole genome DNA of Glioma pink as a template, and the primers used in the PCR process were as follows:

[0023] Forward primer 5′-CCG CTCGAG AGGATGGTGAAGATAATTCCCACTGTGC-3′

[0024] reverse primer 5′-TGC TCTAGA TCATCGCCAG...

Embodiment 2

[0030] Embodiment 2: Recombinant plasmid transforms Aspergillus niger G1 strain and verification

[0031] Draw the Aspergillus niger G1 spore suspension in the center of the CMA plate (9cm petri dish), wait for the colony to cover the entire petri dish, cut 1 / 4 size of the culture based on 200mL CMA liquid medium, cultivate at 30°C and 200rpm for 14~ 16h.

[0032] Collect the bacteria with a sterile Miracloth filter cloth, rinse once with solution A, transfer it to 40ml of lysate with a cotton swab under sterile conditions, lyse at 30°C and 200rpm for two hours, and observe the protoplasts with a microscope the formation of.

[0033] Filter the above lysate with a sterile Miracloth filter cloth, collect protoplasts with two 50ml sterile centrifuge tubes and rinse with solution B to make each tube about 25ml. Centrifuge at 2000rpm for 5min to discard the supernatant, and wash the precipitate twice with 20ml solutionB. Resuspend the pellet in 10 mL of solution B, and count th...

Embodiment 3

[0048] Example 3: Fermentation verification of positive transformant strains.

[0049] Pick the above-mentioned positive transformant strains, inoculate them in 20ml TSB fermentation medium, and cultivate them at 30°C and 200rpm for 5 days; the obtained fermentation broth is filtered with 8 layers of gauze, and the filtrate is centrifuged at 14000g for 10min to collect the supernatant; The supernatant was electrophoresed on a 12% SDS-PAGE gel. Electropherogram as figure 2 .

[0050] TSB fermentation medium: 12g NaNO 3 , 0.5g KCl, 1.5g KH 2 PO 4 , 2.05g MgSO 4 ·7H 2 O, 3.5 g NaH 2 PO 4 ·H 2 O, 45g tryptic soybean broth, 70g sodium citrate, 1g Tween 80, 1mL trace elements (see below), add dlH 2 O to a final volume of 700 mL, after autoclaving, add 300 mL of 40% maltose sterilized by filtration with a 0.22 μm microporous membrane.

[0051] Trace elements: in 250mL dlH 2 Add 1g FeSO to O 4 ·7H 2 O, 8.8g ZnSO 4 · 7 h 2 O, 0.4g CuSO 4 ·5H 2 O, 0.15g MnSO 4 4H 2...

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Abstract

The invention provides glucamylase of which an amino acid sequence is SEQ ID NO:1. The invention discloses a glucamylase gene of gliocladium roseum for the first time. A shake flask fermentation experiment is carried out on recombinant bacteria, and the enzyme activity of a fermentation liquor can be up to 292U / ml. By adopting the glucamylase disclosed by the invention, an alpha-1,4-glucosidic bond can be efficiently decomposed from the non-reductive end of a carbohydrate chain, glucose can be hydrolyzed, and an alpha-1.6 glucoside bond also can be slowly hydrolyzed, and converted into the glucose, so as to be widely applied to the glucose industry.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a glucoamylase and an application thereof. Background technique: [0002] Glucoamylase (Glucoamylase, EC3.2.1.3) is an exocarbase that hydrolyzes starch. It can hydrolyze starch from non-reducing terminal α-1.4 glucosidic bond to produce glucose, and can also slowly hydrolyze α-1.6 glucosidic bond to convert it into glucose. It can also hydrolyze dextrin and release β-D-glucose from the non-reducing end of glycogen. [0003] A variety of bacteria, fungi, yeast and plants produce glucoamylase, and most of the glucoamylase used in industry comes from fungi. For example from the following bacteria: Aspergillus (Aspergillus) (Svensson et al., Carlsberg Res. Commun. 48: 529-544 (1983); Boel et al., Agric. Biol. Chem. 53: 923-929 (1989) ; U.S. Patent No. 5,024,941; U.S. Patent No. 4,794,175 and WO88 / 09795); Talaromyces (U.S. Patent No. 4,247,637; U.S. Patent...

Claims

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Application Information

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IPC IPC(8): C12N9/34C12N15/56C12N15/80C12N1/15C12P19/20C12P19/02C12R1/685
CPCC12N9/2428C12Y302/01003
Inventor 王华明张亮
Owner 山东中科生物创新产业园管理有限公司
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