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Low-temperature alpha-amylase originated from fungus and coding gene and application of low-temperature alpha-amylase

A technology encoding gene and amylase, applied in applications, fungi, genetic engineering, etc., can solve the problems of unresearched enzymatic properties and no separation and purification of amylase.

Inactive Publication Date: 2014-12-03
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has considerable amylase activity on Geomyces pannorum, there is no report about its amylase gene sequence and protein sequence, and due to the limitation of the culture conditions of this bacteria, the amylase of this bacteria has not been isolated and purified so far. not yet studied

Method used

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  • Low-temperature alpha-amylase originated from fungus and coding gene and application of low-temperature alpha-amylase
  • Low-temperature alpha-amylase originated from fungus and coding gene and application of low-temperature alpha-amylase
  • Low-temperature alpha-amylase originated from fungus and coding gene and application of low-temperature alpha-amylase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Amplification of Low Temperature α-Amylase Gene

[0045] 1.1 Strains and their cultivation

[0046] Received Geomyces pannorum AK07KGI1001R1-2(1) from the cooperation project of the Faculty of Biological Sciences, University of Malaysia (bacteria identification NCBI accession number: JF720026.biramy Krishn et al.2011.Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica . Polar Biol, 34:1535–1542).

[0047] Wash G. pannorum spores from the 10-day-cultured PDA slant with sterile water, inoculate liquid medium, and culture at 20°C for 7 days to collect mycelia for genome extraction; inoculate starch solid medium with a toothpick, and culture at 20°C After 5 days, the mycelia were collected for total RNA extraction.

[0048] 1.2 Genome Extraction

[0049] Refer to the instructions of the Omega Fungal Genome Extraction Kit

[0050] 1.3 Total RNA extraction

[0051] Follow the instructions of Takara RNA extraction reagent...

Embodiment 2

[0119] Embodiment 2. Containing the construction of the Aspergillus oryzae recombinant expression vector of low temperature alpha-amylase gene

[0120] 2.1. Extraction of Aspergillus oryzae genome

[0121] Aspergillus oryzae RIB40 (National Research Institute of Brewing Stock Culture and ATCC42149) was inoculated on CM medium and incubated at 30°C for 7 days until the spores matured. Prepare an appropriate amount of spore suspension and inoculate it in CM medium liquid seed medium, and cultivate it at 30°C and 200rpm for 2 days until the mycelium concentration reaches 4-5g / L for genome extraction. The method refers to the instructions of the Omega fungal genome extraction kit.

[0122] 2.2. Isolation of Aspergillus oryzae α-amylase promoter (PamyB) and its signal peptide sequence

[0123] According to the sequence of the amyB promoter published on GeneBank, using the above-mentioned extracted Aspergillus oryzae genomic DNA as a template, specific PCR amplification was perform...

Embodiment 3

[0151] Embodiment 3. Transformation of the Aspergillus oryzae recombinant bacteria containing low temperature α-amylase gene expression vector

[0152] 3.1 Preparation of Aspergillus oryzae uracil auxotrophic RIB40-PF1 protoplasts

[0153] (1) A.oryzae RIB40-PF1 (the bacterial strain RIB40-PF1 used in the present invention was screened and obtained by the inventor himself, and the bacterial strain has been preserved in the General Microorganism Center CGMCC of China Microbiological Culture Collection Management Committee, and the preservation number is CGMCC NO : 9129, the preservation date is 2014.05.08.) The spores on the slant were inoculated into the CM liquid medium, cultivated overnight (12~16h) at 200rpm at 30°C, and formed dense and small bacterial balls (d<2mm), washed with sterile water Next, use sterile Microcloth to filter the culture medium to obtain mycelia, and wash with solution I for 2 to 3 times. During the washing process, centrifuge at 6000 rpm at 4°C for ...

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Abstract

The invention provides low-temperature alpha-amylase originated from a fungus. The low-temperature alpha-amylase originated from the fungus is a protein of (a) or (b): (a) a protein which is formed by the following amino acid sequences shown in SEQ ID NO.19; (b) a protein which has the function of the alpha-amylase and is derived from (a) by means of substituting and / or deleting and / or adding one or more amino acid residues to the amino acid sequences shown in the SEQ ID NO.19. The coding gene provided by the invention is deoxyribonucleotide shown in (1) or (2): (1) a nucleotide sequence shown in SEQ ID NO.17; (b) a nucleotide sequence shown in SEQ ID NO.18. A recombinant host cell provided by the invention comprises a recombinant expression vector with the above coding gene. The optimum temperature of the alpha-amylase according to the invention is 40 DEG C which is 10 DEG C lower than that of normal-temperature amylase. The alpha-amylase is good in low-temperature adaptability, still maintains the activity of over 20% of highest activity at 0-30 DEG C and has a huge application prospect in detergent and textile desizing.

Description

technical field [0001] The invention relates to a fungal alpha-amylase with better activity at low temperature, and also relates to the coding gene and application of the low-temperature amylase. technical background [0002] α-amylase (α-1,4-D-glucan-glucanhydrolase, EC3.2.1.1) belongs to endo-amylase, which can treat amylopectin amylose or other -1,4-glucan molecules The internal -1,4-glucosidic bonds are hydrolyzed in a random manner to generate linear and branched oligosaccharides of different lengths. It is a general term for a class of enzymes. It is the main enzyme in the starch industry. It is used in food processing, detergents, and textiles. Pulp, paper industry and other fields have important commercial value. Low-temperature amylase (cold-adapted amylase) refers to the amylase that can effectively play a catalytic role at low temperature. Generally, the optimum temperature is 35-45°C. In addition to the above-mentioned applications, it can also be used for biore...

Claims

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Application Information

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IPC IPC(8): C12N9/30C12N15/56C12N15/80C12N1/15C12R1/69
CPCC12N9/242C12Y302/01001
Inventor 高蓓魏东芝毛佑志
Owner EAST CHINA UNIV OF SCI & TECH
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