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Alpha amylase and aspergillus niger strain for expressing same

A technology of α-amylase and Aspergillus, which is applied in the field of filamentous fungal host Aspergillus niger cells, can solve the problems of low expression of production strains and restrict the wide application of amylase, and achieve the effect of broadening the market space

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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  • Abstract
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AI Technical Summary

Problems solved by technology

[0006] At present, a large number of α-amylases from the genus Aspergillus (Aspergillus.sp) have been reported and used in industrial production. These amylases have excellent enzymatic properties, but the expression level of the production strains is generally low, which seriously restricts the development of amylases. wide application of

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  • Alpha amylase and aspergillus niger strain for expressing same
  • Alpha amylase and aspergillus niger strain for expressing same
  • Alpha amylase and aspergillus niger strain for expressing same

Examples

Experimental program
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Effect test

Embodiment 1

[0073] Cloning of Example 1 α-amylase gene AclaP11

[0074] Genomic DNA was extracted from overnight cultures of Aspergillus clavus CGMCC3.5289 using the Fungal Genomic DNA Extraction Kit (Omega) following the manufacturer's instructions.

[0075] Design PCR primers according to the α-amylase gene sequence of XM_001271888 on the NCBI, and the sequence of the forward primer AclaP11-F for cloning the AclaP11 gene in Aspergillus clavus is as follows:

[0076] 5'-ACGGC CTTAAG AAGATGCCTCGGATTTGGTCCTC

[0077] (the sequence shown in the underline is AflII restriction site),

[0078] The reverse primer AclaP11-R sequence is:

[0079] 5'-ATT GCGGCCGC AGCTCAAAGCACAGATCTTGCTTC

[0080] (The underlined sequence is the NotI restriction site).

[0081] The gene was amplified from A. claviculare genomic DNA using Phusion DNA polymerase (Thermo scientific).

[0082] Amplification conditions:

[0083] Step 1: Keep at 98°C for 3 minutes.

[0084] The second step: hold at 98°C for 10...

Embodiment 2

[0088] Embodiment 2: PEG-mediated protoplast fusion transforms Aspergillus niger

[0089]Draw the Aspergillus niger G1 spore suspension in the center of the CMA plate (9cm petri dish), wait for the colony to cover the whole petri dish, cut 1 / 4 size of the culture based on 200mL CMA liquid medium, culture at 30°C, 200rpm for 14~ 16h.

[0090] Collect the mycelium with a sterile Miracloth filter cloth, and wash it once with solution A, transfer the washed mycelium to 40mL protoplastization solution under aseptic conditions, and incubate at 30°C and 200rpm for 1-2h, The progress of protoplastization was detected by microscopic observation.

[0091] Filter the above-mentioned warm bath liquid with a sterile Miracloth filter cloth, and the obtained filtrate is the protoplast solution. The protoplast solution was divided into two 50mL sterile disposable centrifuge tubes, and the volume of each tube was adjusted to 45mL with solution B, centrifuged at 4000rpm for 8min to obtain the...

Embodiment 3

[0104] Embodiment 3: the ultraviolet mutagenesis and screening of starting bacterial strain Aspergillus niger 1-B

[0105] Aspergillus niger 1-B was used as the starting strain for ultraviolet mutagenesis and screening.

[0106] Inoculate the starting strain Aspergillus niger 1-B on CMA solid medium, culture at 30°C for 5 days until black spores grow, wash the spores with sterile water, and dilute to a spore concentration of 10 6 level, the spore suspension was irradiated under an ultraviolet lamp for 4 minutes, the irradiation distance was 22 cm, and the power of the ultraviolet lamp was 30w. The resulting spore suspension was diluted 100 times, coated on a CMA solid medium plate, and after 48 to 72 hours, a small and dense colony with a small colony radius was selected, streaked and inoculated on a CMA solid medium plate, and after it grew black spores, use After washing with sterile water, inoculate in TSB fermentation medium, measure enzyme activity after 5 days of fermen...

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Abstract

The invention aims to provide alpha amylase and an aspergillus niger strain for expressing the same. An amino acid sequence of alpha amylase is SEQ ID NO:1. A coding mRNA (messenger ribonucleic acid) sequence of alpha amylase is SEQ ID NO:2. The mutant strain aspergillus niger 1-B5 obtained by an ultraviolet mutagenesis method can efficiently express alpha amylase AclaP11 derived from aspergillus clavatus, with strain number of CGMCC (China General Microbiological Culture Collection Center) No.8443. The enzyme activity of aspergillus niger 1-B5 is 1.97 times that of an original strain. The optimum pH value and optimum temperature of alpha amylase AclaP11 expressed by aspergillus niger 1-B5 are respectively 6.0 and 50 DEG C. Compared with the original strain, aspergillus niger 1-B5 has the advantage that the enzymatic properties are not changed. Besides, aspergillus niger 1-B5 is a food safety fungus (GRAS(generally recognized as safe)), so that alpha amylase expressed by aspergillus niger 1-B5 in a secretory manner can be applied to the field of food additives, thus further broadening the market space of alpha amylase.

Description

technical field [0001] The invention belongs to the technical field of separation and expression of amylase, and in particular relates to an α-amylase derived from Aspergillus clavatus and a filamentous fungal host Aspergillus niger cell used for recombinantly expressing the α-amylase. Background technique [0002] α-amylases (E.C.3.2.1.1) are capable of catalyzing the endohydrolysis of 1,4-α-glycosidic linkages in oligo- and polysaccharides, acting in a random fashion on starch, glycogen and related poly- and oligosaccharides, releasing α- Configuration of the reducing group. At present, α-amylase is widely used in a variety of industrial applications, such as bakery, wine, corn steep liquor and ethanol production, and vinegar fermentation and other fields. [0003] The sources of α-amylase are very extensive and can be isolated from animals, plants and microorganisms. Among them, amylase derived from microorganisms has the characteristics of abundant sources, diverse per...

Claims

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Application Information

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IPC IPC(8): C12N9/30C12N15/56C12N1/15C12R1/685
CPCC12N9/242C12Y302/01001
Inventor 王华明闫真
Owner 山东中科生物创新产业园管理有限公司
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