Optimized high-temperature trehalase MS-Tre capable of being expressed efficiently in aspergillus niger and encoding gene and application of optimized high-temperature trehalase MS-Tre

A trehalase and high-efficiency expression technology, applied in the field of genetic engineering, can solve the problems of low expression level of recombinant trehalase, and achieve the effects of good stability, reduced cooling energy consumption, and improved utilization rate

Active Publication Date: 2019-08-09
SOUTH CHINA UNIV OF TECH
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

In the current literature reports, the research on trehalase mainly focuses on the determination of enzymatic properties, and the expr...

Method used

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  • Optimized high-temperature trehalase MS-Tre capable of being expressed efficiently in aspergillus niger and encoding gene and application of optimized high-temperature trehalase MS-Tre
  • Optimized high-temperature trehalase MS-Tre capable of being expressed efficiently in aspergillus niger and encoding gene and application of optimized high-temperature trehalase MS-Tre
  • Optimized high-temperature trehalase MS-Tre capable of being expressed efficiently in aspergillus niger and encoding gene and application of optimized high-temperature trehalase MS-Tre

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Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1, the construction of general expression vector

[0047] Using the Aspergillus niger genome as a template, perform PCR amplification (Prime STAR premix HS (purchased from takara company)), using primer Ttef-fw (as shown in SEQ ID NO.8, Primer means primer, the same below) and primer Ttef-rev (as shown in SEQ ID NO.9) amplifies the tef terminator (as shown in SEQ ID NO.3), using primer amyA-fw (as shown in SEQ ID NO.12) and primer amyA-rev (as shown in SEQ ID NO.12) Shown in SEQ ID NO.13) to amplify the last 1000bp (as shown in SEQ ID NO.5) of the gene encoding neutral amylase amyA, use primer pyrG-fw (as shown in SEQ ID NO.10) as a template with the Aspergillus nidulans genome shown) and primer pyrG-rev (shown in SEQ ID NO.11) to amplify the pyrG marker (shown in SEQ ID NO.4), and the PCR products obtained are respectively named as Ttef, amyA, pyrG (shown in SEQ ID NO. .3, as shown in SEQ ID NO.5, as shown in SEQ ID NO.4), use NEBuilder HiFi DNA Assembly Cl...

Embodiment 2

[0048] Embodiment 2, containing the target gene expression vector construction

[0049]Using the optimized MS-Tre gene nucleotide sequence of the synthetic amino acid sequence, the non-optimized MSre gene nucleotide sequence of the synthetic amino acid sequence, and the Aspergillus niger genome as templates, primer Tre1-fw (such as SEQ ID NO.18 shown) and primer Tre-rev (shown in SEQ ID NO.19) amplified to obtain the MS-Tre gene sequence (shown in SEQ ID NO.2) after amino acid sequence optimization, with primer Tre2-fw (shown in SEQ ID NO.2) ID NO.20) and primer Tre-rev (as shown in SEQ IDNO.19) amplified to obtain the unoptimized MSre gene sequence of amino acid sequence (as shown in SEQ ID NO.2), with primer PamyA-fw (as shown in SEQ ID NO.16) and the primer PamyA-rev (shown in SEQ ID NO.17) were amplified to obtain the Aspergillus niger neutral amylase promoter PamyA sequence (shown in SEQ ID NO.7). The 3 PCR-amplified PCR fragments were connected to the linearized univers...

Embodiment 3

[0050] Embodiment 3, transformation of expression vector pMD20-PamyA-MS-Tre-Ttef-pyrG-amyA and pMD20-PamyA-MSre-Ttef-pyrG-amyA plasmid in Aspergillus niger

[0051] Prepared according to the steps provided in (Gomi K, Iimura Y, Hara S. Integrative transformation of Aspergillusoryzae with a plasma containing the Aspergillus nidulans argB gene [J]. Agricultural and biological chemistry, 1987, 51 (9): 2549-2555.) The protoplasts of the host fungus Aspergillus niger (ΔpyrG), the expression vectors pMD20-PamyA-MS-Tre-Ttef-pyrG-amyA and pMD20-PamyA-MSre-Ttef-pyrG-amyA obtained above were transformed into the protoplasts respectively , coated hypertonic CD medium (containing 1M sucrose, 0.3% (w / v) NaNO 3 , 0.2% (w / v) KCl, 0.05% (w / v) MgSO 4 .7H 2 O, 0.1% (w / v) K 2 HPO 4 .3H 2 O, 0.001% (w / v) FeSO 4 .7H 2 (0, 2% (w / v) agar powder, pH 5.5, the unit of w / v is g / mL), put it into a 30° C. incubator, and observe the growth of transformants after 5 days.

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Abstract

The invention discloses optimized high-temperature trehalase MS-Tre capable of being expressed efficiently in aspergillus niger and an encoding gene and application of the optimized high-temperature trehalase MS-Tre. The amino acid sequence of the optimized high-temperature trehalase MS-Tre is shown in SEQ ID NO.1, and the amino acid sequence of the encoding gene of the optimized high-temperaturetrehalase MS-Tre is shown in SEQ ID NO.2. Compared with a to-be-optimized encoding gene, the encoding gene of the optimized high-temperature trehalase MS-Tre can be expressed more efficiently in the aspergillus niger, and a recombinant aspergillus niger strain can produce trehalase efficiently. The trehalase has the advantages that the stability is good, disaccharide can also be hydrolyzed into monosaccharide at the high temperature, the cooling energy consumption after starch liquefaction is reduced, the utilization rate of starchy raw materials is increased, the utilization efficiency of biological energy sources is improved, and the production cost is reduced. The trehalase MS-Tre and the encoding gene thereof have certain practical significance and application value for achieving industrial production of the trehalase.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an optimized high-temperature trehalase MS-Tre capable of being highly expressed in Aspergillus niger, its coding gene and its application. Background technique [0002] Trehalose trehalose (α-D-glucopyranosyl-α-D-glucopyranoside) is a non-reducing disaccharide composed of two glucose molecules with 1,1-glycosidic bonds, widely present in bacteria, fungi , protozoa, plants, and mammals. [0003] Trehalase (trehalase) is a kind of glucoside hydrolase, which belongs to hydrolase in terms of enzyme classification, and its classification number is EC3.2.1.28. It can specifically hydrolyze one molecule of trehalose to generate two molecules of glucose, and it exists widely. In bacteria, molds, plants and animals. According to the different pH environment where trehalase exerts its enzymatic activity, trehalase can be divided into acid trehalase and neutral trehalase. The optimum p...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/80C12N1/15C12P7/06C12P13/14C12R1/685
CPCC12N9/2402C12N15/80C12P7/06C12P13/14C12Y302/01028Y02E50/10
Inventor 潘力董良波王斌
Owner SOUTH CHINA UNIV OF TECH
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