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An optimized high-temperature trehalase ms-tre that can be highly expressed in Aspergillus niger and its encoding gene and application

A high-efficiency expression technology of trehalase, which is applied in the field of genetic engineering, can solve the problems of low expression level of recombinant trehalase, achieve good stability, improve utilization rate, and increase production

Active Publication Date: 2021-05-14
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the current literature reports, the research on trehalase mainly focuses on the determination of enzymatic properties, and the expression level of recombinant trehalase is very low, and there is no special literature report on the high-efficiency expression of trehalase.

Method used

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  • An optimized high-temperature trehalase ms-tre that can be highly expressed in Aspergillus niger and its encoding gene and application
  • An optimized high-temperature trehalase ms-tre that can be highly expressed in Aspergillus niger and its encoding gene and application
  • An optimized high-temperature trehalase ms-tre that can be highly expressed in Aspergillus niger and its encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the construction of universal 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 ...

Embodiment 2

[0048] Embodiment 2, containing 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 universal e...

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 an optimized high-temperature trehalase MS-Tre capable of high-efficiency expression in Aspergillus niger, its coding gene and application. The amino acid sequence of the optimized high-temperature trehalase MS-Tre is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2. Compared with the coding gene before optimization, the optimized high-temperature trehalase MS-Tre coding gene of the present invention can be more efficiently expressed in Aspergillus niger, and the recombinant Aspergillus niger strain can produce high trehalase. The trehalase provided by the invention has good stability, can also hydrolyze disaccharides into monosaccharides under high temperature conditions, reduce cooling energy consumption after starch liquefaction and improve the utilization rate of starchy raw materials, improve the utilization efficiency of bioenergy, reduce Production cost, the trehalase MS-Tre provided by the present invention and its coding gene have certain practical significance and application value for realizing the industrialized production of 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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Patent Type & Authority Patents(China)
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