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Optimized high-temperature acid trehalinase TreMT1 and coding gene and application thereof

A technology of trehalase and coding gene, 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 yield

Active Publication Date: 2019-04-19
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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  • Optimized high-temperature acid trehalinase TreMT1 and coding gene and application thereof
  • Optimized high-temperature acid trehalinase TreMT1 and coding gene and application thereof
  • Optimized high-temperature acid trehalinase TreMT1 and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the construction of universal expression vector

[0048] 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) and primer Ttef-rev (as shown in SEQ ID NO. 9) to amplify the tef terminator (as shown in SEQ ID NO.3), use primer amyA-fw (as shown in SEQ ID NO.12) and primer amyA-rev (as shown in SEQ ID NO.13) to amplify Increase the last 1000bp of the gene encoding neutral amylase amyA (shown in SEQ ID NO.5), use the primer pyrG-fw (shown in SEQ ID NO.10) and primer pyrG-rev (shown in Aspergillus nidulans genome) as a template As shown in SEQ ID NO.11) to amplify the pyrG marker (as shown in SEQ ID NO.4), the obtained PCR products are respectively named as Ttef, amyA, pyrG (respectively as SEQ ID NO.3, such as SEQ ID NO.5 , shown in SEQ ID NO.4), using the NEBuilder HiFiDNA Assembly Cloning Kit kit, the four fragments of Ttef, pyrG, am...

Embodiment 2

[0049] Embodiment 2, containing target gene expression vector construction

[0050] Using the optimized nucleotide sequence of the synthetic TreMT1 gene and the Aspergillus niger genome as templates, amplified with primers Tre-fw (shown in SEQ ID NO.16) and primers Tre-rev (shown in SEQ ID NO.17) Obtain the TreMT1 gene sequence (as shown in SEQ ID NO.2), amplify with primer PamyA-fw (as shown in SEQ ID NO.14) and primer PamyA-rev (as shown in SEQ ID NO.15) to obtain Aspergillus niger Amylase promoter PamyA sequence (as shown in SEQ ID NO.7). The two PCR-amplified PCR fragments were connected with the linearized universal expression vector by fusion PCR. The ligation product was transformed into Escherichia coli Match1T1 (purchased from takara company) competent, after 12 hours of culture at 37°C, the transformants were picked and placed in liquid LB+Amp (final concentration 100 μg / ml) medium, and cultured at 37°C with a shaker speed of 200rpm After 12 hours, electrophoresis ...

Embodiment 3

[0051] Embodiment 3, transformation of expression vector pMD20-PamyA-TreMT1-Ttef-pyrG-amyA plasmid in Aspergillus niger

[0052] Prepared according to the procedure provided in (Gomi K, Iimura Y, Hara S. Integrative transformation of Aspergillus oryzae 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 vector pMD20-PamyA-TreMT1-Ttef-pyrG-amyA plasmid obtained above was transformed into the protoplasts, and the hyperosmotic CD medium (comprising 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 O, 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 relates to optimized high-temperature acid trehalinase TreMT1 and a coding gene and application thereof. The amino acid sequence of the optimized high-temperature acid trehalinase TreMT1is shown as SEQ ID NO.1; the nucleotide sequence of the encoding gene of the optimized high-temperature acid trehalinase TreMT1 is shown as SEQ ID NO.2. Targeted high-temperature acid trehalinase TreMT1 can be generated at high yield through a constructed high-temperature acid trehalinase TreMT1 strain. The trehalase stability is high, disaccharide can be hydrolyzed into monosaccharide under thehigh-temperature acidic condition, the cooling energy consumption of starch liquefacation is reduced, the utilization rate of starchy raw materials is increased, resource waste is reduced, the utilization efficiency of biological energy sources is increased, the production cost is reduced, and certain practical significance and application value are achieved on efficient expression and even industrial production of trehalase.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an optimized high-temperature acidic trehalase TreMT1 and its coding gene and application. Background technique [0002] Trehalose (α-D-glucopyranosyl-α-D-glucopyranoside) is a non-reducing disaccharide composed of two glucose molecules with a 1,1-glycosidic bond, widely present in bacteria, fungi , protozoa, plants, and mammals. [0003] Trehalase (trehalase) is a kind of glucoside hydrolase, which belongs to hydrolase in enzyme classification, and its classification number is EC3.2.1.28. It can specifically hydrolyze one molecule of trehalose into 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 pH of acid trehalase is about 4.5-5.5, and it is generally secreted to the ...

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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