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Maltooligosyl trehalose synthase as well as expression gene and application thereof

A technology based on trehalose synthase and maltooligosaccharides, which is applied in the field of genetic engineering, can solve the problems of low enzyme activity and achieve high yield and broad application prospects

Active Publication Date: 2017-02-22
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

MTSase and MTHase ​​from different sources have different enzymatic properties. Arthrobactor sp.StrainQ36 has an optimum reaction temperature of 40°C, pH 6.5, and a conversion rate of up to 80%. Sulfolobus acidocaldarius ATCC33909 has a higher optimum reaction temperature of 75°C and a higher Low pH5.0, but the enzyme activities are very low

Method used

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  • Maltooligosyl trehalose synthase as well as expression gene and application thereof
  • Maltooligosyl trehalose synthase as well as expression gene and application thereof
  • Maltooligosyl trehalose synthase as well as expression gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Extraction of total genome DNA of Arthrobacter oxydans.

[0023] Inoculate the Arthrobacter oxydans strain stored at -80°C into LB liquid medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L) for activation and culture for 24 hours, and then inoculate at 1% The amount was inoculated into fresh LB medium and cultured for 24 hours, and 10 mL of bacterial liquid was taken to extract the total DNA of the bacterial genome according to the method provided by Shanghai Sangong Bacterial Genome Extraction Kit.

Embodiment 2

[0024] Embodiment 2: the acquisition of MTSase, MTHase ​​gene

[0025] The acquisition of MTSase and MTHase ​​genes is as follows: figure 1 As shown, degenerate primers F1 and R1, F2 and R2 were designed according to the homologous alignment, respectively.

[0026] F1: GGTTCCGSGTGSGASGTGAAGAACTGCCA

[0027] R1: TTGGCCATSACCATKCCSGAGGTCTGCTGGAA

[0028] F2: ATCTACGARCTSCACSTGGGCACCTT

[0029] R2: GGTTCCGSGTGSGASGTGAAGAACTGCCA

[0030] Using the total genomic DNA prepared in Example 1 as a template, using F1 (upstream primer), R1 (downstream primer) and F2 (upstream primer), R2 (downstream primer) as primers respectively, using the Ex TaqTM kit from TaKaRa Company according to Product instructions for PCR amplification;

[0031] The PCR amplification system is as follows:

[0032] Genomic DNA 2 μL, upstream primer 2 μL, downstream primer 2 μL, Taq enzyme 25 μL, ddH 2 O 19 μL.

[0033] PCR conditions were: denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, a...

Embodiment 3

[0057] Embodiment 3: Construction and transformation of recombinant plasmid

[0058] Ligate the purified target gene MTSase and MTHase ​​products with the linearized vector pET-22b(+) / (Nde I, Xho I) respectively, the ligation system is shown in Table 1, and transform the ligated products into Escherichia coli BL21 competent In normal cells, culture at 200r / min at 37°C for 1 hour, then spread the transformed cells on LB plates containing 100 μg / mL ampicillin, culture overnight at 37°C, pick single colonies and screen for positive clones by colony PCR, Sent to Shanghai Sangon for sequencing, the results showed that the inserts contained an open reading frame (ORF) with a length of 2328bp (as shown in SEQ ID NO: 1) and 1770bp (as shown in SEQ ID NO: 3), respectively, encoding by 775 and a protein encoded by 589 amino acids.

[0059] Table 1: Connection system

[0060]

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Abstract

The invention relates to maltooligosyl trehalose synthase as well as an expression gene and an application thereof. A nucleotide sequence of the expression gene MTSase of the maltooligosyl trehalose synthase is shown as SEQ ID NO.1; and an amino acid sequence of maltooligosyl trehalose synthase MTSase is shown as SEQ ID NO.2. The invention also relates to the application of the maltooligosyl trehalose synthase MTSase in preparing and producing trehalose. The maltooligosyl trehalose synthase expression gene MTSase, which is extracted from arthrobacter oxydans, is discovered by the invention for the first time; the maltooligosyl trehalose synthase MTSase, which is expressed by virtue of the gene, is significantly better than existing known maltooligosyl trehalose synthase MTSase; and the enzyme (the maltooligosyl trehalose synthase), when acts together with MTHase derived from the arthrobacter oxydans in producing the trehalose, is high in yield; therefore, the enzyme has a broad application prospect.

Description

technical field [0001] The invention relates to maltooligosaccharide-based trehalose synthetase and its expression gene and application, belonging to the technical field of genetic engineering. Background technique [0002] Trehalose is a safe non-reducing disaccharide formed by combining two molecules of glucose through α-1,1 glycosidic bonds, and widely exists in plants, animals and microorganisms. Trehalose has a wide range of biological significance. It is a good stabilizer in medicine and can be used to protect hormones, vitamins, antibiotics, biological agents, enzymes, antiserum, vaccines and other easily inactivated substances; it can be used in cosmetics Maintaining cell viability, with moisturizing and anti-radiation effects; in agriculture, it can maintain the normal growth of crops under high temperature, high drought, and high salt conditions; it is used as a natural additive to improve quality and flavor in food, and it also has freshness preservation Therefor...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N9/90C12N15/70C12N1/21C12P19/12
CPCC12N9/90C12P19/12C12Y504/99015
Inventor 王瑞明薛乐平王腾飞汪俊卿
Owner QILU UNIV OF TECH
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