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Trehalose synthase mutant and gene thereof

A technology of trehalose synthase and mutants, applied in genetic engineering, glycosyltransferase, plant gene improvement, etc., can solve the problem of low activity of trehalose synthase

Active Publication Date: 2017-02-22
NANJING UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is that the existing Mycobacterium smegmatis The activity of trehalose synthase is too low, in order to improve the activity of the enzyme, the present invention provides a method derived from mycobacterium smegmatis Mycobacterium smegmatis The trehalose synthase mutant, the amino acid sequence of the enzyme is shown in SEQ ID NO: 2, 4, 6, 8 or 10

Method used

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  • Trehalose synthase mutant and gene thereof

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Embodiment 1

[0018] Example 1 Preparation of Trehalose Synthase Mutants

[0019] according to mycobacterium smegmatis The gene sequence of the source trehalose synthase was designed and synthesized sequentially to introduce five mutants: R169A, R169A-D174A, R169A-D174A-D167A, R169A-D174A-D167A-T175A, R169A-D174A-D167A-T175A-E176A The primers were used to carry out site-directed mutagenesis on the trehalose synthase in five steps, and the DNA coding sequence was determined.

[0020] The Arg codon at position 169 was identified as an Ala codon.

[0021] On the basis of the previous step, change the 174th Asp codon to Ala codon.

[0022] On the basis of the previous step, change the 167th Asp codon to Ala codon.

[0023] On the basis of the previous step, the 175th Thr codon was changed to Ala codon.

[0024] On the basis of the previous step, change the 176th Glu codon to Ala codon.

[0025] The mutant gene was connected with an appropriate expression vector and introduced into Escheri...

Embodiment 2

[0045] Example 2 Expression and Purification of Trehalose Synthase

[0046]The constructed plasmid pET22b-TreS was transformed into E.coliBL21 (DE3) host for expression. A single clone was inoculated in 5 mL of LB liquid medium with an ampicillin concentration of 100 mg / L, and cultured in a shaker at 200 rpm at 37°C for 4 h. Transfer the cultured strains to 1 L of LB liquid medium with an ampicillin concentration of 100 mg / L, culture at 37°C until OD600=0.6, cool down to 30°C and add IPTG (final concentration 0.2 mM) to induce expression for 14 h, 6000 rpm The cells were collected by centrifugation for 10 min and stored in a -80°C refrigerator.

[0047] Take 10 g of frozen bacteria and resuspend in BufferA (25mM Tris, 300Mm Nacl, pH8.0). Use a high-pressure cell disruptor to break the cells, centrifuge at 16,000 rpm for 30 min after breaking, and collect the supernatant; secondly, Ni-NTA affinity chromatography: first use Buffer A to equilibrate the Ni-NTA column, and slowly...

Embodiment 3

[0048] Example 3 Expression of trehalose synthase mutants in Bacillus subtilis

[0049] (1) Construction of Bacillus subtilis expression vector

[0050] Through molecular biological operations such as double enzyme digestion, gel recovery, and T4 ligase connection, five mutants of trehalose synthase were cloned into the pMA5 vector to obtain five different pMA5-TreS, which were prepared for use in Bacillus subtilis. expression.

[0051] (2) Expression of recombinant strain WB600( pMA5-tres)

[0052] Introduce the constructed recombinant vector into the competent Bacillus subtilis B. subtilis WB600, activate the recombinant bacteria WB600, and carry out dilution coating, and then pick a ring of recombinant bacteria with a moderate size of a single colony to further activate in LB, 37°C, 250r / min for overnight incubation. The next day, microscopic examination was performed to see if there was bacterial infection, and then inoculated in LB medium at an inoculation volume of 3...

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Abstract

The invention discloses a trehalose synthase mutant and a gene thereof. The enzyme is formed through mutation of mycobacterium smegmatis trehalose synthase. Particularly, amino acids at the 167th site, the 169th site, the 174th site, the 175th site and the 176th site of the emzyme are mutated into alanine, wherein the alanine mutated from the amino acids at the 169th site is freely selected to be combined with the alanine mutated from the amino acids at the 167th site, the 174th site, the 175th site and the 176th site. After mutation, compared with a wild type enzyme, enzyme activity is improved by 2.56 times at most.

Description

technical field [0001] The present invention belongs to the field of biotechnology, and specifically relates to the field of enzyme engineering, a mutant of trehalose synthase, a gene, a recombinant vector, a transgenic cell and the application of the composition in catalyzing the synthesis of trehalose. Background technique [0002] Trehalose (Trehalose), the molecular formula is C 12 h 22 o 11 • 2H 2 O is a disaccharide formed by combining two glucose molecules with α,α-1,1 glycosidic bonds; trehalose has no hemiacetal hydroxyl group, is a non-reducing sugar, and has stable chemical properties. Because trehalose can form a unique protective film on the cell surface under harsh environmental conditions such as high temperature, high cold, high osmotic pressure, and dehydration, effectively protecting protein molecules from denaturation and inactivation, it is known as the "sugar of life". This characteristic makes trehalose not only can be used as an active protective a...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12P19/12
CPCC12N9/1051C12P19/12C12Y204/01245
Inventor 黄和王东生江凌周家海徐晴
Owner NANJING UNIV OF TECH
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