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Cubilose acid aldolase mutant as well as coding gene and application thereof

An acid aldolase and mutant technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of high production cost, low conversion rate, low catalytic activity of bird's nest acid aldolase, etc., and reduce production costs. , the effect of improving market competitiveness

Active Publication Date: 2014-06-25
BONTAC BIO ENG SHENZHEN
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0010] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a mutant of sialic acid aldolase and its coding gene and application, aiming to solve the problem of low catalytic activity of sialic acid aldolase in the current enzymatic production of sialic acid, and the conversion The problem of low production rate and high production cost

Method used

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  • Cubilose acid aldolase mutant as well as coding gene and application thereof
  • Cubilose acid aldolase mutant as well as coding gene and application thereof
  • Cubilose acid aldolase mutant as well as coding gene and application thereof

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preparation example Construction

[0027] The general process of the preparation of the bird's nest acid aldolase mutant is as follows: first construct the carrier plasmid containing the parent bird's nest acid aldolase gene, then set the site for site-directed mutation and the amino acid species after mutation, and then synthesize appropriate primers , using the carrier plasmid containing the parent bird's nest acid aldolase gene as a template, PCR amplifies the DNA fragment, assembles the amplified DNA fragment and PCR amplifies the full-length mutant gene. Then the full-length mutated gene is cloned into an appropriate vector and transformed into an appropriate host cell, and a positive clone with bird's nest acid aldolase activity is selected through culturing. Finally, plasmid DNA is extracted from the positive clones, and DNA sequence analysis is performed to determine the introduced mutation. After confirming that the target fragment is inserted into the vector, it can be screened by LB medium to obtain b...

Embodiment 1

[0036] Amplification and cloning of parent bird's nest acid aldolase gene:

[0037] Primers EC-F and EC-R were designed according to the gene sequence of GenBank (GenBank NC_012971). The gene encoding bird's nest acid aldolase was amplified from Escherichia coli BL21(DE3) with primer pair EC-F and EC-R.

[0038] Amplification conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer EC-F, 400 nM primer EC-R, 1.0 U Pfu DNA polymerase (Promega, USA), Pick a little Escherichia coli BL21 (DE3) cells with an inoculation loop, and adjust the reaction volume to 50 ml with sterile water.

[0039] The PCR amplification reaction program was: 95°C for 3 minutes, 40 cycles: 95°C for 50 seconds, 50°C for 30 seconds and 72°C for 1 minute, and finally 72°C for 10 minutes. The amplified product was digested with restriction endonucleases NdeI and AscI and then ligated with ve...

Embodiment 2

[0043] Site-directed mutagenesis of site 25 of bird's nest acid aldolase

[0044] In order to obtain the mutant A25E by mutating Ala(A) at the 25th position in the parental amino acid sequence to Glu(E), the plasmid pRSET-EC in Example 1 was used as a template to design primer pairs 25EF and 25ER (as shown in Table 1 Show).

[0045] The primer pair EC-F and 25ER was used to amplify the F-ER fragment, and the primer pair 25EF and EC-R was used to amplify the EF-R fragment. The specific sequences of primers EC-F and EC-R are shown in Table 1.

[0046] The above amplification reaction conditions are: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer EC-F and 400 nM primer 25ER, or 400 nM primer 25EF and 400 nM primer EC-R, 1.5 U Pfu DNA polymerase (Promega, USA), 20 ng pRSET-EC, the reaction volume was adjusted to 50 microliters with sterile water.

[0047] The PCR ampli...

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Abstract

The invention discloses a cubilose acid aldolase mutant as well as a coding gene and application thereof. The cubilose acid aldolase mutant is obtained from sequence 2 in a sequence list through point mutation, and the point mutation is at least one mutation at the 25th site and 275th site of the sequence. Through site directed mutation on the cubilose acid aldolase, cubilose acid aldolase mutant with high catalytic activity is finally obtained. Besides, the mutant uses N-acetylmannosamine, sodium pyruvate and trinosin (ATP) as the substrate and has the cubilose acid aldolase catalytic activity which is at least 50% higher than that of the parent. Thus, the cubilose acid aldolase mutant can be used for producing cubilose acid (sialic acid), the production cost is lowered, and the market competitiveness of the corresponding product is enhanced.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a mutant of bird's nest acid aldolase and its coding gene and application. Background technique [0002] Sialic acid (English name: Sialic acid) is also known as sialic acid or N-acetylneuraminic acid. Its structure is shown in the figure below. It is a kind of natural sugar compound widely present in biological systems. , plays an indispensable role in the process of biochemical reactions. The structure of N-acetylneuraminic acid (silicate acid) is as figure 1 shown. [0003] Bird's nest acid is the main biologically active ingredient in bird's nest, a traditional rare food in China. It is also one of the important components provided by colostrum for the early brain development and immune system improvement of infants. Due to the high content of bird's nest acid in bird's nest, it is reported that the detection of bird's nest acid is used for quality contr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12P19/26
CPCC12N9/88C12P19/26C12Y401/03003
Inventor 傅荣昭
Owner BONTAC BIO ENG SHENZHEN
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