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Method for preparing sialyllactose

A technology of sialyllactose and sialic acid, which is applied in the direction of fermentation, etc., can solve the problems of long fermentation period of sialyllactose, failure to meet the needs of industrialization, waste of fermentation and time cost, etc., to save fermentation and time cost, and transform system components Simple, short-cycle effects

Inactive Publication Date: 2019-11-01
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI +1
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Problems solved by technology

However, the main disadvantage of using the whole-cell one-pot method to catalyze the preparation of sialyllactose is that the enzymes with different functions are expressed in different engineering strains, which consumes a lot of fermentation and time costs.
The preparation of sialyllactose by fermentation has a long fermentation period and low yield, which cannot meet the needs of industrialization.

Method used

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  • Method for preparing sialyllactose
  • Method for preparing sialyllactose
  • Method for preparing sialyllactose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The method for preparing sialyllactose provided in this embodiment comprises the following steps:

[0067] 1 Construction of a multi-enzyme co-expression system (1) Construct the CMP-sialic acid synthetase (CMP-NeuAc synthetase) gene (SEQ ID NO.1) on the pACYCDuet-1 vector MCS1 using the restriction endonuclease sites BamHI and HindIII (See Figure 10 ), forming the recombinant plasmid pACYCDuet-CMPNs.

[0068] (2) Design primers with NdeI and AvrII restriction sites so that the 3' end of the α-2,3-sialyltransferase gene (SEQ ID NO.2) is amplified with a 6×His histidine tag. The amplified product and the recombinant plasmid pACYCDuet-CMPNs were digested with endonucleases NdeI and AvrII respectively, and the α-2,3-sialyltransferase gene containing histidine tag was constructed into the plasmid under the action of T4 ligase On pACYCDuet-CMPNs MCS2, the recombinant plasmid pACYCDuet-CMPNs-ST was formed.

[0069] (3) Transfer the recombinant plasmid into BL21(DE3) compe...

Embodiment 2

[0085]After the reaction in Example 1 is over, use a magnet to absorb the magnetic nanoparticles and discard the reaction solution. After washing the immobilized enzyme twice with a buffer, continue to put it into the reaction kettle. The reaction raw materials are put in the same way as in Example 1. Phase analysis detected that 44.5g / L sialyllactose was obtained, and the conversion rate was 88%, which indicated that the co-immobilized enzyme realized the reuse of biocatalyst.

Embodiment 3

[0087] This example provides a method for preparing 6'-sialyllactose, which is basically the same as in Example 1, except that 6'-sialyllactose is prepared using α-2,6-sialyltransferase instead of α-2,3 - sialyltransferase. That is, the α-2,6-sialyltransferase gene (SEQ ID NO.5) is used to replace the α-2,3-sialyltransferase gene in Example 1, and other methods for preparing immobilized enzymes according to Example 1 And transformation conditions, after transformation 12, 6'-sialyllactose content is 46.6g / L in the system, and transformation rate is 92% ( Figure 13 ).

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Abstract

The invention discloses a method for preparing sialyllactose, and relates to the technical field of preparation of sialyllactose. The method for preparing sialyllactose disclosed by the invention comprises the steps of adding kinase to a reaction system containing a substrate, and performing an enzyme catalysis reaction to prepare the sialyllactose, wherein the kinase comprises CMP kinase, polyphosphate sialic acid synthase and sialytransferase; and the substrate comprises CMP, polyphosphate, sialic acid and lactose. The method adopts various kinase namely the CMP kinase, the polyphosphate kinase, the CMP-sialic acid synthetase and sialytransferase to catalyze the corresponding substrate to prepare the sialyllactose. The method has the characteristics of being high in yield, low in cost, short in cycle and the like.

Description

technical field [0001] The invention relates to the technical field of sialyllactose preparation, in particular to a method for preparing sialyllactose. Background technique [0002] Human milk oligosaccharides (HMOs, human milk oligosaccharides) are the third largest solid component in human breast milk after lactose and fat. The oligosaccharide content of breast milk of other animals such as goat milk and goat milk is far inferior. It has been confirmed that HMOs have the effect of prebiotics, which can selectively stimulate the growth of beneficial bacteria in the intestine, indirectly inhibit the growth of harmful bacteria, and maintain the balance of intestinal microecology, thereby minimizing the occurrence of digestive tract diseases and improving the health of the host baby. This step-by-step, three-dimensional and all-round protection and cultivation of sexual function provides precious time and a good environment for the development and maturity of the digestive s...

Claims

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Application Information

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IPC IPC(8): C12P19/26C12P19/14C12P19/18
CPCC12P19/14C12P19/18C12P19/26
Inventor 李忠奎陈祥松李翔宇吴金勇倪志坚袁丽霞王纪姚建铭
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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