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Cyclodextrin glycosyl transferase for improving substrate specificity of soluble starch

A technology of glycosyltransferase and substrate specificity, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of substrate specificity (low conversion rate, etc.), and achieve the effect of improving substrate specificity

Active Publication Date: 2014-02-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low substrate specificity (conversion rate) of cyclodextrin glucosyltransferase (CGTase) to soluble starch at present, improving its substrate specificity to soluble starch through molecular modification of CGTase technology will promote and The rapid development of related industries of L-AA glycosyl derivatives

Method used

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  • Cyclodextrin glycosyl transferase for improving substrate specificity of soluble starch
  • Cyclodextrin glycosyl transferase for improving substrate specificity of soluble starch
  • Cyclodextrin glycosyl transferase for improving substrate specificity of soluble starch

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Example 1 Short peptide fusion cyclodextrin glucosyltransferase with improved substrate specificity

[0019] The short peptide fusion cyclodextrin glycosyltransferase of the present invention is fused to the N-terminus of the dextrin glycosyltransferase published in GenBank AF047363.1 with six different self-organized amphiphilic short peptides (SAP) (see Table 1). , six fusion enzymes were constructed: SAP1-CGTase to SAP6-CGTase. The above six fusion enzymes can be constructed by chemical total synthesis or fusion PCR. The short peptide fusion cyclodextrin glycosyltransferase was derived from Paenibacillus macerans (P.macerans) JFB05-01 (CCTCC NO: M208063).

[0020] Table 1 Sequences of six different self-organizing amphipathic short peptides

[0021]

Embodiment 2

[0022] Example 2 Preparation method of short peptide fusion cyclodextrin glucosyltransferase with improved substrate specificity

[0023] 1) Construction of short peptide fusion cyclodextrin glucosyltransferase

[0024] In this example, the total chemical synthesis method is used as an example to illustrate, but the protection of the invention is not limited to the method of obtaining the fusion enzyme only by the chemical total synthesis method. The preparation method of fusion enzyme SAP1-CGTase to SAP6-CGTase is as follows:

[0025] like figure 1 As shown, the above 6 short peptides were respectively linked to the N-terminus of CGTase by PT linker, and a histidine tag was inserted into the N-terminus of the short peptides to facilitate purification. Then, the constructed short peptide fusion CGTase was connected to the pET-22b(+) expression plasmid through NdeI and XhoI restriction sites. The constructed recombinant plasmid pET-22b(+) / SAP-CGTase was transformed into the ...

Embodiment 3

[0029] Example 3 Enzyme activity analysis and synthesis and detection of AA-2G.

[0030] 1) Enzyme activity assay method:

[0031]Method for measuring α-cyclization activity by methyl orange method: Take 0.1mL of appropriately diluted enzyme solution, add it to 0.9mL of 3% soluble starch solution prepared in advance with 50mM phosphate buffer (pH6.5), at 40°C After reacting for 10 min, add 1.0 mL of 1.0 M hydrochloric acid to stop the reaction, then add 1.0 mL of 0.1 mM methyl orange prepared with 50 mM phosphate buffer, incubate at 16°C for 20 min, and measure the absorbance at 505 nm. One enzyme activity unit defines the amount of enzyme required to produce 1 μmol α-cyclodextrin per minute under the conditions.

[0032] Determination of starch hydrolysis activity: Add appropriate amount of enzyme solution to 50mM phosphate buffer (pH6.5) containing 1% soluble starch, react at 50°C for 10min, and then use DNS method to measure the concentration of reducing sugar. One unit o...

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Abstract

The invention discloses cyclodextrin glycosyl transferase (CGTase) for improving substrate specificity of soluble starch, and belongs to the field of genetic engineering and enzyme engineering. According to the invention, the CGTase from P.macerans strain JFB05-01(CCTCC (China Center For Type Culture Collection) NO:M208063) is taken as initial CGTase, and two self-organizing amphiphilic short peptides (SAP) are respectively fused at the N end of the initial CGTase to create two fusion enzymes-SAP1-CGTase and SAP2-CGTase. Compared with the initial CGTase, when the soluble starch is taken as a glycosyl donor, the yields of synthetic AA-2G (2-O-alpha-D-pyran glucosyl-L-ascorbic acid) are increased by about 1.33 times and 2.36 times respectively. Therefore, the fusion enzymes are more favorable for utilizing the soluble starch as the glycosyl donor for producing the AA-2G in comparison with the initial CGTase.

Description

technical field [0001] The invention relates to a cyclodextrin glycosyltransferase with improved soluble starch substrate specificity, in particular to a short peptide fusion cyclodextrin glycosyltransferase with improved soluble starch substrate specificity and a preparation method, belonging to genetic engineering and the field of enzyme engineering. Background technique [0002] L-ascorbic acid (L-AA, vitamin C) is a water-soluble vitamin that participates in many physiological activities in the body, plays an important role in maintaining and promoting human health, and is an essential nutrient that the human body cannot synthesize by itself. However, L-AA is extremely unstable, and is easily oxidized to dehydroascorbic acid in the air, destroying the conjugated system in the molecule and causing an irreversible cleavage reaction, especially in the presence of air, light, heat and metal ions, the reaction is more rapid , so that the physiological activity of L-AA weaken...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/70C12N1/21C12P19/60C12R1/19
CPCC12N9/1074C12P19/60C12Y204/01019
Inventor 陈坚堵国成刘龙李江华韩瑞枝
Owner JIANGNAN UNIV
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