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L-rhamnulose-1-phosphate aldolase and application thereof in catalytic synthesis of rare sugar D-psicose

A phosphate aldolase and amino acid technology, applied in the biological field, can solve the problems of difficult product separation and purification, low yield, difficult refining and purification, etc., and achieve the effects of high synthesis efficiency, cost saving and high enzyme activity

Active Publication Date: 2018-07-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether it is chemical synthesis or biotransformation to prepare D-psicose, there is a big problem, product separation and purification are very difficult
The D-psicose in the crude product is often mixed with other sugars, not only the yield is reduced, but also the physical and chemical properties of D-psicose and its mixed sugars are almost identical, so it is difficult to refine and purify

Method used

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  • L-rhamnulose-1-phosphate aldolase and application thereof in catalytic synthesis of rare sugar D-psicose
  • L-rhamnulose-1-phosphate aldolase and application thereof in catalytic synthesis of rare sugar D-psicose

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The RhaD coding gene was inserted into the expression vector pET-28a by double digestion with BamHI and SacI to obtain the recombinant expression plasmid pET-28a-rhaD. Using the plasmid pET-28a-rhaD as a template, saturation site-directed mutations were performed on the active region of L-rhamnosan-1-phosphate aldolase RhaD. See SEQ ID NO.1 for the gene sequence of wild-type L-rhamnosan-1-phosphate aldolase.

[0043] The primer sequences are:

[0044] Wild-type rhaD amplification primers:

[0045] Upstream primer rhaD-F GCGC GGATCC ATGCAAAACATTACTCAG (the underlined sequence is the BamHI restriction site);

[0046] downstream primer rhaD-R GCGC GAGCTC TTACAGCGCCAGCGCACTG (the underlined sequence is the SacI restriction site).

[0047] As well as carrying out combined site mutations of more than two RhaD active region sites, the wild-type RhaD recombinant expression plasmid pET-28a-rhaD was used as a blank control in this study. The sites of the saturation site-d...

Embodiment 2

[0063] RhaD aldolase wild type and mutant enzyme activity detection:

[0064] For the determination of enzyme activity, the 50 μL total volume reaction system is as follows: DHAP (0.2M, 5 μL, 0.02M); D-glyceraldehyde (0.5M, 2 μL, 0.02M), aldolase RhaD wild type or mutant (10mg / mL , 2.5 μL, 0.5 mg / mL) was supplemented with 50 mM Tris-HCl (pH 8.0) to a volume of 50 μL. The reaction was allowed to stand at 30°C overnight, and the pH was adjusted to 5 with 3M HCl, then 0.25 μL of acid phosphatase AP was added, and the reaction was left to stand at 30°C overnight. At the end of the reaction, 3M NaOH was used to adjust the pH to 7 to terminate the reaction, and then centrifuged at 15000 g for 10 min to collect the supernatant. HPLC detection, such as figure 1 and figure 2 shown.

[0065] The chromatographic conditions of the present invention are: a Hitachi differential detector (HITACHI RI), a chromatographic column Bio-Rad Aminex HPX-87H, a mobile phase of 5 mmol / L dilute sul...

Embodiment 3

[0070] Stereoselective analysis of RhaD aldolase:

[0071] This study found that the L-rhamnosugar-1-phosphate aldolase (RhaD) of the dihydroxyacetone phosphate (DHAP)-dependent aldolase family derived from Escherichia coli MG1655 catalyzed two reaction substrates: the first One is dihydroxyacetone phosphate (DHAP) and the second is aldehydes. When D-glyceraldehyde is used as the aldehyde acceptor, the stereoselectivity of the aldehyde acceptor is lost, and two rare sugars, D-psicose and D-sorbose, are produced, and the ratio of the two is close to 1:1. When the site-directed mutant strains N29I, N29K, P188C, P188T, T266A, and Y152A of MG1655 RhaD used DHAP and D-glyceraldehyde as substrates, D-psicose was mainly produced, while the RhaD site-directed mutant strain of Escherichia coli MG1655 strain P188F can completely synthesize D-psicose, and the specific experimental results are shown in Table 1.

[0072] In this study, site-directed saturation mutations were performed on...

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Abstract

The invention discloses L-rhamnulose-1-phosphate aldolase and application thereof in catalytic synthesis of rare sugar D-psicose. The gene sequence of the L-rhamnulose-1-phosphate aldolase is obtainedby performing saturation site-directed mutagenesis of the 29th site, the 188th site, the 266th site or the 152nd site respectively on the amino acid of the wild type L-rhamnulose-1-phosphate aldolasecoded by SEQ ID NO.1. The invention overcomes the defect that wild type RhaD which produces D-psicose and D-sorbose in a ratio of 1 to 1 does not have stereoselectivity, the catalytic reaction only produces the D-psicose or takes the D-psicose as a main component, and the mutated RhaD has higher enzyme activity. By preparing the D-psicose from RhaD of rite-directed mutagenesis by the preparationmethod disclosed by the invention, the synthetic efficiency is high, the process is simple, the cost is reduced, and the maximum catalytic efficiency of the D-psicose reaches 100%; and the method disclosed by the invention is utilized for realizing synthesis of a lot of D-psicose by catalysis of D-glyceraldehyde by industrialized RhaD.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to L-rhamnosan-1-phosphate aldolase and its application in catalyzing the synthesis of rare sugar D-psicose. Background technique [0002] D-psicose (D-psicose) is a rare monosaccharide, used as a zero-energy, non-digestible substitute for edible sugar. Since D-psicose has zero calories, it is difficult to be digested and absorbed by the intestines It has been evaluated as the most potential sucrose substitute by the US Food Navigation Network, and is now mostly cited as a new type of low-calorie sweetener. At the same time, D-psicose can also act as an inhibitor of liver lipase and intestinal α-glucosidase, reducing fat accumulation. Many studies have shown that D-psicose prevents testicular damage induced by bis(2-ethyl)ethyl phthalate by inhibiting the production of ROS in testicular tissue. In addition, D-psicose, in addition to its neuroprotective effect on 6-hydroxyd...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/60C12N15/70C12P19/02
CPCC12N9/80C12N15/70C12P19/02C12Y401/02019
Inventor 高晓冬李子杰汪马燕
Owner JIANGNAN UNIV
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