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A d-psicose 3-epimerase mutant and its application

A technology of epimerase and psicose, which is applied in the field of D-psicose 3-epimerase mutants, can solve rare problems and achieve good application prospects

Active Publication Date: 2021-05-04
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few sources of DPE enzymes for the synthesis of D-psicose, and enzymes that can meet high-temperature catalysis to prepare high-concentration D-psicose are even rarer.

Method used

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  • A d-psicose 3-epimerase mutant and its application
  • A d-psicose 3-epimerase mutant and its application
  • A d-psicose 3-epimerase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Screening and activity determination of novel DPE

[0026] 1. Source of enzyme and construction of recombinant bacteria

[0027] The new DPEs were obtained from the NCBI database, which were derived from Rhizobiales bacterium (GenBank No. WP_112533378.1), Martelella sp. (GenBank No. MAU19484.1), Novibacillus thermophilus (GenBank No. WP_077721022.1), and named RbDPE, MsDPE and NtDPE. According to the amino acid sequence, the codon was optimized according to the codon preference of Escherichia coli, and three selected nucleotide sequences were synthesized by the method of total synthesis through the conventional operation of genetic engineering, such as SEQ ID NO.2, SEQ ID NO.4 and shown in SEQ ID NO.6; the amino acid sequences encoding the enzyme are shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5 respectively. Add 6×his-tag tags at the end of the nucleic acid sequence, add restriction sites Xba I and Xho I at both ends, clone the gene into the Xba I and X...

Embodiment 2

[0044] Example 2: Construction and screening of NtDPE single point mutants

[0045] 1. Mutant construction

[0046] Design the mutation primers for site-directed mutation according to the parental sequence of NtDPE, use the rapid PCR technology, and use the recombinant vector pET28b / NtDPE as a template to introduce a single mutation at position 242. The primers are:

[0047] Forward primer GATGGTTATGTG NNN ATGGAACCG (the underline is the mutated base)

[0048] reverse primer CGGTTCCATCAC NNN ATAACCATC (the underline is the mutated base)

[0049] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μL.

[0050] The PCR amplification conditions were 95° C. for 3 min; (95° C. for 15 s, 55° C. for 15 s, 72° C. for 6.5 min) 30 cycles; 72° C. for 5 min.

[0051] 2. Transformation and expression of mutants

[0052] Take 5 μL of ...

Embodiment 3

[0062] Example 3: Construction and screening of NtDPE double-site mutants

[0063] According to the single mutant NtDPE-1 sequence constructed in Example 2, the mutation primers for site-directed mutation were designed, and the rapid PCR technology was used to use the recombinant vector pET28b / NtDPE-1 as a template to introduce a single mutation at position 105. The primers were:

[0064] Forward primer CATTGATCGTGTG NNN GGTACCGTGTAT (the underline is the mutated base)

[0065] reverse primer ATACACGGTACCA NNN ACACGATCAATG (the underline is the mutated base)

[0066] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μL.

[0067] The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 58°C for 15s, 72°C for 6.5min) for 30 cycles; 72°C for 5min.

[0068] The PCR product was transformed into E.coliBL21(DE3) comp...

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Abstract

The invention relates to a mutant of D-psicose 3-epimerase and its application in preparation of D-psicose by microbial catalyzed isomerization of D-fructose. The D-psicose 3-epimerase mutant is obtained by site-directed mutation of the indicated amino acids, and the point mutation site is one or more of the following: (1) 242nd, (2) 105th, (3) 210th, (4) 147th, (5) 184th. The beneficial effects of the present invention are mainly reflected in: the present invention provides a brand-new D-psicose 3-epimerase and its mutant, which has a high optimum reaction temperature of 85°C, solving the problem of existing Enzymes cannot produce D‑psicose at high temperature due to technical difficulties. Using the mutant to produce D-psicose, the product yield can reach up to 40.1%, which is better than the transformation effect of the original enzyme and other mutant enzymes, and has good industrial application prospects.

Description

[0001] (1) Technical field [0002] The invention relates to a mutant of D-psicose 3-epimerase and its application in preparation of D-psicose by catalyzing the isomerization of D-fructose by microorganisms. [0003] (2) Background technology [0004] D-psicose is the C-3 epimer of D-fructose, which belongs to the rare sugar family. Because of its high sweetness and low energy, it is an ideal sucrose substitute. D-psicose can inhibit the absorption of D-fructose and D-glucose by competing for the absorption and excretion of transport glycoproteins, thereby reducing body fat accumulation and reducing the risk of diabetes. Long-term and short-term dietary supplementation with D-psicose has a significant benefit on the cells of patients with type 2 diabetes and pancreatic β-cell dysfunction. D-psicose is rare in nature, and it is only found in some plants (such as wheat and Alpinia spp.), and its direct extraction leads to waste of resources and damage to the environment. With t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90C12P19/24C12P19/02
CPCC12N9/90C12P19/02C12P19/24C12Y501/03
Inventor 柳志强贾东旭孙晨奕彭晨金利群郑裕国陈德水廖承军程新平李勉毛宝兴
Owner ZHEJIANG UNIV OF TECH
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