Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Mutant of epimerase and application of mutant

An epimerase and mutant technology, applied in the field of enzyme engineering, can solve the problems of poor temperature stability, unsuitable for industrial production, low fructose catalytic activity, etc.

Active Publication Date: 2020-10-20
TIANJIN UNIV OF SCI & TECH +1
View PDF13 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current DAE enzymes have low catalytic activity to fructose and poor temperature stability are not suitable for industrial production of D-psicose

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Mutant of epimerase and application of mutant
  • Mutant of epimerase and application of mutant
  • Mutant of epimerase and application of mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction of mutants of D-psicose-3-epimerase

[0030] With the wild-type D-psicose-3-epimerase gene (GenBank: BAW27657.1) derived from Arthrobacter globiformis, the nucleic acid sequence (coding The amino acid sequence shown in SEQ ID NO: 2). The nucleic acid sequence was connected to the pET28a(+) expression vector to obtain the wild-type recombinant vector AgDAE-pET-28a. Using the wild-type recombinant vector as a template, V105I-F (SEQ ID NO: 5) and V105I-R (SEQ ID NO: 6) as primers, the KOD-Plus mutant kit was used to obtain the nucleus of mutant V105I by PCR site-directed mutation. nucleotide sequence. Concrete reaction steps and conditions are as follows:

[0031] (1) PCR reaction, the reaction system and primer sequences are shown in Table 1 and Table 2 respectively. The underlined bases in Table 2 correspond to mutation points.

[0032] Table 1 PCR reaction system

[0033]

[0034]

[0035] Table 2 V105I-F and V105I-R primer sequences ...

Embodiment 2

[0045] Example 2: Expression of D-psicose-3-epimerase mutants

[0046] (1) The recombinant vectors AgDAE-pET-28a and V105I-pET-28a were transformed into Escherichia coli BL21(DE3) to obtain BL21 / AgDAE recombinant bacteria and BL21 / V105I recombinant bacteria respectively.

[0047] (2) The above two kinds of recombinant bacteria were respectively inoculated into 5 mL LB seed medium (containing 50 μg / mL kanamycin), and cultured at 220 r / min at 37° C. for 12 hours. Inoculate 100mL of LB fermentation medium (containing 50μg / mL kanamycin) with 1% inoculum amount, prepare bacteria concentration OD600=0.6-0.8, add IPTG, final solubility is 0.5mM, at 16°C, 160r / min Under induction for 24h.

[0048] (3) Collect the cells by centrifugation at 5000r / min for 15min, resuspend the cells with Lysis Buffer (20mM Tris-HCl, 500mMNaCl, 20mM imidazole, 2mM DTT pH=7.4), add lysozyme (final solubility is 200μg / mL) , IPTG (final solubility: 1mM) placed on ice for 30min. The cell suspension was pla...

Embodiment 3

[0050] Embodiment 3: HPLC identification of D-psicose product

[0051] Add 20 mM Tirs-HCl (pH=7.4), 150 mM NaCl, 1% (w / v) fructose and an appropriate amount of enzyme to the 200 μL reaction system, and react at 60° C. for 10 min. After the reaction solution was boiled for 5 minutes, it was centrifuged to remove the precipitate, and the reaction solution was diluted to a suitable concentration into a liquid phase vial, and the product was quantitatively analyzed by HPLC. The determination conditions were:

[0052] Chromatograph: Agilent1260;

[0053] Detector: Evaporative Light Scattering Detector (Alltech Chrom, ELSD6000)

[0054] Injection: Agilent autosampler; injection volume 20 μL;

[0055] Chromatographic column: Prevail Carbohydrate ES column-W (5μm, 4.6×250mm, Agela Technologies, China); column temperature 40°C;

[0056] Mobile phase: 85% acetonitrile; flow rate 1 mL / min.

[0057] The result is as figure 1 As shown, the retention time of D-fructose is 18min, the re...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a method for constructing and applying a mutant of a D-allulose-3-epimerase (DAE). The method for constructing the mutant of the enzyme comprises the steps as follows: a DAE gene from Arthrobacter globiformis is used as a parent, valine (105th amino acid residue) in an activity catalytic center of the enzyme is changed into isoleucine with homology comparison and site-directed mutagenesis technologies; and then, an expression vector is constructed, and the mutant of the enzyme is obtained with an E. Coli BL21 (DE3) as a heterologous expression host. The activity determination shows that the temperature stability of the obtained mutant enzyme is significantly increased. The t1 / 2 values at 60 DEG C and 70 DEG C reach 12.5 h and 0.5 h respectively, and the half-life at70 DEG C is 6 times longer than that of a wild type. The activity of the mutant enzyme is also improved. The mutant enzyme obtained by screening can more efficiently catalyze the isomerization of D-fructose into D-allulose at high temperature, provides broad prospects for the industrial production of the D-allulose and has important industrial applications value.

Description

Technical field: [0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a mutant of D-psicose-3-epimerase and its application. Background technique: [0002] D-psicose-3-epimerase (DAE) can catalyze the epimerization of various ketose C3 positions. It is a good catalyst for the production of rare sugars. It can produce D with fructose as a substrate with high additional value. - Allulose. [0003] D-psicose is an important member of the rare sugar family and a new type of low-energy sweetener. D-psicose belongs to ketohexose, is a six-carbon sugar, and is the C-3 epimer of D-fructose. Because of its high sweetness and low energy, it is considered to be an ideal sweetener and an effective substitute for sucrose. D-psicose can undergo Maillard reaction, which can improve the flavor, color, appearance, and taste of food. On the one hand, it can ensure the taste while reducing calories and reducing the worry of getting fat. On ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12P19/24C12P19/02C12R1/19
CPCC12N9/90C12Y501/03C12N15/70C12P19/24C12P19/02C12N2800/22
Inventor 秦慧民毛淑红路福平高鑫
Owner TIANJIN UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com