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

Phosphatase mutant and application thereof in preparation of fructose by catalyzing maltodextrin

A maltodextrin and mutant technology, applied in the field of enzyme engineering, can solve the problems of increased cost of separation and purification, accumulation of by-products, etc.

Pending Publication Date: 2021-11-05
ZHEJIANG UNIV OF TECH
View PDF8 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently known phosphatases have a broad substrate spectrum and can catalyze the dephosphorylation of various phosphate monosaccharides such as glucose-1-phosphate and glucose-6-phosphate, resulting in the accumulation of by-products in the fructose production process and increased downstream separation and purification costs

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
  • Phosphatase mutant and application thereof in preparation of fructose by catalyzing maltodextrin
  • Phosphatase mutant and application thereof in preparation of fructose by catalyzing maltodextrin
  • Phosphatase mutant and application thereof in preparation of fructose by catalyzing maltodextrin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Obtaining of Phosphatase Gene Sequence and Vector Construction

[0027] The polypeptide sequence (GenBank: WP_073073090.1) derived from T.atlanticus was obtained by mining NCBI and KEGG databases. The sequence has not been annotated, and its function is not yet clear. Under the premise of not changing the amino acid sequence of the polypeptide, the codons of the above genes are replaced with codons preferred by Escherichia coli (high frequency usage), and the nucleotide sequence of the phosphatase (M6PP) gene after codon optimization is shown as SEQ ID As shown in NO: 2, the amino acid sequence of the encoded protein is shown in SEQ ID NO: 1.

[0028] The gene sequence shown in SEQ ID NO: 2 was connected to the pET-28b(+) vector, located between the restriction sites BamHI and HindIII, and the recombinant plasmid was named pET-28b(+)-M6PP.

Embodiment 2

[0029] Example 2 Expression and purification of wild-type phosphatase M6PP

[0030] (1) Construction of recombinant bacteria: the recombinant plasmid pET-28b(+)-M6PP in Example 1 was transformed into E. coli BL21(DE3) competent cells to obtain wild-type recombinant bacteria.

[0031] (2) Recombinant bacteria culture: pick the wild-type recombinant bacteria from step (1) into 10mL LB liquid medium, and cultivate to OD at 37°C and 200rpm 600 0.6-0.8; transfer 10mL culture medium to 100mL LB medium, add kanamycin to a final concentration of 50mg / L, and cultivate to OD at 37°C and 180rpm 600 When the temperature is 0.6-0.8, lower the temperature to 28°C, add IPTG to a final concentration of 0.1mM, and induce expression for 12h. The above culture solution was centrifuged at 8000×g for 10 min, the supernatant was discarded, and the pellet was resuspended with HEPES buffer (pH 7.2) to obtain the bacterial suspension.

[0032] (3) Preparation of crude enzyme solution: Step (2) Bacte...

Embodiment 3

[0033] The preparation of embodiment 3 α-GP, PGM, PGI enzyme solution

[0034] Using the method of Example 2, the glucan phosphorylase α-GP (GenBank: AHD18925.1, whose amino acid sequence is shown in SEQ ID NO: 11) derived from Thermotoga maritima MSB8, source Glucose phosphomutase PGM (GenBank: BAD42440.1, amino acid sequence shown in SEQ ID NO: 12) from Thermococcus kodakarensis KOD1, glucose from Thermococcus kodakarensis KOD1 (Thermusthermophilus HB8) Phosphate isomerase PGI (GenBank: BAD70100.1, the amino acid sequence is shown in SEQ ID NO: 13), connected to the pET-28b (+) carrier, the connection sites are both BamHI and HindIII, to obtain the corresponding expression vector, and then Transformed into E.coli BL21(DE3) respectively, using the method of Example 2 to prepare α-GP enzyme solution, PGM enzyme solution, and PGI enzyme solution respectively, and using the BCA protein content detection kit to detect the target protein content, respectively α-GP Enzyme solution...

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 discloses a phosphatase mutant and application thereof in preparation of fructose by catalyzing maltodextrin with multiple enzymes in vitro. The mutant is obtained by performing single-point mutation on the 173th site, 175th site or 179th site of amino acid shown in SEQ ID NO.1. A reaction system is formed by taking enzyme liquid extracted by performing ultrasonic disruption on wet thalli obtained by fermentation culture of engineering bacteria containing a phosphatase mutant coding gene as a catalyst, taking maltodextrin as a substrate, combining glucan phosphorylase, glucose phosphate mutase and glucose phosphate isomerase, adding MgCl2 and phosphate and taking a buffer solution with the pH of 7.5-8.5 as a reaction medium to prepare fructose, wherein the conversion rate reaches 70% or above, and the ratio of fructose to byproduct glucose in the product is 11:1. The phosphatase mutant after substrate specificity regulation and control has important industrial application potential in green synthesis of fructose production by an in-vitro multi-enzyme synthesis system.

Description

(1) Technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to a phosphatase mutant derived from Thermosiphoatlanticus with improved specificity to fructose-6-phosphate and its application in in vitro multi-enzyme synthesis of fructose. (2) Background technology [0002] Fructose is widely used in the fields of food, medicine and chemical industry, and its efficient production has attracted widespread attention. Enzymatic hydrolysis of starch to prepare glucose, followed by isomerization by glucose isomerase is the main method for industrial production of fructose. However, controlled by reaction thermodynamics, this process can only obtain a mixture of glucose and fructose, and further complicated chromatographic separation is required to prepare high-purity fructose. The in vitro biosynthesis system will bring about major changes in the production modes of chemicals, energy and materials due to its advantages of diverse pat...

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
IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21C12N9/10C12N9/90C12N9/92C12P19/24C12P19/02C12R1/19
CPCC12N9/16C12N9/1051C12N9/90C12N9/92C12N15/70C12P19/24C12P19/02C12Y204/01097C12Y504/02002C12Y503/01009Y02A50/30
Inventor 郑仁朝汤晓玲王文豪郑裕国
Owner ZHEJIANG UNIV OF 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