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

Mutant xylose isomerase and its gene

A technology of xylose isomerase and xylose, which is applied in the field of enzyme genetic engineering and enzyme biochemical engineering, can solve the problems of less research and achieve the effect of improving specificity

Inactive Publication Date: 2006-12-27
NANJING UNIV OF TECH
View PDF1 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are a large number of literature reports, the catalytic activity of xylose isomerase to the substrate D-glucose in bacterial species such as Thermoanaerobacterium thermosulfurigenes, Thermotoga neapolitana, and Thermotoga neapolitana has been improved by directed evolution or site-directed mutation methods, but for improving xylose isomerization Enzyme specificity for substrate D-xylose is less studied

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 xylose isomerase and its gene
  • Mutant xylose isomerase and its gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Cloning of Thermus thermophilus xylA gene and mutation of Asn91 site Thermus thermophilus HB8 was purchased from American Type Culture Collection (ATCC) ATCC27634.

[0025] Culture medium preparation: Yeast powder 4g / L, peptone 8g / L, NaCl 2.0g / L, pH adjusted to 7.0, autoclaved at 121°C for 15min.

[0026] The Thermus thermophilus HB8 was inoculated in the above medium, placed on a shaker at 75° C., 200 rpm, and cultivated for 12-16 hours. 8000rpm, 4°C, centrifuge for 15min, collect the bacteria.

[0027] The genome of Thermus Thermophilus was extracted using a genome extraction kit (Shanghai Huashun Bioengineering Co., Ltd.). Using the genome as a template, use the following PCR primers (synthesized by Shanghai Boya Company):

[0028] Forward primer 5'-GGAATTCCATATGTACGAGCCCAAACCGGAGCACAGG-3' (SEQ ID NO.3)

[0029] Reverse primer 5'-AAGGAAAAAAGCGGCCGCTCACCCCCGCACCCCCAG-3' (SEQ ID NO.4)

[0030] PCR amplification of xylose isomerase gene xylA. The PCR cyc...

Embodiment 2

[0033] Example 2: Construction, screening and identification of expression recombinant plasmids

[0034] Forward primer 5'-CCATATGTACGAACCAAAACCGGAACATCGCTTTACCTTTT-3'(SEQ ID NO.5)

[0035] Reverse primer 5'-AAGGAAAAAAGCGGCCGCTCAACCACGCACACCCAGGAG-3' (SEQ ID NO.6)

[0036] The mutant gene xylA was amplified by PCR from the plasmid containing the mutant gene in Example 1, the mutant gene xylA and the carrier pET-22b(+) were cut with restriction endonucleases NdeI and NotI, and the above-mentioned double enzymes were digested after recovery. The mutant gene xylA and the pET-22b(+) carrier were connected, and the connection solution was transformed into E. coli DH5α competent cells by conventional genetic engineering technology, and the plasmid was extracted. The above-mentioned primers were used to amplify and identify the mutant gene xylA to be connected to the expression vector pET-22b(+).

Embodiment 3

[0037] Example 3: Expression of Thermus thermophilus xylose isomerase in Escherichia coli Rosetta

[0038] Transform the pET-22b(+)-xylA recombinant plasmid containing the mutant gene xylA into the commercialized expression host Escherichia coli Rosetta (DE3), and pick a single colony to contain 75 μg / mL ampicillin and 34 μg / ml chloramphenicol LB culture medium, cultured overnight at 37°C with shaking, then inoculated into LB culture medium containing 75 μg / mL ampicillin and 34 μg / mL chloramphenicol at 2% (v / v) inoculum size, and cultivated to OD at 37°C600 At about 0.6, add IPTG to a final concentration of 0.9 mM, induce expression for 7 hours, take a certain volume of bacterial liquid at 8000 rpm, and centrifuge at 4°C for 15 minutes to collect the bacterial cells.

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 discontinuous xylo-pfan isomerase and its genes. The discontinuous xylo-pfan isomerase selects form Thermus thermophilus and its amino acid sequence is indicated by SEQ ID NO.2. The said discontinuous xylo-pfan isomerase has improved specificity to the substrate D-wood sugar without changing original heat endurance. The discontinuous xylo-pfan isomerase can be used in miscible liquids including wood sugar and amylaceum to specially detect the wood sugar content and can be used in constructing recombination bacteria with wood sugar.

Description

technical field [0001] The invention belongs to the fields of genetic engineering of enzymes and enzyme biochemical engineering. Specifically, the present invention relates to a mutant xylose isomerase that can isomerize D-xylose to D-xylulose but cannot catalyze D-glucose to generate D -fructose. The present invention also relates to the gene encoding the mutant xylose isomerase. Background technique [0002] Xylose isomerase (Xylose Isomerase, XI) (EC 5.3.1.5), also known as glucose isomerase, can catalyze the isomerization reaction of D-xylose to D-xylulose and D-glucose to D-fructose, It plays an important role in the process of sugar metabolism in microorganisms and is a key enzyme for the industrial production of high fructose syrup. [0003] According to sequence homology, xylose isomerase can be divided into two categories, the first category consists of about 390 amino acid residues, and the second category consists of about 440 amino acid residues. Xylose isome...

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): C12N9/90C12N15/61C12N15/63C12N1/21C12N1/19C12Q1/533
Inventor 严明许伟丁莉许琳欧阳平凯
Owner NANJING 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