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Mutant xylose isomerase and its gene and application

A xylose isomerase and amino acid technology, which is applied in the fields of enzyme genetic engineering and enzyme biochemical engineering to achieve the effect of improving activity

Inactive Publication Date: 2007-05-23
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using bioinformatics tools for protein design, combined with site-directed mutagenesis to improve the activity of xylose isomerase in Thermus thermophilus HB8, has not been reported in the literature

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction, screening and identification of recombinant plasmids expressing wild-type Thermus thermophilus HB8 xylose isomerase.

[0028] Thermus thermophilus HB8 was purchased from the American Type Culture Collection (ATCC), ATCC number 27634. Vector pET-22b(+) was purchased from Novagen.

[0029] Yeast powder 4g / L, peptone 8g / L, NaCl 2.0g / L, adjust the pH to 7.0, and autoclave at 121°C for 15min. Thermus thermophilus HB8 was inoculated in the above medium, placed on a shaker at 75°C, 200rpm, and cultured for 12-16 hours. 8000rpm, centrifuge for 15min, collect the bacteria.

[0030]The genome of Thermus Thermophilus was extracted using a genome extraction kit (Shanghai Huashun Bioengineering Co., Ltd.). Using the total DNA of Thermus Thermophilus HB8 as a template, use the following PCR primers (synthesized by Shanghai Boya Company):

[0031] Forward primer: 5'-CCATATGTACGAACCAAAACCGGAACATCGCTTTACCTTT-3' (SEQ ID NO.9)

[0032] Reverse primer: 5'-AAGG...

Embodiment 2

[0035] Example 2: Site-directed Mutagenesis of Xylose Isomerase Gene

[0036] According to the requirements of the multi-point mutation kit (QuikChange® Multi Site-Directed Mutagenesis Kit, purchased from STRATAGENE Company), design the N91D primer, 5'-ATGGTCACCGCC GAC CTCTTCTCCGAC-3'(SEQ ID NO.11) was carried out by PCR using the pET-22b(+)-xylA plasmid as a template to mutate Asn91 into Asp91. According to the instructions of the multi-point mutation kit, the original plasmid template pET-22b(+)-xylA was digested with restriction endonuclease Dpn I, and the PCR product was transformed into competent cells with high efficiency. The transformed competent cells were cultured using conventional genetic engineering techniques, the plasmids were extracted for sequencing and identification, and the recombinant plasmid pET-22b(+)-xylA-N91D containing the mutated gene was obtained.

[0037] Using D375G primer 5'-TGGAACGCCTG GGC CAGCTGGCGGTG-3' (SEQ ID NO.12), PCR was carried out u...

Embodiment 3

[0040] Example 3: Expression of mutant xylose isomerase in Escherichia coli Rosseta

[0041] Transform the recombinant plasmid containing the mutant gene prepared in Example 2 into the commercialized expression host Escherichia coli Rosetta (DE3), pick a single colony into the LB culture fluid containing 75 μg / mL ampicillin and 34 μg / ml chloramphenicol , cultivate overnight at 37°C with shaking, then inoculate into LB medium containing 75 μg / mL ampicillin and 34 μg / mL chloramphenicol at 2% (v / v) inoculum size, and cultivate to OD at 37°C 600 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 SDS-PAGE analysis of the expression product is shown in Figure 2.

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Abstract

The invention discloses mutated xylose isomerase, its coding gene and application. The amino acid sequence of the xylose isomerase is SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7. The xylose isomerase has higher activity than wild one, and can be used for high fructose syrup production. The coding gene of the mutated xylose isomerase can be used for the construction of recombinant bacterium utilizing xylose.

Description

technical field [0001] The invention belongs to the fields of genetic engineering of enzymes and enzyme biochemical engineering. The present invention relates to a mutant xylose isomerase with improved activity, and also relates to a 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 metabolism of sugar in microorganisms and has been used in the industrial production of high fructose syrup [Bhosale SH, Rao MB and Deshpande VV. Molecular and Industrial Aspects of Glucose Isomerase. Microbiol. Rev., 1996, 60 : 280-300]. Xylose isomerase from Thermus thermophilus HB8 [Zeikus JG, Vieille C, Savchenko A. Thermozymes: biotechnology and structure function relationships. Extremophiles, 1998, 2(3): 179-183], the optimum reaction temperature is abou...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12P19/02C12N1/21C12N1/19
Inventor 严明许伟许琳丁莉欧阳平凯
Owner NANJING UNIV OF TECH
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