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Beta-xylosidase in vivo enzyme aggregate and preparation method thereof

A technology of xylosidase and enzyme aggregates, applied in biochemical equipment and methods, glycosylases, enzymes and other directions, can solve problems such as no change, and achieve the effect of improving catalytic efficiency, promoting industrial application, and improving catalytic properties

Active Publication Date: 2017-03-22
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods of expressing short peptides by fusion at the end of the target protein can only partially improve the stability of the enzyme, but have no effect on the optimum reaction temperature of the enzyme, that is, the optimum enzymatic reaction temperature has not changed.

Method used

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  • Beta-xylosidase in vivo enzyme aggregate and preparation method thereof
  • Beta-xylosidase in vivo enzyme aggregate and preparation method thereof
  • Beta-xylosidase in vivo enzyme aggregate and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction of expression vectors containing encoding short peptides

[0034] According to the amino acid sequence of ELK16 (LELELKLKLELELKLK, see SEQ ID: 2), a corresponding DNA sequence containing a stop codon was synthesized at a gene synthesis company. Among them, a PT-linker (PTPPTTPTPPTTPTPTP) sequence was introduced upstream of the ELK16 sequence, and HindIII and XhoI restriction sites were introduced at both ends of the synthetic sequence. The complete gene sequence is shown in SEQ ID: 1. Using the plasmid pET-30a(+) as the basic plasmid, it was digested with HindIII and XhoI, ligated into the corresponding digested ELK16 DNA sequence, and the ligated product was transformed into E.coli DH5α competent cells, and analyzed by colony PCR, restriction enzyme digestion and sequencing Positive transformants were identified to obtain the expression vector pET-ELK, such as figure 1 shown.

Embodiment 2

[0036] Construction of β-xylosidase expression strain

[0037] 1. Amplification of the β-xylosidase gene

[0038] Using the genomic DNA of Thermoanaerobacterium aotearoense SCUT27 as a template, the β-xylosidase gene (GenBank: KX372717) was amplified, and the PCR primers used were as follows:

[0039] XylC-F: 5′-TCGGCT CATATG GAATACCATGTGGCTAAAA-3' (see SEQ ID: 3), the underlined base is the NdeI restriction site.

[0040] XylC-R: 5′-TAGCAA CTCGAG AGAAGAGCCCCAAACTTTTATGTAATTATTTCCT-3' (see SEQ ID: 4), the underlined base is the XhoI restriction site.

[0041] XylC-E-R: 5'-CTGTTC AAGCTT CCAAACTTTTTATGTAATTATTTCCT-3' (see SEQ ID: 5), the underlined base is the HindIII restriction site.

[0042] The primer pairs XylC-F / XylC-R and XylC-F / XylC-E-R were amplified respectively to obtain β-xylosidase genes xylC1 and xylC2 with a length of about 2 kb.

[0043] 2. Construction of recombinant expression vector

[0044] The purified PCR products xylC1 and pET30a(+) were double-d...

Embodiment 3

[0047] Obtainment of routinely expressed soluble β-xylosidase (ThXylC) and active β-xylosidase aggregates (ThXylC-ELK16).

[0048] The overnight cultured recombinant engineered bacteria E.coli BL21(DE3) / pET-xylC1 and E.coli BL21(DE3) / pET30-xylC2-ELK were respectively inoculated at a ratio of 1:100 in fresh water containing 50 μg / ml kanamycin In LB liquid medium, culture at 37°C and 250rpm until the cell density OD 600 About 0.5-0.7, add IPTG with a final concentration of 1 mmol / L to induce the expression of β-xylosidase, and then continue to culture at 30° C. and 180 rpm for 24 hours.

[0049] The bacteria were collected by centrifugation at 4°C and 8000 g, and 10 ml of PB buffer (50 mmol / L phosphate buffer, 50 mmol / L sodium chloride, pH 7.5) was added to each 1 g of wet bacteria to resuspend, and the cells were sonicated. 15000g high-speed centrifugation for 20 minutes.

[0050] For E.coli BL21(DE3) / pET-xylC1, discard the precipitate after centrifugation, and first incubate...

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Abstract

The invention discloses a preparation method of a beta-xylosidase in vivo enzyme aggregate. The preparation method comprises the following steps: (1) splicing a connecting peptide to a biparental short peptide to construct an expression vector, wherein the biparental short peptide is ELK16; (2) connecting a beta-xylosidase coded gene to the expression vector and transforming the mixture to a recipient bacterium to obtain an engineering bacterium which can express beta- xylosidase-short peptide fused protein; and (3) performing induced expression on the engineering bacterium, breaking the walls of the cells, and performing centrifugalization and / or filtration to obtain a precipitate to obtain the beta-xylosidase in vivo enzyme aggregate. Compared with conventionally expressed ThXylC, the ThXylC-ELK16 active enzyme aggregate obtained by the invention is improved in specific activity and catalytic efficiency, and the heat resistance thereof is further remarkably improved. The method has a huge potential of popularization and application in production.

Description

technical field [0001] The invention belongs to the fields of bioengineering and biocatalysis. The invention specifically relates to a method for greatly improving the specific enzyme activity, catalytic efficiency and heat tolerance of the β-xylosidase (ThXylC) by adding an amphipathic short peptide at the end of the β-xylosidase (ThXylC). Background technique [0002] In industrial and agricultural production, enzymes with better heat tolerance are often required as catalysts for reactions. This is because compared with normal temperature enzymes, enzymes with good heat tolerance have the following advantages: (1) The reaction rate is accelerated; At 10°C, the reaction rate response increases 2-4 times. (2) The catalytic process is carried out at high temperature, which can improve the solubility and availability of substrates such as starch, cellulose, lipids, etc., reduce the viscosity of the compound and facilitate the diffusion and mixing of substances; (3) when the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/70
CPCC12N9/2434C12N15/70C12Y302/01037
Inventor 李爽徐天旺杨晓锋
Owner SOUTH CHINA UNIV OF TECH
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