Acidic high-temperature-resistant recombinant cellulase and application thereof

A technology of cellulase and high temperature resistance, applied in the field of genetic engineering technology and biomass refining, can solve the problem of Avicel's inactivity, etc., and achieve the effect of broad substrate spectrum, easy expression, and stable acquisition

Active Publication Date: 2019-10-22
HUAIYIN INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, Endo5 is inactive against Avicel

Method used

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  • Acidic high-temperature-resistant recombinant cellulase and application thereof
  • Acidic high-temperature-resistant recombinant cellulase and application thereof
  • Acidic high-temperature-resistant recombinant cellulase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Gene preparation of endoglucanase (Endo5), carbohydrate binding domain (CBM) and exoglucanase (Exo5).

[0042] The original endo5-cbm28, endo5, cbm3b-exo5 and exo5 genes in the prior art can be prepared artificially, or the total DNA of Caldicellulosiruptor saccharolyticus (purchased from the American Type Culture Collection) can be used as a template to obtain it by PCR.

[0043] The brand-new endo5-cbm28, endo5, cbm3b-exo5 and exo5 genes in this example are based on the characteristics of enzymes Endo5-CBM28, Endo5, CBM3b-Exo5 and Exo5 binding to substrates, after sequence alignment, prediction and design of new sequences , and sent the new sequence to Shanghai Sangong for gene synthesis. The newly designed gene has better activity on substrates such as CMC-Na, filter paper, Avicel, and natural lignocellulose, and better enzymatic hydrolysis effect.

[0044] Using the new sequence synthesized from the whole gene as a template, the following new primer pairs (SEQ ID N...

Embodiment 2

[0057] Construction and verification of recombinant clones and expression vectors pET28a-endo5-2cbm-exo5, pET28a-endo5-cbm3b-exo5 and pET28a-endo5-cbm28-exo5

[0058] The purified PCR products endo5-cbm28, endo5 (prepared in Example 1) and pET28a (Novagen) were double digested with NcoI and SacI, respectively, and the digested PCR fragments and large vector fragments were recovered by agarose gel electrophoresis. The target fragment and carrier recovered by tapping the gel were concentrated and added to 8 μL sterile water for resuspension. Add 1 μL 10×LigaseBuffer and 1 μL Ligase, and ligate overnight at 16°C. The ligation product was transformed into Escherichia coli (E.coliDH5a), and then spread on LB solid medium containing 50 μg / mL kanamycin (Kan), and cultured at 37°C for 13-15 hours.

[0059] Pick multiple single colonies from the transformation plate, and use the Shanghai Sangon Plasmid Extraction Kit to extract the plasmid. The extracted plasmids were verified by dou...

Embodiment 3

[0062] Expression and purification of recombinant cellulase Endo5-2CBM-Exo5, Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5.

[0063] Recombinant clones, expression vectors pET28a-endo5-2cbm-exo5, pET28a-endo5-cbm3b-exo5 and pET28a-endo5-cbm28-exo5 (prepared in Example 2) were chemically transformed into expression host E.coliBL21(DE3) (Novagen), Obtain the recombinant bacteria containing the recombinant plasmid. Positive recombinants were inoculated in 5 mL of Luria-Bertanibroth (LB) liquid medium containing 50 μg / mL Kan, cultured overnight at 37° C. and 200 rpm. Transfer 4 mL of bacterial liquid to 500 mL of LB liquid medium containing 50 μg / mL Kan, and cultivate the bacterial concentration (OD 600 ) to 0.6-0.8. Add IPTG with a final concentration of 0.5mM, and induce expression at 25°C and 200rpm for 12-14h. The culture solution was centrifuged in a high-speed refrigerated centrifuge at 4°C, 10,000 rpm for 8 minutes to collect the bacterial cells. Add 40 mL of sterile water to w...

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Abstract

The invention discloses three kinds of acidic high-temperature-resistant recombinant cellulase and application thereof. Amino acid sequences of the three kinds of recombinant cellulase are shown in SEQIDNO:1-3 respectively. The three kinds of recombinant cellulase have the highest enzyme activity of 1.54 U/mg to CMC-Na and the highest vitality of 0.167 U/mg to Avicel under the conditions that thepH value is 4.0-5.0 and the temperature is 60-80 DEG C. The vitality of the three kinds of recombinant cellulase basically remains unchanged under the conditions that the temperature is 75 DEG C and the pH value is 4.0-4.5 after heat preservation is carried out for 2 hours. The three kinds of recombinant cellulase can directly degrade the CMC-Na, crystalline cellulose, filter paper and other natural lignocellulose such as rice straw and wheat straw, and the highest vitality of the three kinds of recombinant cellulase to the rice straw reaches 0.106 U/mg. Compared with the prior art, the threekinds of recombinant cellulose have greater superiority, can effectively degrade the natural lignocellulose, and have potential industrial application value.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and biomass refining technology, and specifically relates to three acidic high-temperature-resistant recombinant cellulase and applications thereof. Background technique [0002] As a renewable resource, lignocellulose can be used to produce biofuels or other high value-added products. However, the cellulose in lignocellulose is wrapped by hemicellulose and lignin, and is covalently linked and the microfibrils of cellulose itself are cross-linked so that it is difficult to be degraded by cellulase, so it is difficult to convert it into fermentable sugars. Pretreatment of lignocellulose can break the hydrogen bonds, remove lignin and hemicellulose, and enhance the accessibility of cellulase. However, conventional acid-base, ionic liquid and other pretreatments not only increase the production cost, but also easily lead to serious environmental pollution. In addition, artificially sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/02C12P19/12C12P19/14C12R1/19
CPCC12N9/2437C12N15/70C12P19/02C12P19/12C12P19/14
Inventor 李相前夏继林朱晓燕时号
Owner HUAIYIN INSTITUTE OF TECHNOLOGY
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